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Biology stem cells-nih-2001!! Biology stem cells-nih-2001!! Document Transcript

  • Stem Cells: Scientific Progress and Future Research Directions© 2001 Terese Winslow
  • STEM CELLS:SCIENTIFIC PROGRESS AND FUTURERESEARCH DIRECTIONS June 2001
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  • TABLE OF CONTENTSPreface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iExecutive Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ES-1Chapter 1: The Stem Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Chapter 2: The Embryonic Stem Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Chapter 3: The Human Embryonic Stem Cell and The Human Embryonic Germ Cell . . . . . . . . . . . . . . . . . . . . . . . . . 11Chapter 4: The Adult Stem Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Chapter 5: Hematopoietic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Chapter 6: Autoimmune Diseases and the Promise of Stem Cell-Based Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59Chapter 7: Stem Cells and Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67Chapter 8: Rebuilding the Nervous System with Stem Cells . . . . . . . . . . . . . . . . . . . 77Chapter 9: Can Stem Cells Repair a Damaged Heart? . . . . . . . . . . . . . . . . . . . . . . 87Chapter 10: Assessing Human Stem Cell Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93Chapter 11: Use of Genetically Modified Stem Cells in Experimental Gene Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99Appendix A: Early Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1Appendix B: Mouse Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1Appendix C: Human Embryonic Stem Cells and Embryonic Germ Cells . . . . . . . . . . C-1Appendix D: Stem Cell Tables i. Published Reports on Isolation and Differentiation of Mouse Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2 ii. Published Reports on Isolation and Differentiation of Human Fetal Tissue Germ Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-13 iii. Published Reports on Isolation and Differentiation of Human Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-14 iv. Published Reports on Isolation and Differentiation of Human Embryonic Carcinoma Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . D-16 v. Published Reports on Isolation and Differentiation of Human Adult Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-18 vi. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-22
  • Appendix E: Stem Cell Markers i. Markers: How Do Researchers Use Them to Identify Stem Cells? . . . . . E-1 ii. Commonly Used Markers to Identify Stem Cells and Characterize Differentiated Cell Types . . . . . . . . . . . . . . . . . . . . . . . E-5Appendix F: Glossary and Terms i. Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-1 ii. Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-11Appendix G: Informational Resources i. Persons Interviewed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-1 ii. Special Contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-4 iii. Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-5
  • OPPORTUNITIES AND CHALLENGES: A FOCUS ON FUTURE STEM CELL APPLICATIONSBrain The makings of future news headlines about tomorrow’s life saving therapies starts in the Blood biomedical research laboratory. Ideas abound; early vessels successes and later failures and knowledge gained from both; the rare lightning bolt of an unexpected Lungs breakthrough discovery — this is a glimpse of the behind the scenes action of some of the world’s Heart most acclaimed stem cell scientists’ quest to solve Pancreas some of the human body’s most challengingLiver mysteries. Kidney Stem cells — what lies ahead? The following chapters explore some of the cutting edge research featuring stem cells. Disease and disorders with no therapies or at best, partially effective ones, are the lure of the pursuit of stem cell research. Described here are Cartilage examples of significant progress that is a prologue to an era of medical discovery of cell-based therapies that will one day restore function to those whose lives Bone are now challenged every day — but perhaps in the future, no longer. Muscle
  • REPORT PREPARED BY THE NATIONAL INSTITUTES OF HEALTH Ruth Kirschstein, M.D. Acting Director Office of Science Policy Lana R. Skirboll, Ph.D. Director
  • PREFACEOn February 28, 2001, Tommy G. Thompson, How and whether stem cells derived from any ofSecretary of Health and Human Services, requested these sources can be manipulated to replace cellsthat the National Institutes of Health prepare a in diseased tissues, used to screen drugs and toxins,summary report on the state of the science on stem or studied to better understand normal developmentcells. This report was developed in response to his depends on knowing more about their basic proper-request. It provides the current information about the ties. In this respect, stem cell research is in manybiology of stem cells derived from all sources— ways no different than many other areas of modernembryo, fetal tissue, and adult. biology; it is advancing because new tools and new knowledge are providing the opportunities for newSince 1998, when human pluripotent stem cells were insights. Like all fields of scientific inquiry, research onfirst isolated, research on stem cells has received stem cells raises as many questions as it answers. Thismuch public attention, both because of its extraordi- report describes the state of the science of stem cellnary promise and because of relevant legal and biology and gives some clues as to the many andethical issues. Underlying this recent public scrutiny is varied questions that remain to be answered.decades of painstaking work by scientists in manyfields, who have been deciphering some of the mostfundamental questions about life with the goal of WHAT IS THE SCOPE OF THEimproving health. REPORT?In the last several decades, investments in basic The report is a review of the state of the science ofresearch have yielded extensive knowledge about stem cell research as of June 17, 2001. Included inthe many and complex processes involved in the this report is subject matter addressing stem cellsdevelopment of an organism, including the control from adult, fetal tissue, and embryonic sources.of cellular development. But many questions remain. Because so much of the progress made to date wasHow does a single cell—the fertilized egg—give rise dependent on animal models, a significant emphasisto a complex, multi-cellular organism? The question is placed on understandings gained from mouserepresents a fundamental challenge in develop- models of development and mouse stem cellmental biology. Researchers are now seeking to research. The report also devotes substantial attentionunderstand in greater detail the genetic factors that to scientific publications on the characterization ofregulate cell differentiation in early development. specialized cells developed from embryonic stem cells and the plasticity of adult stem cells. A generalPut simply, stem cells are self-renewing, unspecial- overview of early development is provided in theized cells that can give rise to multiple types all of Appendix to assist the reader in understanding thespecialized cells of the body. The process by which key events in formation of cells, tissues, and the wholedividing, unspecialized cells are equipped to per- organism.form specific functions—muscle contraction or nervecell communication, for example—is called differen- Both scientific and lay publications use a variety oftiation, and is fundamental to the development of terms to describe stem cells and their properties. Forthe mature organism. It is now known that stem cells, this reason, this report adopts a lexicon of terms andin various forms, can be obtained from the embryo, it is used consistently throughout. To aid the reader, athe fetus, and the adult. glossary and terms section is provided. In several i
  • Prefaceplaces in the report, discovery timelines are provided. HOW WAS THE REPORTThe various sources of stem cells are described, as DEVELOPED?are the techniques used to isolate and developthem. A comprehensive listing of various stem cell The report was prepared under the auspices of theisolation and characterizations is also included. Office of Science Policy, Office of the Director, NIH. Several approaches were taken to obtain relevantIn order to ensure the reader is provided information scientific information for the report. A thorough reviewboth about the basic biology of stem cells, and their of the extant literature, including more than 1200therapeutic potential, the report contains several scientific publications was conducted. Scientificchapters focused on particular diseases which might experts (both domestic and international) from allbenefit from stem cell research. These chapters on areas of relevant biomedical research in stem cellsthe use of hematopoietic stem cells, followed by were interviewed in depth. While the majority of thefocus features on specific nervous system diseases, work presented in this report emanates fromdiabetes, heart disease, and autoimmune diseases investigators in academic laboratories, extensiveserve merely as examples of the many applications discussions were held with scientists in the privateof stem cells that are being pursued. Also included pharmaceutical and biotechnology sectors. Thus, theare features that review aspects of stem cells as report makes every effort to encompass what istherapeutic delivery tools for gene therapy and, known and not known about stem cell biology and is,importantly, the safety considerations for developing therefore, not limited to research that is or has beenstem cell-based therapies. funded by the NIH.WHAT IS NOT IN THE SCOPE OF In recent months, there have been many reports inTHE REPORT? the lay press regarding scientific discoveries on various types of stem cells. The science representedNIH recognizes the compelling ethical and legal in this report focuses exclusively on scientificissues surrounding human pluripotent stem cell publications or public presentations. In cases whereresearch. Because extensive discussions regarding technical or logistical information key to the under-these issues have been presented in various forums standing of the details of science was needed,elsewhere, they are not part of this review of the state personal communications with the informationof the science. Also, the report does not make sources were cited.recommendations pertaining to the policies govern-ing Federal funding of such research. ii
  • EXECUTIVE SUMMARYINTRODUCTION At about the same time as scientists were beginning to explore human pluripotent stem cells fromA stem cell is a special kind of cell that has a unique embryos and fetal tissue, a flurry of new informationcapacity to renew itself and to give rise to specialized was emerging about a class of stem cells that havecell types. Although most cells of the body, such as been in clinical use for years—so-called adult stemheart cells or skin cells, are committed to conduct a cells. An adult stem cell is an undifferentiated cellspecific function, a stem cell is uncommitted and that is found in a differentiated (specialized) tissue inremains uncommitted, until it receives a signal to the adult, such as blood. It can yield the specializeddevelop into a specialized cell. Their proliferative cell types of the tissue from which it originated. In thecapacity combined with the ability to become spe- body, it too, can renew itself. During the past decade,cialized makes stem cells unique. Researchers have scientists discovered adult stem cells in tissues thatfor years looked for ways to use stem cells to replace were previously not thought to contain them, such ascells and tissues that are damaged or diseased. the brain. More recently, they reported that adultRecently, stem cells have received much attention. stem cells from one tissue appear to be capable ofWhat is “new” and what has brought stem cell bio- developing into cell types that are characteristic oflogy to the forefront of science and public policy? other tissues. For example, although adult hematopoietic stem cells from bone marrow haveScientists interested in human development have long been recognized as capable of developing intobeen studying animal development for many years. blood and immune cells, recently scientists reportedThis research yielded our first glimpse at a class of that, under certain conditions, the same stem cellsstem cells that can develop into any cell type in the could also develop into cells that have many of thebody. This class of stem cells is called pluripotent, characteristics of neurons. So, a new concept and ameaning the cells have the potential to develop new term emerged-adult stem cell plasticity.almost all of the more than 200 different known celltypes. Stem cells with this unique property come from Are human adult and embryonic stem cells equiva-embryos and fetal tissue. lent in their potential for generating replacement cells and tissues? Current science indicates that, althoughIn 1998, for the first time, investigators were able to both of these cell types hold enormous promise,isolate this class of pluripotent stem cell from early adult and embryonic stem cells differ in importanthuman embryos and grow them in culture. In the few ways. What is not known is the extent to which theseyears since this discovery, evidence has emerged that different cell types will be useful for the developmentthese stem cells are, indeed, capable of becoming of cell-based therapies to treat disease.almost all of the specialized cells of the body and,thus, may have the potential to generate replace- Some considerations are noteworthy regarding thisment cells for a broad array of tissues and organs, report. First, in recent months, there have been manysuch as the heart, the pancreas, and the nervous discussions in the lay press about the anticipatedsystem. Thus, this class of human stem cell holds the abilities of stem cells from various sources and pro-promise of being able to repair or replace cells or jected benefits to be realized from them in replacingtissues that are damaged or destroyed by many of cells and tissues in patients with various diseases. Theour most devastating diseases and disabilities. terminology used to describe stem cells in the lay ES-1
  • Executive Summaryliterature is often confusing or misapplied. Second, cells are not themselves embryos. In fact, evidence iseven among biomedical researchers, there is a lack emerging that these cells do not behave in the labo-of consistency in common terms to describe what ratory as they would in the developing embryo—thatstem cells are and how they behave in the research is, the conditions in which these cells develop in cul-laboratory. Third, the field of stem cell biology is ture are likely to differ from those in the developingadvancing at an incredible pace with new discover- embryo.ies being reported in the scientific literature on a Embryonic germ cell. An embryonic germ cell isweekly basis. derived from fetal tissue. Specifically, they are isolatedThis summary begins with common definitions and from the primordial germ cells of the gonadal ridgeexplanations of key concepts about stem cells. It of the 5- to 10-week fetus. Later in development, theends with an assessment of how adult, embryonic gonadal ridge develops into the testes or ovaries andand fetal stem cells are similar and how they are dif- the primordial germ cells give rise to eggs or sperm.ferent. In between lie important details that describe Embryonic stem cells and embryonic germ cells arewhat researchers have discovered about stem cells pluripotent, but they are not identical in their proper-and how they are being used in the laboratory. ties and characteristics. Differentiation. Differentiation is the process by whichDEFINITIONS AND GENERAL an unspecialized cell (such as a stem cell) becomesCONCEPTS ABOUT STEM CELLS specialized into one of the many cells that make upIn developing this report, some conventions were the body. During differentiation, certain genesestablished to describe consistently what stem cells become activated and other genes become inacti-are, what characteristics they have, and how they are vated in an intricately regulated fashion. As a result, aused in biomedical research. Here are some of the differentiated cell develops specific structures andkey definitions that are used throughout this report. performs certain functions. For example, a mature, differentiated nerve cell has thin, fiber-like projectionsStem cell. A stem cell is a cell from the embryo, that send and receive the electrochemical signalsfetus, or adult that has, under certain conditions, the that permit the nerve cell to communicate with otherability to reproduce itself for long periods or, in the nerve cells. In the laboratory, a stem cell can becase of adult stem cells, throughout the life of the manipulated to become specialized or partiallyorganism. It also can give rise to specialized cells that specialized cell types (e.g., heart muscle, nerve, ormake up the tissues and organs of the body. Much pancreatic cells) and this is known as directedbasic understanding about embryonic stem cells has differentiation.come from animal research. In the laboratory, thistype of stem cell can proliferate indefinitely, a proper- Adult stem cell. An adult stem cell is an undifferenti-ty that is not shared by adult stem cells. ated (unspecialized) cell that occurs in a differentiat- ed (specialized) tissue, renews itself, and becomesPluripotent stem cell. A single pluripotent stem cell specialized to yield all of the specialized cell types ofhas the ability to give rise to types of cells that devel- the tissue from which it originated. Adult stem cellsop from the three germ layers (mesoderm, endo- are capable of making identical copies of them-derm, and ectoderm) from which all the cells of the selves for the lifetime of the organism. This property isbody arise. The only known sources of human pluripo- referred to as “self-renewal.” Adult stem cells usuallytent stem cells are those isolated and cultured from divide to generate progenitor or precursor cells,early human embryos and from fetal tissue that was which then differentiate or develop into “mature” celldestined to be part of the gonads. types that have characteristic shapes and special- ized functions, e.g., muscle cell contraction or nerveEmbryonic stem cell. An embryonic stem cell is cell signaling. Sources of adult stem cells includederived from a group of cells called the inner cell bone marrow, blood, the cornea and the retina ofmass, which is part of the early (4- to 5-day) embryo the eye, brain, skeletal muscle, dental pulp, liver, skin,called the blastocyst. Once removed from the blasto- the lining of the gastrointestinal tract, and pancreas.cyst, the cells of the inner cell mass can be cultured The most abundant information about adult humaninto embryonic stem cells. These embryonic stem ES-2
  • Executive Summarystem cells comes from studies of hematopoietic in the starting tissue; such results from fat cells may, in(blood-forming) stem cells isolated from the bone fact, be due to the presence of hematopoietic stemmarrow and blood. These adult stem cells have been cells in the fat tissue. The importance of showing thatextensively studied and applied therapeutically for one cell type can reproducibly become another andvarious diseases. At this point, there is no isolated self-replicate cannot be overemphasized.population of adult stem cells that is capable of Progenitor or precursor cell. A progenitor or pre-forming all the kinds of cells of the body. Adult stem cursor cell occurs in fetal or adult tissues and is par-cells are rare. Often they are difficult to identify, iso- tially specialized; it divides and gives rise to differenti-late, and purify. There are insufficient numbers of cells ated cells. Researchers often distinguish precursor/available for transplantation and adult stem cells do progenitor cells from adult stem cells in the followingnot replicate indefinitely in culture. way: when a stem cell divides, one of the two newPlasticity. Plasticity is the ability of an adult stem cell cells is often a stem cell capable of replicating itselffrom one tissue to generate the specialized cell again. In contrast, when a progenitor/precursor celltype(s) of another tissue. A recently reported example divides, it can form more progenitor/precursor cells orof plasticity is that, under specific experimental condi- it can form two specialized cells, neither of which istions, adult stem cells from bone marrow generated capable of replicating itself. Progenitor/precursor cellscells that resemble neurons and other cell types that can replace cells that are damaged or dead, thusare commonly found in the brain. The concept of maintaining the integrity and functions of a tissueadult stem cell plasticity is new, and the phenome- such as liver or brain. Progenitor/precursor cells givenon is not thoroughly understood. Evidence suggests rise to related types of cells-lymphocytes such as Tthat, given the right environment, some adult stem cells, B cells, and natural killer cells, for example—butcells are capable of being “genetically repro- in their normal state do not generate a wide varietygrammed” to generate specialized cells that are of cell types.characteristic of different tissues.Clonality or clonally derived stem cell. A cell is said CHALLENGES IN STEM CELLto be clonally derived or to exhibit clonality if it was RESEARCHgenerated by the division of a single cell and is It is important to understand some of the difficultiesgenetically identical to that cell. In stem cell that researchers have had in isolating various types ofresearch, the concept of clonality is important for stem cells, working with the cells in the laboratory,several reasons. For researchers to fully understand and proving experimentally that the cells are trueand harness the ability of stem cells to generate stem cells. Most of the basic research discoveries onreplacement cells and tissues, the exact identity of embryonic and adult stem cells come from researchthose cells’ genetic capabilities and functional quali- using animal models, particularly mice.ties must be known. Human pluripotent stem cellsfrom embryos and fetal tissue are by their nature In 1981, researchers reported methods for growingclonally derived. However, very few studies have mouse embryonic stem cells in the laboratory, and itshown clonal properties of the cells that are devel- took nearly 20 years before similar achievementsoped from adult stem cells. It is crucial to know could be made with human embryonic stem cells.whether a single cell is capable of developing an Much of the knowledge about embryonic stem cellsarray of cell types, or whether multiple stem cell has emerged from two fields of research: appliedtypes, that when grown together, are capable of reproductive biology, i.e., in vitro fertilization technolo-forming multiple cell types. For instance, recent gies, and basic research on mouse embryology.research has shown that a mixture of cells removed There have been many technical challenges thatfrom fat tissue or umbilical cord blood are capable have been overcome in adult stem cell research asof developing into blood cells, bone cells, and well. Some of the barriers include: the rare occur-perhaps others. Researchers have not shown that a rence of adult stem cells among other, differentiatedsingle cell is responsible for giving rise to other cell cells, difficulties in isolating and identifying the cellstypes or, if so, what kind of cell it is. These results may (researchers often use molecular “markers” to identifywell be attributable to multiple types of precursor cells ES-3
  • Executive Summaryadult stem cells), and in many cases, difficulties in At present, there have been multiple human adultgrowing adult stem cells in tissue culture. Much of the stem cell lines that have been created through aresearch demonstrating the plasticity of adult stem combination of public and private resources (e.g.,cells comes from studies of animal models in which a hematopoietic stem cells). Substantial adult stem cellmixture of adult stem cells from a donor animal is research has been underway for many years, and ininjected into another animal, and the development recent years this has included basic studies on theof new, specialized cells is traced. “plasticity” of such cells.In 1998, James Thomson at the University ofWisconsin-Madison isolated cells from the inner cell WHAT KINDS OF RESEARCH MIGHTmass of the early embryo, called the blastocyst, and BE CONDUCTED WITH STEM CELLS?developed the first human embryonic stem cell lines. There has been much written about the newAt the same time, John Gearhart at Johns Hopkins discoveries of various stem cell types and theirUniversity reported the first derivation of human properties. Importantly, these cells are research toolsembryonic germ cells from an isolated population of and they open many doors of opportunity for bio-cells in fetal gonadal tissue, known as the primordial medical research.germ cells, which are destined to become the eggsand sperm. From both of these sources, the Transplantation Research—Restoring Vitalresearchers developed pluripotent stem cell “lines,” Body Functionswhich are capable of renewing themselves for long Stem cells may hold the key to replacing cells lost inperiods and giving rise to many types of human cells many devastating diseases. There is little doubt thator tissues. Human embryonic stem cells and embry- this potential benefit underpins the vast interest aboutonic germ cells differ in some characteristics, stem cell research. What are some of thesehowever, and do not appear to be equivalent. diseases? Parkinson’s disease, diabetes, chronic heartWhy are the long-term proliferation ability and disease, end-stage kidney disease, liver failure, andpluripotency of embryonic stem cells and embryonic cancer are just a few for which stem cells have thera-germ cells so important? First, for basic research peutic potential. For many diseases that shorten lives,purposes, it is important to understand the genetic there are no effective treatments but the goal is toand molecular basis by which these cells continue to find a way to replace what natural processes havemake many copies of themselves overlong periods taken away. For example, today, science has broughtof time. Second, if the cells are to be manipulated us to a point where the immune response can beand used for transplantation, it is important to have subdued, so that organs from one person can besufficient quantities of cells that can be directed to used to replace the diseased organs and tissues ofdifferentiate into the desired cell type(s) and used to another. But, despite recent advances in transplanta-treat the many patients that may be suffering from a tion sciences, there is a shortage of donor organsparticular disease. that makes it unlikely that the growing demand for lifesaving organ replacements will be fully metIn recent months, other investigators have been through organ donation strategies.successful in using somewhat different approaches toderiving human pluripotent stem cells. At least 5 other The use of stem cells to generate replacement tissueslaboratories have been successful in deriving pluri- for treating neurological diseases is a major focus ofpotent stem cells from human embryos and one research. Spinal cord injury, multiple sclerosis, Parkin-additional laboratory has created cell lines from fetal son’s disease, and Alzheimer’s disease are amongtissue. In each case, the methods for deriving pluri- those diseases for which the concept of replacingpotent stem cells from human embryos and embry- destroyed or dysfunctional cells in the brain or spinalonic germ cells from fetal tissue are similar, yet they cord is a practical goal. This report features severaldiffer in the isolation and culture conditions as initially recent advances that demonstrate the regenerativedescribed by Thomson and Gearhart, respectively. It properties of adult and embryonic stem cells.is not known to what extent U.S.-based researchers Another major discovery frontier for research on adultare using these additional sources of embryonic stem and embryonic stem cells is the development ofand germ cells. ES-4
  • Executive Summarytransplantable pancreatic tissues that can be used to research that links developmental biology and stemtreat diabetes. Scientists in academic and industrial cell biology is understanding the genes and mole-research are vigorously pursuing all possible avenues cules, such as growth factors and nutrients, thatof research, including ways to direct the specializa- function during the development of the embryo sotion of adult and embryonic stem cells to become that they can be used to grow stem cells in thepancreatic islet-like cells that produce insulin and can laboratory and direct their development intobe used to control blood glucose levels. Researchers specialized cell types.have recently shown that human embryonic stemcells to be directly differentiated into cells that pro- Therapeutic Delivery Systemsduce insulin. Stem cells are already being explored as a vehicle for delivering genes to specific tissues in the body.There are common misconceptions about both adult Stem cell-based therapies are a major area of inves-and human embryonic stem cells. First, the lines of tigation in cancer research. For many years, restora-unaltered human embryonic stem cells that exist will tion of blood and immune system function has beennot be suitable for direct use in patients. These cells used as a component in the care of cancer patientswill need to be differentiated or otherwise modified who have been treated with chemotherapeuticbefore they can be used clinically. Current chal- agents. Now, researchers are trying to devise morelenges are to direct the differentiation of embryonic ways to use specialized cells derived from stem cellsstem cells into specialized cell populations, and also to target specific cancerous cells and directly deliverto devise ways to control their development or prolif- treatments that will destroy or modify them.eration once placed in patients.A second misconception is that adult stem cells are Other Applications of Stem Cellsready to use as therapies. With the exception of the Future uses of human pluripotent cell lines mightclinical application of hematopoietic stem cells to include the exploration of the effects of chromoso-restore the blood and immune system, this is not the mal abnormalities in early development. This mightcase. The therapeutic use of this mixture of cells has include the ability to monitor the development ofproven safe because the mixture is place back into early childhood tumors, many of which are embryon-the environment from which it was taken, e.g., the ic in origin. Another future use of human stem cellsbone marrow. In fact, many of the adult stem cell and their derivatives include the testing of candidatepreparations currently being developed in the labora- therapeutic drugs. Although animal model testing is atory represent multiple cell types that are not fully mainstay of pharmaceutical research, it cannotcharacterized. In order to safely use stem cells or cells always predict the effects that a developmental drugdifferentiated from them in tissues other than the tissue may have on human cells. Stem cells will likely befrom which they were isolated, researchers will need used to develop specialized liver cells to evaluatepurified populations (clonal lines) of adult stem cells. drug detoxifying capabilities and represents a newIn addition, the potential for the recipient of a stem type of early warning system to prevent adverse reac-cell transplant to reject these tissues as foreign is very tions in patients. The coupling of stem cells with thehigh. Modifications to the cells, to the immune sys- information learned from the human genome projecttem, or both will be a major requirement for their use. will also likely have many unanticipated benefits inIn sum, with the exception of the current practice of the future.hematopoietic stem cell transplantation, much basic Critical Evidence and Questions about Stemresearch lies ahead before direct patient application Cell Researchof stem cell therapies is realized. What is the evidence that specialized cells generatedBasic Research Applications from human stem cells can replace damaged orEmbryonic stem cells will undoubtedly be key diseased cells and tissues? Currently, there are moreresearch tools for understanding fundamental events questions than answers.in embryonic development that one day may Most of the evidence that stem cells can be directedexplain the causes of birth defects and approaches to differentiate into specific types of cells suitable forto correct or prevent them. Another important area of ES-5
  • Executive Summarytransplantation—for example, neurons, heart muscle and they can give rise to mature cell types thatcells, or pancreatic islet cells—comes from experi- have characteristic shapes and specializedments with stem cells from mice. And although more functions of a particular tissue.is known about mouse stem cells, not all of that infor- • Adult stem cells are rare. Often they are difficultmation can be translated to the understanding of to identify, isolate, and purify.human stem cells. Mouse and human cells differ in • One important, limiting factor for the use of adultsignificant ways, such as the laboratory conditions stem cells in future cell-replacement strategies isthat favor the growth and specialization of specific that there are insufficient numbers of cellscell types. available for transplantation. This is becauseAnother important aspect of developing therapies most adult stem cell lines when grown in abased on stem cells will be devising ways to prevent culture dish are unable to proliferate in anthe immune system of recipients from rejecting the unspecialized state for long periods of time. Indonated cells and tissues that are derived from cases where they can be grown under thesehuman pluripotent stem cells. Modifying or evading conditions, researchers have not been able tothe immune rejection of cells or tissues developed direct them to become specialized as function-from embryonic stem cells will not be able to be ally useful cells.done exclusively using mouse models and human • Stem cells from the bone marrow are the most-adult stem cells. studied type of adult stem cells. Currently, theyAs with any new research tool, it will also be important are used clinically to restore various blood andto compare the techniques and approaches that immune components to the bone marrow viavarious laboratories are using to differentiate and use transplantation. There are two major types ofhuman embryonic stem cells. Such research will pro- stem cells found in bone: hematopoietic stemvide a more complete understanding of the cells’ cells which form blood and immune cells, andcharacteristics. One key finding about the directed stromal (mesenchymal) stem cells that normallydifferentiation of pluripotent stem cells learned thus form bone, cartilage, and fat. The restrictedfar is that relatively subtle changes in culture condi- capacity of hematopoietic stem cells to grow intions can have dramatic influences on the types of large numbers and remain undifferentiated incells that develop. the culture dish is a major limitation to their broader use for research and transplantationWhat Is Known About Adult Stem Cells? studies. Researchers have reported that at least • To date, published scientific papers indicate that two other populations of adult stem cells occur adult stem cells have been identified in brain, in bone marrow and blood, but these cells are bone marrow, peripheral blood, blood vessels, not well characterized. skeletal muscle, epithelia of the skin and diges- • Evidence to date indicates that umbilical cord tive system, cornea, dental pulp of the tooth, blood is an abundant source of hematopoietic retina, liver, and pancreas. Thus, adult stem cells stem cells. There do not appear to be any have been found in tissues that develop from all qualitative differences between the stem cells three embryonic germ layers. obtained from umbilical cord blood and those • There is no evidence of an adult stem cell that is obtained from bone marrow or peripheral blood. pluripotent. It has not been demonstrated that • Several populations of adult stem cells have one adult stem cell can be directed to develop been identified in the brain, particularly in a into any cell type of the body. That is, no adult region important in memory, known as the hip- stem cell has been shown to be capable of pocampus. Their function in the brain is unknown. developing into cells from all three embryonic When the cells are removed from the brain of germ layers. mice and grown in tissue culture, their prolifera- • In the body, adult stem cells can proliferate tion and differentiation can be influenced by without differentiating for a long period (the char- various growth factors. acteristic referred to as long-term self-renewal), ES-6
  • Executive Summary • Current methods for characterizing adult stem important for cells to maintain their capacity to cells depend on determining cell-surface replicate. Human pluripotent stem cells appear markers and making observations about their to maintain relatively long telomeres, indicating differentiation patterns in culture dishes. that they have the ability to replicate for many, • Some adult stem cells appear to have the many generations. capability to differentiate into tissues other than • Evidence of structural, genetic, and functional the ones from which they originated; this is cells characteristic of specialized cells referred to as plasticity. Reports of human or developed from cultured human and mouse mouse adult stem cells that demonstrate plastic- embryonic stem cells has been shown for: ity and the cells they differentiate or specialize 1) Pancreatic islet-cell like cells that secrete into include: 1) blood and bone marrow insulin (mouse and human); 2) cardiac muscle (unpurified hematopoietic) stem cells differenti- cells with contractile activity (mouse and ate into the 3 major types of brain cells (neurons, human); 3) blood cells (human and mouse); oligodendrocytes, and astrocytes), skeletal 4) nerve cells that produce certain brain chemi- muscle cells, cardiac muscle cells, and liver cals (mouse). cells; 2) bone marrow (stromal) cells differentiates • At the time of this report, there are approximately into cardiac muscle cells, skeletal muscle cells, 30 cell lines of human pluripotent stem cells that fat, bone, and cartilage; and 3) brain stem cells have been derived from human blastocysts or differentiate into blood cells and skeletal fetal tissue. muscle cells. • Overall, it appears human embryonic cells and • Very few published research reports on the embryonic germ cells are not equivalent in their plasticity of adult stem cells shown that a single, potential to proliferate or differentiate. identified adult stem cell can give rise to a differ- entiated cell type of another tissue. That is, there What are Some of the Questions that Need to is limited evidence that a single adult stem cell be Answered about Stem Cells? or genetically identical line of adult stem cells • What are the mechanisms that allow human demonstrates plasticity. Researchers believe that embryonic stem cells and embryonic germ cells it is most likely that a variety of populations of to proliferate in vitro without differentiating? stem cells may be responsible for the phenom- • What are the intrinsic controls that keep stem ena of developing multiple cell types. cells from differentiating? • A few experiments have shown plasticity of adult • Is there a universal stem cell? That is, could a stem cells by demonstrating the development of kind of stem cell exist (possibly circulating in the mature, fully functional cells in tissues other than blood) that can generate the cells of any organ which they were derived and the restoration of or tissue? lost or diminished function in an animal model. • Do adult stem cells exhibit plasticity as a normalWhat is Known About Human Pluripotent Stem Cells? event in the body or is it an artifact of the culture • Since 1998, research teams have refined the conditions? If plasticity occurs normally, is it a techniques for growing human pluripotent cells in characteristic of all adult stem cells? What are culture systems. Collectively, the studies indicate the signals that regulate the proliferation and that it is now possible to grow these cells for up differentiation of stem cells that demonstrate to two years in a chemically defined medium. plasticity? • The cell lines have been shown to have a • What are the factors responsible for stem cells to normal number of chromosomes and they “home” to sites of injury or damage? generate cell types that originate from all three • What are the intrinsic controls that direct stem primary germ layers. cells along a particular differentiation pathway to • Cultures of human pluripotent stem cells have form one specialized cell over another? How are active telomerase, which is an enzyme that such intrinsic regulators, in turn, influenced by the maintains the length of telomeres and is ES-7
  • Executive Summary microenvironment, or niche, where stem cells COMPARISONS OF ADULT STEM normally reside? CELLS AND EMBRYONIC STEM CELLS • Will the knowledge about the genetic mecha- nisms regulating the specialization of embryonic Biomedical research on stem cells is at an early cells into cells from all embryonic germ layers stage, but is advancing rapidly. After many years of during development enable the scientists to isolating and characterizing these cells, researchers engineer adult stem cells to do the same? are just now beginning to employ stem cells as discovery tools and a basis for potential therapies. • What are the sources of adult stem cells in the This new era of research affords an opportunity to use body? Are they “leftover” embryonic stem cells, what has already been learned to explore the similar- or do they arise in some other way? And if the ities and differences of adult and embryonic stem latter is true—which seems to be the case— cells. (In this discussion, comments about embryonic exactly how do adult stem cells arise, and why stem cells derived from human embryos, and do they remain in an undifferentiated state when embryonic germ cells derived from fetal tissue, will be all the cells around them have differentiated? referred to equally as embryonic stem cells, unless • How many kinds of adult stem cells exist, and in otherwise distinguished.) which tissues do they exist? How are Adult and Embryonic Stem Cells • Is it possible to manipulate adult stem cells to Similar? increase their ability to proliferate in a culture dish so that adult stem cells can be used as a By definition, stem cells have in common the ability sufficient source of tissue for transplants? to self-replicate and to give rise to specialized cells and tissues (such as cells of the heart, brain, bone, • Does the genetic programming status of stem etc.) that have specific functions. In most cases, stem cells play a significant role in maintaining the cells can be isolated and maintained in an unspe- cells, directing their differentiation, or determining cialized state. Scientists use similar techniques (i.e., their suitability for transplant? cell-surface markers and monitoring the expression of • Are the human embryonic stem and germ cells certain genes) to identify or characterize stem cells that appear to be homogeneous and undiffer- as being unspecialized. Scientists then use different entiated in culture, in fact, homogeneous and genetic or molecular markers to determine that the undifferentiated? Or are they heterogeneous cells have differentiated—a process that might be and/or “partially” differentiated? compared to distinguishing a particular cell type by • What are the cellular and molecular signals that reading its cellular barcode. are important in activating a human pluripotent Stem cells from both adult and embryonic sources stem cell to begin differentiating into a special- can proliferate and specialize when transplanted into ized cell type? an animal with a compromised immune system. • Will analysis of genes from human pluripotent (Immune-deficient animals are less likely to reject the stem cells reveal a common mechanism that transplanted tissue). Scientists also have evidence maintains cells in an undifferentiated state? that differentiated cells generated from either stem • Do all pluripotent stem cells pass through a cell type, when injected or transplanted into an ani- progenitor/precursor cell stage while becoming mal model of disease or injury, undergo “homing,” a specialized? If so, can a precursor or progenitor process whereby the transplanted cells are attracted cell stage be maintained as optimal cells for by and travel to the injured site. Similarly, researchers therapeutic transplantation? are finding that the cellular and non-cellular “environ- ment” into which stem cell-derived tissues are placed • What stage of differentiation of stem cells will be (e.g., whether they are grown in a culture dish or best for transplantation? Would the same stage transplanted into an animal) prominently influences be optimal for all transplantation applications, or how the cells differentiate. will it differ on a case-by-case basis? • What differentiation stages of stem cells would Another important area that requires substantially more be best for screening drugs or toxins, or for research concerns the immunologic characteristics of delivering potentially therapeutic drugs? ES-8
  • Executive Summaryhuman adult and embryonic stem cells. If any of these potential as embryonic stem and germ cells? Tostem cells are to be used as the basis for therapy, it is date, there are no definitive answers to these ques-critical understand how the body’s immune system will tions, and the answers that do exist are sometimesrespond to the transplantation of tissue derived from conflicting.these cells. At this time, there is no clear advantage of These sources of stem cells do not seem to have theone stem cell source over the other in this regard. same ability to proliferate in culture and at the sameHow are Adult and Embryonic Stem Cells time retain the capacity to differentiate into function-Different? ally useful cells. Human embryonic stem cells can be generated in abundant quantities in the laboratoryPerhaps the most distinguishing feature of embryonic and can be grown (allowed to proliferate) in theirstem cells and adult stem cells is their source. Most undifferentiated (or unspecialized) state for many,scientists now agree that adult stem cells exist in many generations. From a practical perspective inmany tissues of the human body (in vivo), although basic research or eventual clinical application, it isthe cells are quite rare. In contrast, it is less certain significant that millions of cells can be generatedthat embryonic stem cells exist as such in the from one embryonic stem cell in the laboratory. Inembryo. Instead, embryonic stem cells and embry- many cases, however, researchers have had difficultyonic germ cells develop in tissue culture after they finding laboratory conditions under which some adultare derived from the inner cell mass of the early stem cells can proliferate without becomingembryo or from the gonadal ridge tissue of the fetus, specialized. This problem is most pronounced withrespectively. hematopoietic stem cells isolated from blood orDepending on the culture conditions, embryonic bone marrow. These cells when cultured in thestem cells may form clumps of cells that can differ- laboratory either fail to proliferate or do so to aentiate spontaneously to generate many cell types. limited extent, although they do proliferate if trans-This property has not been observed in cultures of planted into an animal or human. This technicaladult stem cells. Also, if undifferentiated embryonic barrier to proliferation has limited the ability ofstem cells are removed from the culture dish and researchers to explore the capacity of certain typesinjected into a mouse with a compromised immune of adult stem cells to generate sufficient numbers ofsystem, a benign tumor called a teratoma can specialized cells for transplantation purposes.develop. A teratoma typically contains a mixture of These differences in culturing conditions contribute topartially differentiated cell types. For this reason, the contrasts in the experimental systems used toscientists do not anticipate that undifferentiated evaluate the ability to become specialized underembryonic stem cells will be used for transplants or particular laboratory conditions. Much of the infor-other therapeutic applications. It is not known whether mation on the directed differentiation of embryonicsimilar results are observed with adult stem cells. stem cells into cells with specialized function comesStem cells in adult tissues do not appear to have the from studying mouse or human embryonic cell linessame capacity to differentiate as do embryonic grown in laboratory culture dishes. In contrast, moststem cells or embryonic germ cells. Embryonic stem knowledge about the differentiation of adult stemand germ cells are clearly pluripotent; they can cells differentiation are from observations of cells anddifferentiate into any tissues derived from all three tissues in animal models in which mixtures of cellsgerm layers of the embryo (ectoderm, mesoderm, have been implanted.and endoderm). But are adult stem cells also Stem cells also differ in their capacity to specializepluripotent? When they reside in their normal tissue into various cell and tissue types. Current evidencecompartments—the brain, the bone marrow, the indicates that the capability of adult stem cells toepithelial lining of the gut, etc.—they produce the give rise to many different specialized cell types iscells that are specific to that kind of tissue and they more limited than that of embryonic stem cells. Ahave been found in tissues derived from all three single embryonic stem cell has been shown to giveembryonic layers. But can adult stem cells be taken rise to specialized cells from all three embryonicout of their normal environment and be manipulated layers. However, it has not yet been shown that aor otherwise induced to have the same differentiation ES-9
  • Executive Summarysingle adult stem cell can give rise to specialized experiments with different animal models or, in thiscells derived from all three embryonic germ cell case, different cell lines. However, there have beenlayers. Therefore, a single adult stem cell has not very few studies that compare various stem cell linesbeen shown to have the same degree of pluri- with each other. It may be that one source provespotency as embryonic stem cells. better for certain applications, and a different cell source proves better for others.CONCLUSIONS For researchers and patients, there are many practi-Two important points about embryonic and adult cal questions about stem cells that cannot yet bestem cells have emerged so far: the cells are differ- answered. How long will it take to develop therapiesent and present immense research opportunities for for Parkinson’s Disease and diabetes with and withoutpotential therapy. As research goes forward, scientists human pluripotent stem cells? Can the full range ofwill undoubtedly find other similarities and differences new therapeutic approaches be developed usingbetween adult and embryonic stem cells. During the only adult stem cells? How many different sources ofnext several years, it will be important to compare stem cells will be needed to generate the best treat-embryonic stem cells and adult stem cells in terms of ments in the shortest period of time?their ability to proliferate, differentiate, survive and Predicting the future of stem cell applications isfunction after transplant, and avoid immune rejec- impossible, particularly given the very early stage oftion. Investigators have shown that differentiated cells the science of stem cell biology. To date, it isgenerated from both adult and embryonic stem cells impossible to predict which stem cells—those derivedcan repair or replace damaged cells and tissues in from the embryo, the fetus, or the adult—or whichanimal studies. methods for manipulating the cells, will best meet theScientists upon making new discoveries often verify needs of basic research and clinical applications.reported results in different laboratories and under The answers clearly lie in conducting more research.different conditions. Similarly, they will often conduct ES-10
  • 1. THE STEM CELLWHAT IS A STEM CELL? Most scientists use the term pluripotent to describe stem cells that can give rise to cells derived from allA stem cell is a cell that has the ability to divide (self three embryonic germ layers—mesoderm, endo-replicate) for indefinite periods—often throughout the derm, and ectoderm. These three germ layers arelife of the organism. Under the right conditions, or the embryonic source of all cells of the body (seegiven the right signals, stem cells can give rise (differ- Figure 1.1. Differentiation of Human Tissues). All of theentiate) to the many different cell types that make up many different kinds of specialized cells that make upthe organism. That is, stem cells have the potential to the body are derived from one of these germ layersdevelop into mature cells that have characteristic (see Table 1.1. Embryonic Germ Layers From Whichshapes and specialized functions, such as heart cells, Differentiated Tissues Develop). “Pluri”—derived fromskin cells, or nerve cells. the Latin plures—means several or many. Thus, pluripotent cells have the potential to give rise toTHE DIFFERENTIATION POTENTIAL any type of cell, a property observed in the naturalOF STEM CELLS: BASIC CONCEPTS course of embryonic development and under certainAND DEFINITIONS laboratory conditions.Many of the terms used to define stem cells depend Unipotent stem cell, a term that is usually applied toon the behavior of the cells in the intact organism a cell in adult organisms, means that the cells in(in vivo), under specific laboratory conditions (in vitro), question are capable of differentiating along onlyor after transplantation in vivo, often to a tissue that is one lineage. “Uni” is derived from the Latin word unus,different from the one from which the stem cells which means one. Also, it may be that the adult stemwere derived. cells in many differentiated, undamaged tissues are typically unipotent and give rise to just one cell typeFor example, the fertilized egg is said to be under normal conditions. This process would allow fortotipotent—from the Latin totus, meaning entire— a steady state of self-renewal for the tissue. However,because it has the potential to generate all the cells if the tissue becomes damaged and the replacementand tissues that make up an embryo and that sup- of multiple cell types is required, pluripotent stem cellsport its development in utero. The fertilized egg may become activated to repair the damage [2].divides and differentiates until it produces a matureorganism. Adult mammals, including humans, consist The embryonic stem cell is defined by its origin—thatof more than 200 kinds of cells. These include nerve is from one of the earliest stages of the developmentcells (neurons), muscle cells (myocytes), skin (epithe- of the embryo, called the blastocyst. Specifically,lial) cells, blood cells (erythrocytes, monocytes, lym- embryonic stem cells are derived from the inner cellphocytes, etc.), bone cells (osteocytes), and cartilage mass of the blastocyst at a stage before it wouldcells (chondrocytes). Other cells, which are essential implant in the uterine wall. The embryonic stem cellfor embryonic development but are not incorporated can self-replicate and is pluripotent—it can give riseinto the body of the embryo, include the extra- to cells derived from all three germ layers.embryonic tissues, placenta, and umbilical cord. All The adult stem cell is an undifferentiated (unspecial-of these cells are generated from a single, totipotent ized) cell that is found in a differentiated (specialized)cell—the zygote, or fertilized egg. 1
  • The Stem Cell Zygote Blastocyst Ectoderm (external layer) Mesoderm (middle layer) Endoderm (internal layer) Germ cells© 2001 Terese Winslow, Caitlin Duckwall Skin cells Neuron Pigment Cardiac Skeletal Tubule cell Red Smooth Pancreatic Thyroid Lung cell Sperm Egg of of brain cell muscle muscle of the blood muscle cell cell (alveolar epidermis cells kidney cells (in gut) cell) Figure 1.1. Differentiation of Human Tissues. tissue; it can renew itself and become specialized to the dental pulp of the tooth, liver, skin, gastrointestinal yield all of the specialized cell types of the tissue from tract, and pancreas. Unlike embryonic stem cells, at which it originated. Adult stem cells are capable of this point in time, there are no isolated adult stem self-renewal for the lifetime of the organism. Sources cells that are capable of forming all cells of the body. of adult stem cells have been found in the bone That is, there is no evidence, at this time, of an adult marrow, blood stream, cornea and retina of the eye, stem cell that is pluripotent. 2
  • The Stem Cell Table 1.1. Embryonic Germ Layers From Which Differentiated Tissues Develop Embryonic Germ Layer Differentiated Tissue Endoderm Thymus Thyroid, parathyroid glands Larynx, trachea, lung Urinary bladder, vagina, urethra Gastrointestinal (GI) organs (liver, pancreas) Lining of the GI tract Lining of the respiratory tract Mesoderm Bone marrow (blood) Adrenal cortex Lymphatic tissue Skeletal, smooth, and cardiac muscle Connective tissues (including bone, cartilage) Urogenital system Heart and blood vessels (vascular system) Ectoderm Skin Neural tissue (neuroectoderm) Adrenal medulla Pituitary gland Connective tissue of the head and face Eyes, ears[1]REFERENCES 1. Chandross, K.J. and Mezey, E. (2001). Plasticity of adult bone marrow stem cells. Mattson, M.P and Van Zant, G. . eds. (Greenwich, CT: JAI Press). 2. Slack, J.M. (2000). Stem cells in epithelial tissues. Science. 287, 1431-1433. 3
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  • 2. THE EMBRYONIC STEM CELLAs stated in the first chapter, an embryonic stem cell For research purposes, the definition of an ES cell is(ES cell) is defined by its origin. It is derived from the more than a self-replicating stem cell derived fromblastocyst stage of the embryo. The blastocyst is the the embryo that can differentiate into almost all ofstage of embryonic development prior to implanta- the cells of the body. Scientists have found it neces-tion in the uterine wall. At this stage, the preimplan- sary to develop specific criteria that help them bettertation embryo of the mouse is made up of 150 cells define the ES cell. Austin Smith, whose studies ofand consists of a sphere made up of an outer layer mouse ES cells have contributed significantly to theof cells (the trophectoderm), a fluid-filled cavity field, has offered a list of essential characteristics that(the blastocoel), and a cluster of cells on the interior define ES cells [18, 32].(the inner cell mass).Studies of ES cells derived from mouse blastocysts DEFINING PROPERTIES OF ANbecame possible 20 years ago with the discovery of EMBRYONIC STEM CELLtechniques that allowed the cells to be grown in the • a Derived from the inner cell mass/epiblast of thelaboratory. Embryonic–like stem cells, called embry- blastocyst.onic germ (EG) cells, can also be derived from • a Capable of undergoing an unlimited numberprimordial germ (PG) cells (the cells of the developing of symmetrical divisions without differentiatingfetus from which eggs and sperm are formed) of the (long-term self-renewal).mouse [20] and human fetus [30]. • Exhibit and maintain a stable, full (diploid), nor-In this chapter the discussion will be limited to mouse mal complement of chromosomes (karyotype).embryonic stem cells. Chapter 3 describes the • Pluripotent ES cells can give rise to differentiatedhuman embryonic stem cell. cell types that are derived from all three primary germ layers of the embryo (endoderm, meso-DO EMBRYONIC STEM CELLS derm, and ectoderm).ACTUALLY OCCUR IN THE EMBRYO? • a,bCapable of integrating into all fetal tissuesSome scientists argue that ES cells do not occur in during development. (Mouse ES cells maintainedthe embryo as such. ES cells closely resemble the in culture for long periods can still generate anycells of the preimplantation embryo [3], but are not in tissue when they are reintroduced into anfact the same [32]. An alternative perspective is that embryo to generate a chimeric animal.)the embryos of many animal species contain stem • a,b Capable of colonizing the germ line andcells. These cells proliferate extensively in the embryo, giving rise to egg or sperm cells.are capable of differentiating into all the types of • aClonogenic, that is a single ES cell can givecells that occur in the adult, and can be isolated and rise to a colony of genetically identical cells, orgrown ex vivo (outside the organism), where they clones, which have the same properties as thecontinue to replicate and show the potential to original cell.differentiate [18]. 5
  • The Embryonic Stem Cell • Expresses the transcription factor Oct-4, which cells from one culture dish and replating them into then activates or inhibits a host of target genes fresh culture dishes. Whether the number of passages and maintains ES cells in a proliferative, non- affects the differentiation potential of human ES cells differentiating state. remains to be determined. (For a detailed discussion • Can be induced to continue proliferating or to of the techniques for maintaining mouse ES cells in differentiate. culture, see Appendix B. Mouse Embryonic Stem Cells.) • Lacks the G1 checkpoint in the cell cycle. ES A second method for determining the pluripotency of cells spend most of their time in the S phase of mouse ES cells is to inject the cells into adult mice the cell cycle, during which they synthesize DNA. (under the skin or the kidney capsule) that are either Unlike differentiated somatic cells, ES cells do not genetically identical or are immune-deficient, so the require any external stimulus to initiate DNA tissue will not be rejected. In the host animal, the replication. injected ES cells develop into benign tumors called • Do not show X inactivation. In every somatic cell teratomas. When examined under a microscope, it of a female mammal, one of the two X chromo- was noted that these tumors contain cell types somes becomes permanently inactivated. X derived from all three primary germ layers of the inactivation does not occur in undifferentiated embryo—endoderm, mesoderm, and ectoderm. ES cells. Teratomas typically contain gut-like structures such as layers of epithelial cells and smooth muscle; skeletal[a Not shown in human EG cells. b Not shown in human ES or cardiac muscle (which may contract sponta-cells. All of the criteria have been met by mouse ES cells.] neously); neural tissue; cartilage or bone; and some- times hair. Thus, ES cells that have been maintainedARE EMBRYONIC STEM CELLS for a long period in vitro can behave as pluripotentTRULY PLURIPOTENT? cells in vivo. They can participate in normal embryo- genesis by differentiating into any cell type in thePluripotency—that is the ability to give rise to differen- body, and they can also differentiate into a widetiated cell types that are derived from all three range of cell types in an adult animal. However,primary germ layers of the embryo, endoderm, normal mouse ES cells do not generate trophoblastmesoderm, and ectoderm—is what makes ES cells tissues in vivo [32].unique. How do we know that these cells are, indeed,pluripotent? Laboratory-based criteria for testing the A third technique for demonstrating pluripotency ispluripotent nature of ES cells derived from mice to allow mouse ES cells in vitro to differentiate sponta-include three kinds of experiments [19]. One test is neously or to direct their differentiation along specificconducted by injecting ES cells derived from the pathways. The former is usually accomplished byinner cell mass of one blastocyst into the cavity of removing feeder layers and adding leukemia inhibi-another blastocyst. The “combination” embryos are tory factor (LIF) to the growth medium. Within a fewthen transferred to the uterus of a pseudopregnant days after changing the culture conditions, ES cellsfemale mouse, and the progeny that result are aggregate and may form embryoid bodies (EBs).chimeras. Chimeras are a mixture of tissues and In many ways, EBs in the culture dish resembleorgans of cells derived from both donor ES cells and teratomas that are observed in the animal. EBs con-the recipient blastocyst. sist of a disorganized array of differentiated or partially differentiated cell types that are derived from theThis test has been extended in studies designed to three primary germ layers of the embryo—the endo-test whether cultured ES cells can be used to replace derm, mesoderm, and ectoderm [32].the inner cell mass of a mouse blastocyst and pro-duce a normal embryo. They can, but the process is The techniques for culturing mouse ES cells from thefar less efficient than that of using cells taken directly inner cell mass of the preimplantation blastocyst werefrom the inner cell mass. Apparently, the ability of ES first reported 20 years ago [9, 19], and versions ofcells to generate a complete embryo depends on these standard procedures are used today inthe number of times they have been passaged laboratories throughout the world. It is striking that, toin vitro [21, 22]. A passage is the process of removing date, only three species of mammals have yielded 6
  • The Embryonic Stem Celllong-term cultures of self-renewing ES cells: mice, the ES cells are growing. For example, the plasticmonkeys, and humans [27, 34, 35, 36] (see Appendix culture dishes used to grow both mouse and humanB. Mouse Embryonic Stem Cells). ES cells can be treated with a variety of substances that allow the cells either to adhere to the surface ofHOW DOES A MOUSE EMBRYONIC the dish or to avoid adhering and instead float in theSTEM CELL STAY UNDIFFERENTIATED? culture medium. In general, an adherent substrate helps prevent them from interacting and differentiat-As stated earlier, a true stem cell is capable of main- ing. In contrast, a nonadherent substrate allows the EStaining itself in a self-renewing, undifferentiated state cells to aggregate and thereby interact with eachindefinitely. The undifferentiated state of the embry- other. Cell-cell interactions are critical to normalonic stem cell is characterized by specific cell mark- embryonic development, so allowing some of theseers that have helped scientists better understand how “natural” in vivo interactions to occur in the cultureembryonic stem cells—under the right culture condi- dish is a fundamental strategy for inducing mouse ortions—replicate for hundreds of population doublings human ES cell differentiation in vitro. In addition,and do not differentiate. To date, two major areas adding specific growth factors to the culture mediumof investigation have provided some clues. One triggers the activation (or inactivation) of specificincludes attempts to understand the effects of genes in ES cells. This initiates a series of molecularsecreted factors such as the cytokine leukemia events that induces the cells to differentiate along ainhibitory factor on mouse ES cells in vitro. The sec- particular pathway.ond area of study involves transcription factors suchas Oct-4. Oct-4 is a protein expressed by mouse and Another way to direct differentiation of ES cells is tohuman ES cells in vitro, and also by mouse inner cell introduce foreign genes into the cells via transfectionmass cells in vivo. The cell cycle of the ES also seems or other methods [6, 39]. The result of these strategiesto play a role in preventing differentiation. From stud- is to add an active gene to the ES cell genome,ies of these various signaling pathways, it is clear that which then triggers the cells to differentiate along amany factors must be balanced in a particular way particular pathway. The approach appears to be afor ES cells to remain in a self-renewing state. If the precise way of regulating ES cell differentiation, but itbalance shifts, ES cells begin to differentiate [18, 31]. will work only if it is possible to identify which gene(For a detailed discussion of how embryonic stem must be active at which particular stage of differen-cells maintain their pluripotency, see Appendix B. tiation. Then, the gene must be activated at theMouse Embryonic Stem Cells.) right time—meaning during the correct stage of differentiation—and it must be inserted into the genome at the proper location.CAN A MOUSE EMBRYONIC STEM CELLBE DIRECTED TO DIFFERENTIATE Another approach to generate mouse ES cells usesINTO A PARTICULAR CELL TYPE cloning technology. In theory, the nucleus of aIN VITRO? differentiated mouse somatic cell might be repro- grammed by injecting it into an oocyte. The resultantOne goal for embryonic stem cell research is the pluripotent cell would be immunologically com-development of specialized cells such as neurons, patible because it would be genetically identical toheart muscle cells, endothelial cells of blood vessels, the donor cell [25].and insulin secreting cells similar to those found in thepancreas. The directed derivation of embryonic stem All of the techniques just described are still highlycells is then vital to the ultimate use of such cells in experimental. Nevertheless, within the past severalthe development of new therapies. years, it has become possible to generate specific, differentiated, functional cell types by manipulatingBy far the most common approach to directing the growth conditions of mouse ES cells in vitro. It isdifferentiation is to change the growth conditions of not possible to explain how the directed differentia-the ES cells in specific ways, such as by adding tion occurs, however. No one knows how or whengrowth factors to the culture medium or changing gene expression is changed, what signal-transductionthe chemical composition of the surface on which systems are triggered, or what cell-cell interactions 7
  • The Embryonic Stem Cellmust occur to convert undifferentiated ES cells into Table 2.1. Reported differentiated cell typesprecursor cells and, finally, into differentiated cells from mouse embryonic stem cells in vitro*that look and function like their in vivo counterparts. Cell Type ReferenceEmbryonic stem cells have been shown to differen- Adipocyte [7]tiate into a variety of cell types. For example, mouse Astrocyte [11]ES cells can be directed in vitro to yield vascularstructures [40], neurons that release dopamine and Cardiomyocyte [8, 17]serotonin [14], and endocrine pancreatic islet cells Chondrocyte [13][16]. In all three cases, proliferating, undifferentiated Definitive hematopoietic [23, 24, 38]mouse ES cells provide the starting material and Dendritic cell [10]functional, differentiated cells were the result. Also,the onset of mouse ES cell differentiation can be Endothelial cell [28, 40]triggered by withdrawing the cytokine LIF, which pro- Keratinocyte [1, 40]motes the division of undifferentiated mouse ES cells. Lymphoid precursor [26]In addition, when directed to differentiate, ES cells Mast cell [37]aggregate, a change in their three-dimensional Neuron [2, 33]environment that presumably allowed some of thecell-cell interactions to occur in vitro that would occur Oligodendrocyte [4, 15]in vivo during normal embryonic development. Osteoblast [5]Collectively, these three studies provide some of the Pancreatic islets [16]best examples of directed differentiation of ES cells. Primitive haematopoietic [8, 23]Two of them showed that a single precursor cell cangive rise to multiple, differentiated cell types [16, 40], Smooth muscle [40]and all three studies demonstrated that the resulting Striated muscle [29]differentiated cells function as their in vivo counter- Yolk sac endoderm [8]parts do. These two criteria—demonstrating that a sin- Yolk sac mesoderm [8]gle cell can give rise to multiple cells types and the *Adapted with permission from reference [32].functional properties of the differentiated cells—formthe basis of an acid test for all claims of directeddifferentiation of either ES cells or adult stem cells.Unfortunately, very few experiments meet these 3. Brook, F.A. and Gardner, R.L. (1997). The origin and efficient derivation of embryonic stem cells in the mouse. Proc. Natl.criteria, which too often makes it impossible to assess Acad. Sci. U. S. A. 94, 5709-5712.whether a differentiated cell type resulted from theexperimental manipulation that was reported. (For a 4. Brustle, O., Jones, K.N., Learish, R.D., Karram, K., Choudhary, K., Wiestler, O.D., Duncan, I.D., and McKay, R.D. (1999).detailed discussion of the methods used to differen- Embryonic stem cell-derived glial precursors: a source oftiate mouse embryonic stem cells, see Appendix B. myelinating transplants. Science. 285, 754-756.Mouse Embryonic Stem Cells.) 5. Buttery, L.D., Bourne, S., Xynos, J.D., Wood, H., Hughes, F.J., Hughes, S.P Episkopou, V., and Polak, J.M. (2001). .,Table 2.1 provides a summary of what is known today Differentiation of osteoblasts and in vitro bone formationabout the types of cells that can be differentiated from murine embryonic stem cells. Tissue Eng. 7, 89-99.from mouse embryonic stem cells. 6. Call, L.M., Moore, C.S., Stetten, G., and Gearhart, J.D. (2000). A cre-lox recombination system for the targetedREFERENCES integration of circular yeast artificial chromosomes into embryonic stem cells. Hum. Mol. Genet. 9, 1745-1751. 1. Bagutti, C., Wobus, A.M., Fassler, R., and Watt, F.M. (1996). Differentiation of embryonal stem cells into keratinocytes: 7. Dani, C., Smith, A.G., Dessolin, S., Leroy, P Staccini, L., ., comparison of wild-type and ß(1) integrin-deficient cells. Villageois, P Darimont, C., and Ailhaud, G. (1997). ., Dev. Biol. 179, 184-196. Differentiation of embryonic stem cells into adipocytes in vitro. J. Cel. Sci. 110, 1279-1285. 2. Bain, G., Kitchens, D., Yao, M., Huettner, J.E., and Gottlieb, D.I. (1995). Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168, 342-357. 8
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  • The Embryonic Stem Cell33. Strubing, C., Ahnert-Hilger, G., Shan, J., Wiedenmann, B., 38. Wiles, M.V. and Keller, G. (1991). Multiple hematopoietic Hescheler, J., and Wobus, A.M. (1995). Differentiation of lineages develop from embryonic stem (ES) cells in culture. pluripotent embryonic stem cells into the neuronal lineage Development. 111, 259-267. in vitro gives rise to mature inhibitory and excitatory neurons. Mech. Dev. 53, 275-287. 39. Wiles, M.V., Vauti, F., Otte, J., Fuchtbauer, E.M., Ruiz, P., Fuchtbauer, A., Arnold, H.H., Lehrach, H., Metz, T., von34. Thomson, J.A., Kalishman, J., Golos, T.G., Durning, M., Harris, Melchner, H., and Wurst, W. (2000). Establishment of a C.P Becker, R.A., and Hearn, J.P (1995). Isolation of a pri- ., . gene-trap sequence tag library to generate mutant mice mate embryonic stem cell line. Proc. Natl. Acad. Sci. U. S. from embryonic stem cells. Nat. Genet. 24, 13-14. A. 92, 7844-7848. 40. Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M.,35. Thomson, J.A. and Marshall, V.S. (1998). Primate embryonic Nishikawa, S., Yurugi, T., Naito, M., Nakao, K., and Nishikawa, stem cells. Curr. Top. Dev. Biol. 38, 133-165. S. (2000). Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature. 408, 92-96.36. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998). Embryonic stem cell lines derived from human blastocysts. Science. 282, 1145-1147.37. Tsai, M., Wedemeyer, J., Ganiatsas, S., Tam, S.Y., Zon, L.I., and Galli, S.J. (2000). In vivo immunological function of mast cells derived from embryonic stem cells: an approach for the rapid analysis of even embryonic lethal mutations in adult mice in vivo. Proc. Natl. Acad. Sci. U. S. A. 97, 9186-9190. 10
  • 3. THE HUMAN EMBRYONIC STEM CELL AND THE HUMAN EMBRYONIC GERM CELLA new era in stem cell biology began in 1998 with culture of ES cells from the blastocysts of rhesus mon-the derivation of cells from human blastocysts and keys [46] and marmosets [47], and methods used byfetal tissue with the unique ability of differentiating IVF clinics to prepare human embryos for transplanti-into cells of all tissues in the body, i.e., the cells are ng into the uterus to produce a live birth [11, 49].pluripotent. Since then, several research teams havecharacterized many of the molecular characteristics TIMELINE OF HUMAN EMBRYONICof these cells and improved the methods for culturing STEM CELL RESEARCHthem. In addition, scientists are just beginning to directthe differentiation of the human pluripotent stem • 1878: First reported attempts to fertilizecells and to identify the functional capabilities of the mammalian eggs outside the body [49].resulting specialized cells. Although in its earliest phas- • 1959: First report of animals (rabbits) producedes, research with these cells is proving to be impor- through IVF in the United States [49].tant to developing innovative cell replacement • 1960s: Studies of teratocarcinomas in the testesstrategies to rebuild tissues and restore critical of several inbred strains of mice indicates theyfunctions of the diseased or damaged human body. originated from embryonic germ cells. The work establishes embryonal carcinoma (EC) cells as aOVERVIEW kind of stem cell [17, 24]. For a more detailed discussion of human embryonal carcinomaIn 1998, James Thomson and his colleagues reported cells, see Appendix C.methods for deriving and maintaining humanembryonic stem (ES) cells from the inner cell mass • 1968: Edwards and Bavister fertilize the firstof human blastocysts that were produced through human egg in vitro [49].in vitro fertilization (IVF) and donated for research • 1970s: EC cells injected into mouse blastocystspurposes [46]. At the same time, another group, led produce chimeric mice. Cultured SC cells areby John Gearhart, reported the derivation of cells explored as models of embryonic development,that they identified as embryonic germ (EG) cells. although their complement of chromosomes isThe cells were cultured from primordial germ cells abnormal [25].obtained from the gonadal ridge and mesenchyma • 1978: Louise Brown, the first IVF baby, is born inof 5- to 9-week fetal tissue that resulted from England [49].elective abortions [41]. • 1980: Australia’s first IVF baby, Candace Reed, isThe two research teams developed their methods born in Melbourne [49].for culturing human ES and EG cells by drawing on • 1981: Evans and Kaufman, and Martin derivea host of animal studies, some of which date back mouse embryonic stem (ES) cells from the inneralmost 40 years: derivations of pluripotent mouse cell mass of blastocysts. They establish cultureES cells from blastocysts [13, 15], reports of the conditions for growing pluripotent mouse ES cellsderivation of EG cells [27, 36], experiments with in vitro. The ES cells yield cell lines with normal,stem cells derived from mouse teratocarcinomas diploid karyotyes and generate derivatives of all[24] and human embryonal carcinomas and three primary germ layers as well as primordialteratocarcinomas [4, 17, 24], the derivation and 11
  • The Human Embryonic Stem Cell and The Human Embryonic Germ Cell germ cells. Injecting the ES cells into mice of human EC cells non-human primate ES cells. induces the formation of teratomas [15, 26]. Several (non-clonal) cell lines are established The first IVF baby, Elizabeth Carr, is born in the that form teratomas when injected into United States [49]. immune-deficient mice. The teratomas include • 1984-88: Andrews et al., develop pluripotent, cell types derived from all three primary germ genetically identical (clonal) cells called embry- layers, demonstrating the pluripotency of human onal carcinoma (EC) cells from Tera-2, a cell line ES cells [48]. Gearhart and colleagues derive of human testicular teratocarcinoma [5]. Cloned human embryonic germ (EG) cells from the human teratoma cells exposed to retinoic acid gonadal ridge and mesenchyma of 5- to differentiate into neuron-like cells and other cell 9-week fetal tissue that resulted from elective types [3, 44]. abortions. They grow EG cells in vitro for approxi- mately 20 passages, and the cells maintain • 1989: Pera et al., derive a clonal line of human normal karyotypes. The cells spontaneously form embryonal carcinoma cells, which yields tissues aggregates that differentiate spontaneously, and from all three primary germ layers. The cells are ultimately contain derivatives of all three primary aneuploid (fewer or greater than the normal germ layers. Other indications of their number of chromosomes in the cell) and their pluripotency include the expression of a panel of potential to differentiate spontaneously in vitro markers typical of mouse ES and EG cells. The is typically limited. The behavior of human EC EG cells do not form teratomas when injected cell clones differs from that of mouse ES or into immune-deficient mice [41]. EC cells [33]. • 2000: Scientists in Singapore and Australia led by • 1994: Human blastocysts created for Pera, Trounson, and Bongso derive human ES reproductive purposes using IVF and donated cells from the inner cell mass of blastocysts by patients for research, are generated from the donated by couples undergoing treatment for 2-pronuclear stage. The inner cell mass of the infertility. The ES cells proliferate for extended blastocyst is maintained in culture and generates periods in vitro, maintain normal karyotypes, aggregates with trophoblast-like cells at the differentiate spontaneously into somatic cell periphery and ES-like cells in the center. The cells lineages derived from all three primary germ retain a complete set of chromosomes (normal layers, and form teratomas when injected into karyotype); most cultures retain a stem cell-like immune-deficient mice. morphology, although some inner cell mass clumps differentiate into fibroblasts. The cultures • 2001: As human ES cell lines are shared and are maintained for two passages [6, 7]. new lines are derived, more research groups report methods to direct the differentiation of • 1995-96: Non-human primate ES cells are the cells in vitro. Many of the methods are derived and maintained in vitro, first from the aimed at generating human tissues for inner cell mass of rhesus monkeys [46], and then transplantation purposes, including pancreatic from marmosets [47]. The primate ES cells are islet cells, neurons that release dopamine, and diploid and have normal karyotypes. They are cardiac muscle cells. pluripotent and differentiate into cells types derived from all three primary germ layers. The primate ES cells resemble human EC cells and DERIVATION OF HUMAN EMBRYONIC indicate that it should be possible to derive and STEM CELLS maintain human ES cells in vitro. The first documentation of the isolation of embryonic • 1998: Thomson et al., derive human ES cells stem cells from human blastocysts was in 1994 [7]. from the inner cell mass of normal human Since then, techniques for deriving and culturing blastocysts donated by couples undergoing human ES cells have been refined [38, 48]. The ability treatment for infertility. The cells are cultured to isolate human ES cells from blastocysts and grow through many passages, retain their normal them in culture seems to depend in large part on the karyotypes, maintain high levels of telomerase integrity and condition of the blastocyst from which activity, and express a panel of markers typical the cells are derived. In general, blastocysts with a 12
  • The Human Embryonic Stem Cell and The Human Embryonic Germ Cell large and distinct inner cell mass tend to yield ES more detailed discussion, see Appendix A. cultures most efficiently [11] (see Figure 3.1. Early Development.) Human Blastocyst Showing Inner Cell Mass and Trophectoderm). Many IVF clinics now transfer day-5 embryos to the uterus for optimal implantation, a stage of develop- Timeline for the Development of a Human ment that more closely parallels the stage at which a blastocyst would implant in the wall of the uterus in vivo. This represents a change—and a greatly improved implantation rate—from earlier IVF procedures in which a 2-cell embryo was used for implantation. Day-5 blastocysts are used to derive ES cell cultures. A normal day-5 human embryo in vitro consists of 200 to 250 cells. Most of the cells comprise the trophectoderm. For deriving ES cell cultures, the trophectoderm is removed, either by microsurgery or immunosurgery (in which antibodies against the trophectoderm help break it down, thus freeing the inner cell mass). At this stage, the inner cell mass is composed of only 30 to 34 cells [10].Photo Credit: Mr. J. Conaghan The in vitro conditions for growing a human embryo to the blastocyst stage vary among IVF clinics and are reviewed elsewhere [6, 8, 14, 16, 18, 21, 39, 49, 50]. However, once the inner cell mass is obtained from either mouse or human blastocysts, the tech- Figure 3.1. Human Blastocyst Showing Inner Cell Mass niques for growing ES cells are similar. (For a detailed and Trophectoderm. discussion see Appendix C. Human Embryonic Stem Cells and Human Embryonic Germ Cells.) Blastocyst In Vitro After a human oocyte is fertilized in vitro by a sperm DERIVATION OF HUMAN EMBRYONIC cell, the following events occur according to a fairly GERM CELLS predictable timeline [9, 12, 16]. At 18 to 24 hours As stated earlier, human embryonic germ (EG) cells after in vitro fertilization of the oocyte is considered share many of the characteristics of human ES cells, day 1. By day 2 (24 to 25 hours), the zygote (fertilized but differ in significant ways. Human EG cells are egg) undergoes the first cleavage to produce a derived from the primordial germ cells, which occur 2-cell embryo. By day 3 (72 hours), the embryo in a specific part of the embryo/fetus called the reaches the 8-cell stage called a morula. It is at this gonadal ridge, and which normally develop into stage that the genome of the embryo begins to mature gametes (eggs and sperm). Gearhart and his control its own development. This means that any collaborators devised methods for growing pluripo- maternal influences—due to the presence of mRNA tent cells derived from human EG cells. The process and proteins in the oocyte cytoplasm—are requires the generation of embryoid bodies from EG significantly reduced. By day 4, the cells of the cells, which consists of an unpredictable mix of embryo adhere tightly to each other in a process partially differentiated cell types [19]. The embryoid known as compaction and by day 5, the cavity of body-derived cells resulting from this process have the blastocyst is completed. The inner cell mass high proliferative capacity and gene expression begins to separate from the outer cells, which patterns that are representative of multiple cell become the trophectoderm that surrounds the lineages. This suggests that the embryoid body- blastocyst. This represents the first observable sign derived cells are progenitor or precursor cells for of cell differentiation in the embryo. (For a 13
  • The Human Embryonic Stem Cell and The Human Embryonic Germ Cella variety of differentiated cell types [19]. (For a more COMPARISONS BETWEEN HUMANdetailed description of the derivation of EG cells, see EMBRYONIC STEM CELLS ANDAppendix C. Human Embryonic Stem Cells andHuman Embryonic Germ Cells.) EMBRYONIC GERM CELLS The ES cells derived from human blastocysts byPLURIPOTENCY OF HUMAN Thomson and his colleagues, and from human EGEMBRYONIC STEM CELLS AND cells derived by Gearhart and his collaborators, areEMBRYONIC GERM CELLS similar in many respects. In both cases, the cells replicate for an extended period of time, show noAs stated earlier, a truly pluripotent stem cell is a cell chromosomal abnormalities, generate both XXthat is capable of self-renewal and of differentiating (female) and XY (male) cultures, and express a setinto most all of the cells of the body, including cells of of markers regarded as characteristic of pluripotentall three germ layers. Human ES and EG cells in vitro cells. When the culture conditions are adjusted toare capable of long-term self-renewal, while retaining permit differentiation (see below for details), botha normal karyotype [1, 38, 41, 42, 48]. Human ES ES and EG cells spontaneously differentiate intocells can proliferate for two years through 300 derivatives of all three primary germ layers—population doublings [29] or even 450 population endoderm, mesoderm, and ectoderm (see Tabledoublings [30]. Cultures derived from embryoid 3.1. Comparison of Mouse, Monkey, and Humanbodies generated by human embryonic germ Pluripotent Stem Cells).cells have less capacity for proliferation. Most willproliferate for 40 population doublings; the maximum However, the ES cells derived from human blastocystsreported is 70 to 80 population doublings [42]. and EG cells differ not only in the tissue sources from which they are derived, they also vary with respect toTo date, several laboratories have demonstrated that their growth characteristics in vitro, and their behaviorhuman ES cells in vitro are pluripotent; they can in vivo [34]. In addition, human ES cells have beenproduce cell types derived from all three embryonic propagated for approximately two years in vitro, forgerm layers [1, 20, 38, 40]. several hundred population doublings [1], whereas human embryoid body-derived cells from cultures ofCurrently, the only test of the in vivo pluripotency of embryonic germ cells have been maintained for onlyhuman ES cells is to inject them into immune- 70 to 80 population doublings [42]. Also, human ESdeficient mice where they generate differentiated cells will generate teratomas containing differentiatedcells that are derived from all three germ layers. cell types, if injected into immunocompromisedThese include gut epithelium (which, in the embryo, mice colonies, while human EG cells will notis derived from endoderm); smooth and striated [20, 37, 38, 41, 48].muscle (derived from mesoderm); and neuralepithelium, and stratified squamous epithelium Several research groups are trying to grow human ES(derived from ectoderm) [20, 38, 48]. cells without feeder layers of mouse embryo fibro- blasts (MEF), which are labor-intensive to generate.However, two aspects of in vivo pluripotency typically At a recent meeting, scientists from the Geronused in animals have not been met by human ES Corporation reported that they have grown human EScells: evidence that cells have the capacity to be cell without feeder layers, in medium conditioned byinjected into a human embryo and form an organ- MEFs and supplemented with basic FGF [51].ism made up of cells from two genetic lineages;and evidence that they have the ability to generategerm cells, the precursors to eggs and sperm in a DIRECTED DIFFERENTIATION OFdeveloping organism. These are theoretical HUMAN EMBRYONIC STEM CELLSconsiderations, however, because such tests using AND EMBRYONIC GERM CELLShuman ES cells have not been conducted. In any IN VITROcase, these two demonstrations of human ES cellpluripotency are not likely to be critical for potential Currently, a major goal for embryonic stem celltherapeutic uses of the cells—in transplants or drug research is to control the differentiation of human ESdevelopment, for example [43]. and EG cell lines into specific kinds of cells—an 14
  • The Human Embryonic Stem Cell and The Human Embryonic Germ Cell Table 3.1. Comparison of Mouse, Monkey, and Human Pluripotent Stem Cells Marker Mouse EC/ Monkey Human Human Human Name ES/EG cells ES cells ES cells EG cells EC cells SSEA-1 + – – + – SSEA-3 – + + + + SEA-4 – + + + + TRA-1-60 – + + + + TRA-1-81 – + + + + Alkaline + + + + + phosphatase Oct-4 + + + Unknown + Telomerase activity + ES, EC Unknown + Unknown + Feeder-cell ES, EG, Yes Yes Yes Some; relatively low dependent some EC clonal efficiency Factors which aid LIF and other Co-culture with Feeder cells + LIF, bFGF, Unknown; in stem cell factors that act feeder cells; other serum; feeder forskolin low proliferative self-renewal through gp130 promoting factors layer + capacity receptor and can have not been serum-free substitute for identified medium + bFGF feeder layer Growth Form tight, Form flat, loose Form flat, loose Form rounded, Form flat, loose characteristics rounded, aggregates; aggregates; multi-layer clumps; aggregates; in vitro multi-layer clumps; can form EBs can form EBs can form EBs can form EBs can form EBs Teratoma + + + – + formation in vivo Chimera + Unknown + – + formation KEY ES cell = Embryonic stem cell TRA = Tumor rejection antigen-1 EG cell = Embryonic germ cell LIF = Leukemia inhibitory factor EC cell = Embryonal carcinoma cell bFGF = Basic fibroblast growth factor SSEA = Stage-specific embryonic antigen EB = Embryoid bodiesobjective that must be met if the cells are to be used POTENTIAL USES OF HUMANas the basis for therapeutic transplantation, testing EMBRYONIC STEM CELLSdrugs, or screening potential toxins. The techniquesnow being tested to direct human ES cell differentia- Many uses have been proposed for humantion are borrowed directly from techniques used to embryonic stem cells. The most-often discussed isdirect the differentiation of mouse ES cells in vitro. For their potential use in transplant therapy—i.e., tomore discussion on directed differentiation of human replace or restore tissue that has been damaged byES and EG cells see Appendix C. disease or injury (see also Chapters 5-9). 15
  • The Human Embryonic Stem Cell and The Human Embryonic Germ CellUsing Human Embryonic Stem Cells for failure, and osteogenesis imperfecta. However, treat-Therapeutic Transplants ments for any of these diseases require that humanDiseases that might be treated by transplanting ES cells be directed to differentiate into specific cellhuman ES-derived cells include Parkinson’s disease, types prior to transplant. The research is occurring indiabetes, traumatic spinal cord injury, Purkinje cell several laboratories, but is limited because so fewdegeneration, Duchenne’s muscular dystrophy, heart laboratories have access to human ES cells. Thus, at Human ES Cells • Lineage selection by cell survival Establish pure cultures or cell sorting (e.g., insulin promoter of specific cell type driving antibiotic resistance gene or GFP) • Induce with supplemental growth factor(s) or inducer cells (e.g., retinoic acid for neural cells) Test physiologic function • In vitro (e.g., stimulated insulin release) Demonstrate Demonstrate Test methods efficacy safety to prevent rejection • In rodent models • In non-human primate model with • Multi-drug immunosuppression rhesus ES cell-derived tissues • In non-human primate model • Create differentiated cells with rhesus ES cell-derived cells • Show absence of tumor formation isogenic to prospective recipient (e.g., diabetes and Parkinson’s using nuclear reprogramming disease models in primates) • Show absence of transmission of infectious agents • Transduce ES cells to express • Evaluate integration into host recipient MHC genes tissue (e.g., cardiomyocytes for treatment of heart failure) • Establish hematopoietic chimera and immunologic tolerance • ? recurrent autoimmunity (e.g., diabetes) Human trialsFigure 3.2. Major Goals in the Development of Transplantation Therapies from Human ES Cell Lines. (Reproduced withpermission from Stem Cells, 2001) 16
  • The Human Embryonic Stem Cell and The Human Embryonic Germ Cellthis stage, any therapies based on the use of human patient’s cells, such as a skin cell, and injecting theES cells are still hypothetical and highly experimental nucleus into an oocyte. The oocyte, thus “fertilized,”[22, 29, 31] (see Figure 3.2. Major Goals in the could be cultured in vitro to the blastocyst stage. ESDevelopment of Transplantation Therapies from cells could subsequently be derived from its inner cellHuman ES Cell Lines). mass, and directed to differentiate into the desired cell type. The result would be differentiated (or partlyOne of the current advantages of using ES cells as differentiated) ES-derived cells that match exactly thecompared to adult stem cells is that ES cells have an immunological profile of the person who donated theunlimited ability to proliferate in vitro, and are more somatic cell nucleus, and who is also the intendedlikely to be able to generate a broad range of cell recipient of the transplant—a labor intensive, but trulytypes through directed differentiation. Ultimately, it will customized therapy [29].also be necessary to both identify the optimalstage(s) of differentiation for transplant, and Other Potential Uses of Human Embryonicdemonstrate that the transplanted ES-derived cells Stem Cellscan survive, integrate, and function in the recipient. Many potential uses of human ES cells have been proposed that do not involve transplantation. ForThe potential disadvantages of the use of human ES example, human ES cells could be used to studycells for transplant therapy include the propensity of early events in human development. Still-unexplainedundifferentiated ES cells to induce the formation of events in early human development can result intumors (teratomas), which are typically benign. congenital birth defects and placental abnormalitiesBecause it is the undifferentiated cells—rather than that lead to spontaneous abortion. By studyingtheir differentiated progeny—that have been shown human ES cells in vitro, it may be possible to identifyto induce teratomas, tumor formation might be the genetic, molecular, and cellular events that leadavoided by devising methods for removing any to these problems and identify methods forundifferentiated ES cells prior to transplant. Also, it preventing them [22, 35, 45].should be possible to devise a fail-safe mechanism—i.e., to insert into transplanted ES-derived cells suicide Such cells could also be used to explore the effectsgenes that can trigger the death of the cells should of chromosomal abnormalities in early development.they become tumorigenic. This might include the ability to monitor the develop- ment of early childhood tumors, many of which areHuman ES derived cells would also be advantageous embryonic in origin [32].for transplantation purposes if they did not triggerimmune rejection. The immunological status of Human ES cells could also be used to test candidatehuman ES cells has not been studied in detail, and it therapeutic drugs. Currently, before candidate drugsis not known how immunogenic ES-derived cells are tested in human volunteers, they are subjected tomight be. In general, the immunogenicity of a cell a barrage of preclinical tests. These include drugdepends on its expression of Class I major screening in animal models—in vitro tests using cellshistocompatability antigens (MHC), which allow the derived from mice or rats, for example, or in vivo testsbody to distinguish its own cells from foreign tissue, that involve giving the drug to an animal to assess itsand on the presence of cells that can bind to foreign safety. Although animal model testing is a mainstayantigens and “present” them to the immune system. of pharmaceutical research, it cannot always predict the effects that a candidate drug may have onThe potential immunological rejection of human human cells. For this reason, cultures of human cellsES-derived cells might be avoided by genetically are often employed in preclinical tests. These humanengineering the ES cells to express the MHC antigens cell lines have usually been maintained in vitro forof the transplant recipient, or by using nuclear transfer long periods and as such often have differenttechnology to generate ES cells that are genetically characteristics than do in vivo cells. These differencesidentical to the person who receives the transplant. can make it difficult to predict the action of a drug inIt has been suggested that this could be accom- vivo based on the response of human cell lines inplished by using somatic cell nuclear transfer vitro. Therefore, if human ES cells can be directed totechnology (so-called therapeutic cloning) in which differentiate into specific cell types that are importantthe nucleus is removed from one of the transplant 17
  • The Human Embryonic Stem Cell and The Human Embryonic Germ Cell A. Genetic manipulation B. Nuclear reprogramming C. Hematopoietic chimera: of MHC genes complete, mixed, micro Mic Enucleated rop ipe oocyte t g pipet Transfer of Holdin ES cells donated nucleus ES cells Deletion of Substitution of MHC genes MHC genes or X X ES-cells derived from nuclear transfer Tissue-specific product Muscle Pancreatic differentiation islet cells Tissue-specific differentiation Hematopoietic differentiation Neuron X X X X Differentiated tissue expressing MHC-deficient Self-MHC donor nucleus Cotransplantation tissue expressing MHC genes tissueFigure 3.3. Genetic Manipulation of Human Embryonic Stem Cells. (Reproduced with permission from Stem Cells, 2001)for drug screening, the ES-derived cells may be more cells, it may be possible to devise better methods forlikely to mimic the in vivo response of the cells/tissues gene therapy [35] (see Chapter 10. Assessing Humanto the drug(s) being tested and so offer safer, and Stem Cell Safety).potentially cheaper, models for drug screening.Human ES cells could be employed to screen SUMMARYpotential toxins. The reasons for using human ES cells What Do We Know About Human Embryonicto screen potential toxins closely resemble those for Stem Cells?using human ES-derived cells to test drugs (above). Since 1998, research teams have refined theToxins often have different effects on different animal techniques for growing human ES cells in vitro [1, 20,species, which makes it critical to have the best 38]. Collectively, the studies indicate that it is nowpossible in vitro models for evaluating their effects possible to grow human ES cells for more than a yearon human cells. in serum-free medium on feeder layers. The cellsFinally, human ES cells could be used to develop new have normal karyotype and are pluripotent; theymethods for genetic engineering (see Figure 3.3. generate teratomas that contain differentiated cellGenetic Manipulation of Human Embryonic Stem types derived from all three primary germ layers. TheCells). Currently, the genetic complement of mouse long-term cultures of human ES cells have activeES cells in vitro can be modified easily by techniques telomerase and maintain relatively long telomeres,such as homologous recombination. This is a method another marker of proliferating cells.for replacing or adding genes, which requires that a Overall, the pluripotent cells that can be generatedDNA molecule be artificially introduced into the in vitro from human ES cells and human EG cells aregenome and then expressed. Using this method, apparently not equivalent in their potential togenes to direct differentiation to a specific cell type proliferate or differentiate. (ES cells are derived fromor genes that express a desired protein product might the inner cell mass of the preimplantation blastocyst,be introduced into the ES cell line. Ultimately, if such approximately 5 days post-fertilization, whereastechniques could be developed using human ES human EG cells are derived from fetal primordial 18
  • The Human Embryonic Stem Cell and The Human Embryonic Germ Cellgerm cells, 5 to 10 weeks post-fertilization.) ES cells Scientists will need to identify which signal transductioncan proliferate for up to 300 population doublings, pathways must be activated to induce human ES cellwhile cells derived from embryoid bodies that are differentiation along a particular pathway. This includesgenerated from embryonic germ cells (fetal tissue) understanding ligand-receptor interaction and thedouble a maximum of 70 to 80 times in vitro. intracellular components of the signaling system, as well as identifying the genes that are activated or inac-ES cells appear to have a broader ability to differenti- tivated during differentiation of specific cell types [29].ate. Both kinds of cells spontaneously generate neuralprecursor-type cells (widely regarded as a default Identifying intermediate stages of human ES cellpathway for differentiation), and both generate cells differentiation will also be important. As human ESthat resemble cardiac myocytes [19, 45]. However, cells differentiate in vitro, do they form distincthuman ES and EG cells in vitro will spontaneously precursor or progenitor cells that can be identifiedgenerate embryoid bodies that consist of cell types and isolated? If ES cells do form such intermediatefrom all three primary germ layers [1, 20, 38, 42]. cell types, can the latter be maintained and expanded? Would such precursor or progenitor cellsWhat Do We Need To Know About Human be useful for therapeutic transplantation [19]?Embryonic Stem Cells?Scientists are just beginning to understand the biology Finally, scientists will need to determine whatof human embryonic stem cells, and many key differentiation stages of human ES-derived cells arequestions remain unanswered or only partly optimal for other practical applications. For example,answered. For example, in order to refine and what differentiation stages of ES-derived cells wouldimprove ES cell culture systems, it is important that be best for screening drugs or toxins, or for deliveringscientists identify the mechanisms that allow human potentially therapeutic drugs?ES cells in vitro to proliferate without differentiating[29]. Once the mechanisms that regulate human ES REFERENCESproliferation are known, it will likely be possible to 1. Amit, M., Carpenter, M.K., Inokuma, M.S., Chiu, C.P Harris, .,apply this knowledge to the long-standing challenge C.P Waknitz, M.A., Itskovitz-Eldor, J., and Thomson, J.A. .,of improving the in vitro self-renewal capabilities of (2000). Clonally derived human embryonic stem cell linesadult stem cells. maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271-278.It will also be important to determine whether the 2. Andrews, P .W., Damjanov, I., Simon, D., Banting, G.S.,genetic imprinting status of human ES cells plays any Carlin, C., Dracopoli, N.C., and Fogh, J. (1984). Pluripotentsignificant role in maintaining the cells, directing their embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivodifferentiation, or determining their suitability for and in vitro. Lab. Invest. 50, 147-162.transplant. One of the effects of growing mouseblastocysts in culture is a change in the methylation 3. Andrews, P (1988). Human teratocarcinomas. Biochim. .W. Biophys. Acta. 948, 17-36.of specific genes that control embryonic growthand development [23]. Do similar changes in gene 4. Andrews, P (1998). Teratocarcinomas and human .W. embryology: pluripotent human EC cell lines. Reviewimprinting patterns occur in human ES cells (or article. APMIS 106, 158-167.blastocysts)? If so, what is their effect on in vitrodevelopment and on any differentiated cell types 5. Andrews, P W., personal communication. .that may be derived from cultured ES cells? 6. Bongso, A., Fong, C.Y., Ng, S.C., and Ratnam, S.S. (1994). Blastocyst transfer in human in vitro: fertilization; the use ofEfforts will need to be made to determine whether embryo co-culture. Cell Biol. Int. 18, 1181-1189.cultures of human ES cells that appear to be 7. Bongso, A., Fong, C.Y., Ng, S.C., and Ratnam, S. (1994).homogeneous and undifferentiated are, in fact, Isolation and culture of inner cell mass cells from humanhomogeneous and undifferentiated. Is it possible blastocysts. Hum. Reprod. 9, 2110-2117.that human ES cells in vitro cycle in and out of 8. Bongso, A., Fong, C.Y., Ng, S.C., and Ratnam, S.S. (1995).partially differentiated states? And if that occurs, how Co-culture techniques for blastocyst transfer and embry-will it affect attempts to direct their differentiation or onic stem cell production. Asst. Reprod. Rev. 5, 106-114.maintain the cells in a proliferating state [28]? 19
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Gardner, D.K. and Schoolcraft, W.B. (1999). Culture and gene therapy. Reprod. Fertil. Dev. 10, 31-47. transfer of human blastocysts. Curr. Opin. Obstet. Gynecol. 11, 307-311. 36. Resnick, J.L., Bixler, L.S., Cheng, L., and Donovan, P (1992). .J. Long-term proliferation of mouse primordial germ cells in19. Gearhart, J., personal communication. culture. Nature. 359, 550-551.20. Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., 37. Reubinoff BE, Pera, M., Fong, C.Y., and Trounson, A. and Yanuka, O., Amit, M., Soreq, H., and Benvenisty, N. (2000). Bongso, A. (2000). Research Errata. Nat. Biotechnol. 18, Differentiation of human embryonic stem cells into embry- 559. oid bodies comprising the three embryonic germ layers. Mol. Med. 6, 88-95. 38. Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A., and Bongso, A. (2000). Embryonic stem cell lines from human21. Jones, G.M., Trounson, A.O., Lolatgis, N., and Wood, C. blastocysts: somatic differentiation in vitro. Nat. Biotechnol. (1998). Factors affecting the success of human blastocyst 18, 399-404. development and pregnancy following in vitro fertilization and embryo transfer. Fertil. Steril. 70, 1022-1029. 39. Sathananthan, A.H. (1997). Ultrastructure of the human egg. Hum. Cell. 10, 21-38.22. Jones, J.M. and Thomson, J.A. (2000). Human embryonic stem cell technology. Semin. Reprod. Med. 18, 219-223. 40. Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D., and Benvenisty, N. (2000). Effects of eight growth factors on the23. Khosla, S., Dean, W., Brown, D., Reik, W., and differentiation of cells derived from human embryonic stem Feil, R. (2001). Culture of preimplantation mouse embryos cells. Proc. Natl. Acad. Sci. U. S. A. 97, 11307-11312. affects fetal development and the expression of imprinted genes. Biol. Reprod. 64, 918-926. 41. Shamblott, M.J., Axelman, J., Wang, S., Bugg, E.M., Littlefield, J.W., Donovan, P Blumenthal, P .J., .D., Huggins,24. Kleinsmith, L.J. and Pierce Jr, G.B. (1964). 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  • The Human Embryonic Stem Cell and The Human Embryonic Germ Cell42. Shamblott, M.J., Axelman, J., Littlefield, J.W., Blumenthal, 48. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., P.D., Huggins, G.R., Cui, Y., Cheng, L., and Gearhart, J.D. Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998). (2001). Human embryonic germ cell derivatives express a Embryonic stem cell lines derived from human blastocysts. broad range of develpmentally distinct markers and Science. 282, 1145-1147. proliferate extensively in vitro. Proc. Natl. Acad. Sci. U. S. A. 98, 113-118. 49. Trounson, A.O., Gardner, D.K., Baker, G., Barnes, F.L., Bongso, A., Bourne, H., Calderon, I., Cohen, J., Dawson, K.,43. Smith, A.G. (2001). Origins and properties of mouse Eldar-Geve, T., Gardner, D.K., Graves, G., Healy, D., Lane, embryonic stem cells. Annu. Rev. Cell. Dev. Biol. M., Leese, H.J., Leeton, J., Levron, J., Liu, D.Y., MacLachlan, V., Munné, S., Oranratnachai, A., Rogers, P Rombauts, L., .,44. Thompson, S., Stern, P Webb, M., Walsh, F.S., Engstrom, .L., Sakkas, D., Sathananthan, A.H., Schimmel, T., Shaw, J., W., Evans, E.P Shi, W.K., Hopkins, B., and Graham, C.F. ., Trounson, A.O., Van Steirteghem, A., Willadsen, S., and (1984). Cloned human teratoma cells differentiate into Wood, C. (2000b). Handbook of in vitro fertilization, (Boca neuron-like cells and other cell types in retinoic acid. J. Cell Raton, London, New York, Washington, D.C.: Sci. 72, 37-64. CRC Press).45. Thomson, J., personal communication. 50. Trounson, A.O., Anderiesz, C., and Jones, G. (2001). Maturation of human oocytes in vitro and their develop-46. Thomson, J.A., Kalishman, J., Golos, T.G., Durning, M., Harris, mental competence. Reproduction. 121, 51-75. C.P Becker, R.A., and Hearn, J.P (1995). Isolation of a ., . primate embryonic stem cell line. Proc. Natl. Acad. Sci. 51. Xu, C., Inokuma, M.S., Denham, J., Golds, K., Kundu, P., U. S. A. 92, 7844-7848. Gold, J.D., and Carpenter, M.K. Keystone symposia. Pluripotent stem cells: biology and applications. Growth of47. Thomson, J.A., Kalishman, J., Golos, T.G., Durning, M., Harris, undifferentiated human embryonic stem cells on defined C.P and Hearn, J.P (1996). Pluripotent cell lines derived ., . matrices with conditioned medium. Poster abstract. 133. from common marmoset (Callithrix jacchus) blastocysts. Biol. Reprod. 55, 254-259. 21
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  • 4. THE ADULT STEM CELLFor many years, researchers have been seeking to characteristic may not be as definitive as onceunderstand the body’s ability to repair and replace thought [82] (see Figure 4.1. Distinguishing Features ofthe cells and tissues of some organs, but not others. Progenitor/Precursor Cells and Stem Cells).After years of work pursuing the how and why ofseemingly indiscriminant cell repair mechanisms, Adult stem cells are rare. Their primary functions arescientists have now focused their attention on adult to maintain the steady state functioning of a cell—stem cells. It has long been known that stem cells called homeostasis—and, with limitations, to replaceare capable of renewing themselves and that they cells that die because of injury or disease [44, 58].can generate multiple cell types. Today, there is new For example, only an estimated 1 in 10,000 to 15,000evidence that stem cells are present in far more cells in the bone marrow is a hematopoietic (blood-tissues and organs than once thought and that these forming) stem cell (HSC) [105]. Furthermore, adultcells are capable of developing into more kinds of stem cells are dispersed in tissues throughout thecells than previously imagined. Efforts are now mature animal and behave very differently,underway to harness stem cells and to take depending on their local environment. For example,advantage of this new found capability, with the goal HSCs are constantly being generated in the boneof devising new and more effective treatments for a marrow where they differentiate into mature types ofhost of diseases and disabilities. What lies ahead for blood cells. Indeed, the primary role of HSCs is tothe use of adult stem cells is unknown, but it is replace blood cells [26] (see Chapter 5. Hemato-certain that there are many research questions to be poietic Stem Cells). In contrast, stem cells in the smallanswered and that these answers hold great promise intestine are stationary, and are physically separatedfor the future. from the mature cell types they generate. Gut epi- thelial stem cells (or precursors) occur at the bases of crypts—deep invaginations between the mature,WHAT IS AN ADULT STEM CELL? differentiated epithelial cells that line the lumen of theAdult stem cells, like all stem cells, share at least two intestine. These epithelial crypt cells divide fairly often,characteristics. First, they can make identical copies but remain part of the stationary group of cells theyof themselves for long periods of time; this ability to generate [93].proliferate is referred to as long-term self-renewal. Unlike embryonic stem cells, which are defined bySecond, they can give rise to mature cell types that their origin (the inner cell mass of the blastocyst),have characteristic morphologies (shapes) and adult stem cells share no such definitive means ofspecialized functions. Typically, stem cells generate characterization. In fact, no one knows the origin ofan intermediate cell type or types before they adult stem cells in any mature tissue. Some haveachieve their fully differentiated state. The intermedi- proposed that stem cells are somehow set asideate cell is called a precursor or progenitor cell. during fetal development and restrained fromProgenitor or precursor cells in fetal or adult tissues are differentiating. Definitions of adult stem cells vary inpartly differentiated cells that divide and give rise to the scientific literature range from a simple descrip-differentiated cells. Such cells are usually regarded as tion of the cells to a rigorous set of experimental“committed” to differentiating along a particular criteria that must be met before characterizing acellular development pathway, although this 23
  • The Adult Stem Cell STEM CELL STEM CELL (e.g., hematopoietic stem cell) SPECIALIZED CELL (e.g., neuron) PROGENITOR CELL SPECIALIZED CELL (e.g., myeloid (e.g., neutrophil) progenitor cell) SPECIALIZED CELL© 2001 Terese Winslow, Lydia Kibiuk (e.g., red blood cell) Figure 4.1. Distinguishing Features of Progenitor/Precursor Cells and Stem Cells. A stem cell is an unspecialized cell that is capable of replicating or self renewing itself and developing into specialized cells of a variety of cell types. The product of a stem cell undergoing division is at least one additional stem cell that has the same capabilities of the originating cell. Shown here is an example of a hematopoietic stem cell producing a second generation stem cell and a neuron. A progenitor cell (also known as a precursor cell) is unspecialized or has partial characteristics of a specialized cell that is capable of undergoing cell division and yielding two specialized cells. Shown here is an example of a myeloid progenitor/precursor undergoing cell division to yield two specialized cells (a neutrophil and a red blood cell). 24
  • The Adult Stem Cellparticular cell as an adult stem cell. Most of the infor- genetically identical (clonal) stem cells. In order tomation about adult stem cells comes from studies of fully characterize the regenerating and self-renewalmice. The list of adult tissues reported to contain stem capabilities of the adult stem cell, and therefore tocells is growing and includes bone marrow, peripheral truly harness its potential, it will be important toblood, brain, spinal cord, dental pulp, blood vessels, demonstrate that a single adult stem cell can,skeletal muscle, epithelia of the skin and digestive indeed, generate a line of genetically identical cells,system, cornea, retina, liver, and pancreas. which then gives rise to all the appropriate, differenti- ated cell types of the tissue in which it resides.In order to be classified as an adult stem cell, the cellshould be capable of self-renewal for the lifetime ofthe organism. This criterion, although fundamental to EVIDENCE FOR THE PRESENCE OFthe nature of a stem cell, is difficult to prove in vivo. It ADULT STEM CELLSis nearly impossible, in an organism as complex as a Adult stem cells have been identified in many animalhuman, to design an experiment that will allow the and human tissues. In general, three methods arefate of candidate adult stem cells to be identified in used to determine whether candidate adult stemvivo and tracked over an individual’s entire lifetime. cells give rise to specialized cells. Adult stem cellsIdeally, adult stem cells should also be clonogenic. In can be labeled in vivo and then they can beother words, a single adult stem cell should be able tracked. Candidate adult stem cells can also beto generate a line of genetically identical cells, which isolated and labeled and then transplanted back intothen gives rise to all the appropriate, differentiated the organism to determine what becomes of them.cell types of the tissue in which it resides. Again, this Finally, candidate adult stem cells can be isolated,property is difficult to demonstrate in vivo; in practice, grown in vitro and manipulated, by adding growthscientists show either that a stem cell is clonogenic in factors or introducing genes that help determinevitro, or that a purified population of candidate stem what differentiated cells types they will yield. Forcells can repopulate the tissue. example, currently, scientists believe that stem cells in the fetal and adult brain divide and give rise to moreAn adult stem cell should also be able to give rise to stem cells or to several types of precursor cells, whichfully differentiated cells that have mature phenotypes, give rise to nerve cells (neurons), of which there areare fully integrated into the tissue, and are capable many types.of specialized functions that are appropriate for thetissue. The term phenotype refers to all the observ- It is often difficult—if not impossible—to distinguishable characteristics of a cell (or organism); its shape adult, tissue-specific stem cells from progenitor cells,(morphology); interactions with other cells and the which are found in fetal or adult tissues and are partlynon-cellular environment (also called the extracellular differentiated cells that divide and give rise to differ-matrix); proteins that appear on the cell surface entiated cells. These are cells found in many organs(surface markers); and the cell’s behavior (e.g., that are generally thought to be present to replacesecretion, contraction, synaptic transmission). cells and maintain the integrity of the tissue. Progenitor cells give rise to certain types of cells—The majority of researchers who lay claim to having such as the blood cells known as T lymphocytes,identified adult stem cells rely on two of these char- B lymphocytes, and natural killer cells—but are notacteristics—appropriate cell morphology, and the thought to be capable of developing into all the celldemonstration that the resulting, differentiated cell types of a tissue and as such are not truly stem cells.types display surface markers that identify them as The current wave of excitement over the existence ofbelonging to the tissue. Some studies demonstrate stem cells in many adult tissues is perhaps fuelingthat the differentiated cells that are derived from claims that progenitor or precursor cells in those tis-adult stem cells are truly functional, and a few studies sues are instead stem cells. Thus, there are reports ofshow that cells are integrated into the differentiated endothelial progenitor cells, skeletal muscle stemtissue in vivo and that they interact appropriately with cells, epithelial precursors in the skin and digestiveneighboring cells. At present, there is, however, a system, as well as some reports of progenitors or stempaucity of research, with a few notable exceptions, in cells in the pancreas and liver. A detailed summarywhich researchers were able to conduct studies of of some of the evidence for the existence of stem 25
  • The Adult Stem Cellcells in various tissues and organs is presented later in cells have adopted key structural and biochemicalthe chapter. characteristics of the new tissue they are claimed to have generated. Ultimately—and most importantly—itADULT STEM CELL PLASTICITY is necessary to demonstrate that the cells can inte- grate into their new tissue environment, survive in theIt was not until recently that anyone seriously consid- tissue, and function like the mature cells of the tissue.ered the possibility that stem cells in adult tissuescould generate the specialized cell types of another In the experiments reported to date, adult stem cellstype of tissue from which they normally reside—either may assume the characteristics of cells that havea tissue derived from the same embryonic germ developed from the same primary germ layer or alayer or from a different germ layer (see Table 1.1. different germ layer (see Figure 4.2. PreliminaryEmbryonic Germ Layers From Which Differentiated Evidence of Plasticity Among Nonhuman Adult StemTissues Develop). For example, studies have shown Cells). For example, many plasticity experimentsthat blood stem cells (derived from mesoderm) may involve stem cells derived from bone marrow, whichbe able to generate both skeletal muscle (also is a mesodermal derivative. The bone marrow stemderived from mesoderm) and neurons (derived from cells may then differentiate into another mesoder-ectoderm). That realization has been triggered by a mally derived tissue such as skeletal muscle [28, 43],flurry of papers reporting that stem cells derived from cardiac muscle [51, 71] or liver [4, 54, 97].one adult tissue can change their appearance and Alternatively, adult stem cells may differentiate into aassume characteristics that resemble those of differ- tissue that—during normal embryonic develop-entiated cells from other tissues. ment—would arise from a different germ layer. ForThe term plasticity, as used in this report, means that example, bone marrow-derived cells may differenti-a stem cell from one adult tissue can generate the ate into neural tissue, which is derived from embryon-differentiated cell types of another tissue. At this time, ic ectoderm [15, 65]. And—reciprocally—neural stemthere is no formally accepted name for this phenom- cell lines cultured from adult brain tissue may differ-enon in the scientific literature. It is variously referred entiate to form hematopoietic cells [13], or even giveto as “plasticity” [15, 52], “unorthodox differentiation” rise to many different cell types in a chimeric embryo[10] or “transdifferentiation” [7, 54]. [17]. In both cases cited above, the cells would be deemed to show plasticity, but in the case of boneApproaches for Demonstrating Adult Stem Cell marrow stem cells generating brain cells, the findingPlasticity is less predictable.To be able to claim that adult stem cells demon-strate plasticity, it is first important to show that a cell In order to study plasticity within and across germpopulation exists in the starting tissue that has the layer lines, the researcher must be sure that he/she isidentifying features of stem cells. Then, it is necessary using only one kind of adult stem cell. The vast major-to show that the adult stem cells give rise to cell types ity of experiments on plasticity have been conductedthat normally occur in a different tissue. Neither of with adult stem cells derived either from the bonethese criteria is easily met. Simply proving the exis- marrow or the brain. The bone marrow-derived cellstence of an adult stem cell population in a differenti- are sometimes sorted—using a panel of surfaceated tissue is a laborious process. It requires that the markers—into populations of hematopoietic stemcandidate stem cells are shown to be self-renewing, cells or bone marrow stromal cells [46, 54, 71]. Theand that they can give rise to the differentiated cell HSCs may be highly purified or partially purified,types that are characteristic of that tissue. depending on the conditions used. Another way to separate population of bone marrow cells is by frac-To show that the adult stem cells can generate other tionation to yield cells that adhere to a growth sub-cell types requires them to be tracked in their new strate (stromal cells) or do not adhere (hematopoieticenvironment, whether it is in vitro or in vivo. In general, cells) [28].this has been accomplished by obtaining the stemcells from a mouse that has been genetically engi- To study plasticity of stem cells derived from the brain,neered to express a molecular tag in all its cells. It is the researcher must overcome several problems.then necessary to show that the labeled adult stem Stem cells from the central nervous system (CNS), 26
  • The Adult Stem Cell Liver Brain CNS stem cells Skeletal Bone muscle marrow Bone Blood cell Bone marrow Blood vessel stromal cell Epithelial cell Fat cell© 2001 Terese Winslow, Lydia Kibiuk, Caitlin Duckwall Cardiac muscle Neuron Glial cell Figure 4.2. Preliminary Evidence of Plasticity Among Nonhuman Adult Stem Cells. 27
  • The Adult Stem Cellunlike bone marrow cells, do not occur in a single, that may be useful for therapeutic transplantation. Ifaccessible location. Instead, they are scattered in the phenomenon of plasticity is to be used as a basisthree places, at least in rodent brain—the tissue for generating tissue for transplantation, the tech-around the lateral ventricles in the forebrain, a migra- niques for doing it will need to be reproducible andtory pathway for the cells that leads from the ventri- reliable (see Chapter 10. Assessing Human Stem Cellcles to the olfactory bulbs, and the hippocampus. Safety). In some cases, debate continues aboutMany of the experiments with CNS stem cells involve observations that adult stem cells yield cells of tissuethe formation of neurospheres, round aggregates of types different than those from which they werecells that are sometimes clonally derived. But it is not obtained [7, 68].possible to observe cells in the center of a neu-rosphere, so to study plasticity in vitro, the cells are EXPERIMENTAL EVIDENCE OF ADULTusually dissociated and plated in monolayers. To STEM CELLS AND PLASTICITYstudy plasticity in vivo, the cells may be dissociatedbefore injection into the circulatory system of the Adult Stem Cells of the Nervous Systemrecipient animal [13], or injected as neurospheres More than 30 years ago, Altman and Das showed[17]. that two regions of the postnatal rat brain, the hip-What is the Evidence for Plasticity? pocampus and the olfactory bulb, contain dividing cells that become neurons [5, 6]. Despite theseThe differentiated cell types that result from plasticity reports, the prevailing view at the time was that nerveare usually reported to have the morphological char- cells in the adult brain do not divide. In fact, theacteristics of the differentiated cells and to display notion that stem cells in the adult brain can generatetheir characteristic surface markers. In reports that its three major cell types—astrocytes and oligoden-transplanted adult stem cells show plasticity in vivo, drocytes, as well as neurons—was not accepted untilthe stem cells typically are shown to have integrated far more recently. Within the past five years, a seriesinto a mature host tissue and assumed at least some of studies has shown that stem cells occur in theof its characteristics [15, 28, 51, 65, 71]. Many plastic- adult mammalian brain and that these cells canity experiments involve injury to a particular tissue, generate its three major cell lineages [35, 48, 63, 66,which is intended to model a particular human 90, 96, 104] (see Chapter 8. Rebuilding the Nervousdisease or injury [13, 54, 71]. However, there is limited System with Stem Cells).evidence to date that such adult stem cells cangenerate mature, fully functional cells or that the cells Today, scientists believe that stem cells in the fetalhave restored lost function in vivo [54]. Most of the and adult brain divide and give rise to more stemstudies that show the plasticity of adult stem cells cells or to several types of precursor cells. Neuronalinvolve cells that are derived from the bone marrow precursors (also called neuroblasts) divide and give[15, 28, 54, 65, 77] or brain [13, 17]. To date, adult rise to nerve cells (neurons), of which there are manystem cells are best characterized in these two tissues, types. Glial precursors give rise to astrocytes or oligo-which may account for the greater number of plas- dendrocytes. Astrocytes are a kind of glial cell, whichticity studies based on bone marrow and brain. lend both mechanical and metabolic support forCollectively, studies on plasticity suggest that stem neurons; they make up 70 to 80 percent of the cellscell populations in adult mammals are not fixed of the adult brain. Oligodendrocytes make myelin,entities, and that after exposure to a new environ- the fatty material that ensheathes nerve cell axonsment, they may be able to populate other tissues and speeds nerve transmission. Under normal, in vivoand possibly differentiate into other cell types. conditions, neuronal precursors do not give rise to glial cells, and glial precursors do not give rise toIt is not yet possible to say whether plasticity occurs neurons. In contrast, a fetal or adult CNS (centralnormally in vivo. Some scientists think it may [14, 64], nervous system—the brain and spinal cord) stem cellbut as yet there is no evidence to prove it. Also, it is may give rise to neurons, astrocytes, or oligodendro-not yet clear to what extent plasticity can occur in cytes, depending on the signals it receives and itsexperimental settings, and how—or whether—the three-dimensional environment within the brain tissue.phenomenon can be harnessed to generate tissues 28
  • The Adult Stem CellThere is now widespread consensus that the adult A recent report indicates that the astrocytes thatmammalian brain does contain stem cells. However, occur in the subventricular zone of the rodent brainthere is no consensus about how many populations act as neural stem cells. The cells with astrocyteof CNS stem cells exist, how they may be related, markers appear to generate neurons in vivo, asand how they function in vivo. Because there are no identified by their expression of specific neuronalmarkers currently available to identify the cells in vivo, markers. The in vitro assay to demonstrate that thesethe only method for testing whether a given popula- astrocytes are, in fact, stem cells involves their abilitytion of CNS cells contains stem cells is to isolate the to form neurospheres—groupings of undifferentiatedcells and manipulate them in vitro, a process that cells that can be dissociated and coaxed to differen-may change their intrinsic properties [67]. tiate into neurons or glial cells [25]. Traditionally, these astrocytes have been regarded as differentiatedDespite these barriers, three groups of CNS stem cells cells, not as stem cells and so their designation ashave been reported to date. All occur in the adult stem cells is not universally accepted.rodent brain and preliminary evidence indicates theyalso occur in the adult human brain. One group A series of similar in vitro studies based on theoccupies the brain tissue next to the ventricles, formation of neurospheres was used to identify theregions known as the ventricular zone and the sub- subependymal zone as a source of adult rodent CNSventricular zone (see discussion below). The ventricles stem cells. In these experiments, single, candidateare spaces in the brain filled with cerebrospinal fluid. stem cells derived from the subependymal zone areDuring fetal development, the tissue adjacent to the induced to give rise to neurospheres in the presenceventricles is a prominent region of actively dividing of mitogens—either epidermal growth factor (EGF) orcells. By adulthood, however, this tissue is much small- fibroblast growth factor-2 (FGF-2). The neurosphereser, although it still appears to contain stem cells [70]. are dissociated and passaged. As long as a mitogen is present in the culture medium, the cells continueA second group of adult CNS stem cells, described in forming neurospheres without differentiating. Somemice but not in humans, occurs in a streak of tissue populations of CNS cells are more responsive to EGF,that connects the lateral ventricle and the olfactory others to FGF [100]. To induce differentiation intobulb, which receives odor signals from the nose. In neurons or glia, cells are dissociated from therodents, olfactory bulb neurons are constantly being neurospheres and grown on an adherent surfacereplenished via this pathway [59, 61]. A third possible in serum-free medium that contains specific growthlocation for stem cells in adult mouse and human factors. Collectively, the studies demonstrate that abrain occurs in the hippocampus, a part of the brain population of cells derived from the adult rodentthought to play a role in the formation of certain kinds brain can self-renew and differentiate to yield theof memory [27, 34]. three major cell types of the CNS cells [41, 69,Central Nervous System Stem Cells in the 74, 102].Subventricular Zone. CNS stem cells found in the fore- Central Nervous System Stem Cells in the Ventricularbrain that surrounds the lateral ventricles are hetero- Zone. Another group of potential CNS stem cells ingeneous and can be distinguished morphologically. the adult rodent brain may consist of the ependymalEpendymal cells, which are ciliated, line the ventri- cells themselves [47]. Ependymal cells, which arecles. Adjacent to the ependymal cell layer, in a ciliated, line the lateral ventricles. They have beenregion sometimes designated as the subependymal described as non-dividing cells [24] that function asor subventricular zone, is a mixed cell population that part of the blood-brain barrier [22]. The suggestionconsists of neuroblasts (immature neurons) that that ependymal cells from the ventricular zone of themigrate to the olfactory bulb, precursor cells, and adult rodent CNS may be stem cells is thereforeastrocytes. Some of the cells divide rapidly, while unexpected. However, in a recent study, in which twoothers divide slowly. The astrocyte-like cells can be molecular tags—the fluorescent marker Dil, and anidentified because they contain glial fibrillary acidic adenovirus vector carrying lacZ tags—were used toprotein (GFAP), whereas the ependymal cells stain label the ependymal cells that line the entire CNSpositive for nestin, which is regarded as a marker of ventricular system of adult rats, it was shown thatneural stem cells. Which of these cells best qualifies these cells could, indeed, act as stem cells. A fewas a CNS stem cell is a matter of debate [76]. 29
  • The Adult Stem Celldays after labeling, fluorescent or lacZ+ cells were Are ventricular and subventricular zone CNS stemobserved in the rostral migratory stream (which leads cells the same population? These studies and otherfrom the lateral ventricle to the olfactory bulb), and leave open the question of whether cells that directlythen in the olfactory bulb itself. The labeled cells in line the ventricles—those in the ventricular zone—orthe olfactory bulb also stained for the neuronal cells that are at least a layer removed from thismarkers ȋIII tubulin and Map2, which indicated that zone—in the subventricular zone are the same popu-ependymal cells from the ventricular zone of the lation of CNS stem cells. A new study, based on theadult rat brain had migrated along the rostral finding that they express different genes, confirmsmigratory stream to generate olfactory bulb neurons earlier reports that the ventricular and subventricularin vivo [47]. zone cell populations are distinct. The new research utilizes a technique called representational differenceTo show that Dil+ cells were neural stem cells and analysis, together with cDNA microarray analysis, tocould generate astrocytes and oligodendrocytes as monitor the patterns of gene expression in the com-well as neurons, a neurosphere assay was performed plex tissue of the developing and postnatal mousein vitro. Dil-labeled cells were dissociated from the brain. The study revealed the expression of a panel ofventricular system and cultured in the presence of genes known to be important in CNS development,mitogen to generate neurospheres. Most of the such as L3-PSP (which encodes a phosphoserineneurospheres were Dil+; they could self-renew and phosphatase important in cell signaling), cyclin D2 (agenerate neurons, astrocytes, and oligodendrocytes cell cycle gene), and ERCC-1 (which is important inwhen induced to differentiate. Single, Dil+ ependymal DNA excision repair). All of these genes in the recentcells isolated from the ventricular zone could also study were expressed in cultured neurospheres, asgenerate self-renewing neurospheres and differen- well as the ventricular zone, the subventricular zone,tiate into neurons and glia. and a brain area outside those germinal zones. ThisTo show that ependymal cells can also divide in vivo, analysis also revealed the expression of novel genesbromodeoxyuridine (BrdU) was administered in the such as A16F10, which is similar to a gene in andrinking water to rats for a 2- to 6-week period. embryonic cancer cell line. A16F10 was expressed inBromodeoxyuridine (BrdU) is a DNA precursor that is neurospheres and at high levels in the subventricularonly incorporated into dividing cells. Through a series zone, but not significantly in the ventricular zone.of experiments, it was shown that ependymal cells Interestingly, several of the genes identified in cultureddivide slowly in vivo and give rise to a population of neurospheres were also expressed in hematopoieticprogenitor cells in the subventricular zone [47]. A cells, suggesting that neural stem cells and blood-different pattern of scattered BrdU-labeled cells was forming cells may share aspects of their geneticobserved in the spinal cord, which suggested that programs or signaling systems [38]. This finding mayependymal cells along the central canal of the cord help explain recent reports that CNS stem cellsoccasionally divide and give rise to nearby ependy- derived from mouse brain can give rise tomal cells, but do not migrate away from the canal. hematopoietic cells after injection into irradiated mice [13].Collectively, the data suggest that CNS ependymalcells in adult rodents can function as stem cells. The Central Nervous System Stem Cells in thecells can self-renew, and most proliferate via asym- Hippocampus. The hippocampus is one of the oldestmetrical division. Many of the CNS ependymal cells parts of the cerebral cortex, in evolutionary terms,are not actively dividing (quiescent), but they can be and is thought to play an important role in certainstimulated to do so in vitro (with mitogens) or in vivo forms of memory. The region of the hippocampus in(in response to injury). After injury, the ependymal cells which stem cells apparently exist in mouse andin the spinal cord only give rise to astrocytes, not to human brains is the subgranular zone of the dentateneurons. How and whether ependymal cells from the gyrus. In mice, when BrdU is used to label dividingventricular zone are related to other candidate popu- cells in this region, about 50% of the labeled cellslations of CNS stem cells, such as those identified in differentiate into cells that appear to be dentatethe hippocampus [34], is not known. 30
  • The Adult Stem Cellgyrus granule neurons, and 15% become glial cells. vary in different species. These cells appear toThe rest of the BrdU-labeled cells do not have a represent different stem cell populations, rather thanrecognizable phenotype [90]. Interestingly, many, if a single population of stem cells that is dispersed innot all the BrdU-labeled cells in the adult rodent multiple sites. The normal development of the brainhippocampus occur next to blood vessels [33]. depends not only on the proliferation and differen- tiation of these fetal stem cells, but also on aIn the human dentate gyrus, some BrdU-labeled cells genetically programmed process of selective cellexpress NeuN, neuron-specific enolase, or calbindin, death called apoptosis [76].all of which are neuronal markers. The labeledneuron-like cells resemble dentate gyrus granule Little is known about stem cells in the human fetalcells, in terms of their morphology (as they did in brain. In one study, however, investigators derivedmice). Other BrdU-labeled cells express glial fibrillary clonal cell lines from CNS stem cells isolated from theacidic protein (GFAP) an astrocyte marker. The study diencephalon and cortex of human fetuses, 10.5involved autopsy material, obtained with family weeks post-conception [103]. The study is unusual,consent, from five cancer patients who had been not only because it involves human CNS stem cellsinjected with BrdU dissolved in saline prior to their obtained from fetal tissue, but also because the cellsdeath for diagnostic purposes. The patients ranged in were used to generate clonal cell lines of CNS stemage from 57 to 72 years. The greatest number of cells that generated neurons, astrocytes, andBrdU-labeled cells were identified in the oldest oligodendrocytes, as determined on the basis ofpatient, suggesting that new neuron formation in the expressed markers. In a few experiments describedhippocampus can continue late in life [27]. as “preliminary,” the human CNS stem cells were injected into the brains of immunosuppressed ratsFetal Central Nervous System Stem Cells. Not where they apparently differentiated into neuron-likesurprisingly, fetal stem cells are numerous in fetal cells or glial cells.tissues, where they are assumed to play an importantrole in the expansion and differentiation of all tissues In a 1999 study, a serum-free growth medium thatof the developing organism. Depending on the included EGF and FGF2 was devised to grow thedevelopmental stage of an animal, fetal stem cells human fetal CNS stem cells. Although most of theand precursor cells—which arise from stem cells— cells died, occasionally, single CNS stem cells sur-may make up the bulk of a tissue. This is certainly true vived, divided, and ultimately formed neurospheresin the brain [48], although it has not been demon- after one to two weeks in culture. The neurospheresstrated experimentally in many tissues. could be dissociated and individual cells replated. The cells resumed proliferation and formed new neu-It may seem obvious that the fetal brain contains rospheres, thus establishing an in vitro system that (likestem cells that can generate all the types of neurons the system established for mouse CNS neurospheres)in the brain as well as astrocytes and oligodendro- could be maintained up to 2 years. Depending oncytes, but it was not until fairly recently that the con- the culture conditions, the cells in the neurospherescept was proven experimentally. There has been a could be maintained in an undifferentiated dividinglong-standing question as to whether or not the same state (in the presence of mitogen), or dissociatedcell type gives rise to both neurons and glia. In stud- and induced to differentiate (after the removal ofies of the developing rodent brain, it has now been mitogen and the addition of specific growth factorsshown that all the major cell types in the fetal brain to the culture medium). The differentiated cells con-arise from a common population of progenitor cells sisted mostly of astrocytes (75%), some neurons (13%)[20, 34, 48, 80, 108]. and rare oligodendrocytes (1.2%). The neurons gen-Neural stem cells in the mammalian fetal brain are erated under these conditions expressed markersconcentrated in seven major areas: olfactory bulb, indicating they were GABAergic, [the major type ofependymal (ventricular) zone of the lateral ventricles inhibitory neuron in the mammalian CNS responsive(which lie in the forebrain), subventricular zone (next to the amino acid neurotransmitter, gamma-to the ependymal zone), hippocampus, spinal cord, aminobutyric acid (GABA)]. However, catecholamine-cerebellum (part of the hindbrain), and the cerebral like cells that express tyrosine hydroxylase (TH, acortex. Their number and pattern of development critical enzyme in the dopamine-synthesis pathway) 31
  • The Adult Stem Cellcould be generated, if the culture conditions were rat neural crest cells develop into neurons and glia,altered to include different medium conditioned by a an indication of their stem cell-like properties [67].rat glioma line (BB49). Thus, the report indicates that However, the ability of rat E14.5 neural crest cellshuman CNS stem cells obtained from early fetuses taken from sciatic nerve to generate nerve and glialcan be maintained in vitro for a long time without cells in chick is more limited than neural crest cellsdifferentiating, induced to differentiate into the three derived from younger, E10.5 rat embryos. At themajor lineages of the CNS (and possibly two kinds of earlier stage of development, the neural tube hasneurons, GABAergic and TH-positive), and engraft (in formed, but neural crest cells have not yet migratedrats) in vivo [103]. to their final destinations. Neural crest cells from early developmental stages are more sensitive toCentral Nervous System Neural Crest Stem Cells. bone morphogenetic protein 2 (BMP2) signaling,Neural crest cells differ markedly from fetal or adult which may help explain their greater differentiationneural stem cells. During fetal development, neural potential [106].crest cells migrate from the sides of the neural tubeas it closes. The cells differentiate into a range of Stem Cells in the Bone Marrow and Bloodtissues, not all of which are part of the nervous system The notion that the bone marrow contains stem cells[56, 57, 91]. Neural crest cells form the sympathetic is not new. One population of bone marrow cells, theand parasympathetic components of the peripheral hematopoietic stem cells (HSCs), is responsible fornervous system (PNS), including the network of nerves forming all of the types of blood cells in the body.that innervate the heart and the gut, all the sensory HSCs were recognized as a stem cells more than 40ganglia (groups of neurons that occur in pairs along years ago [9, 99]. Bone marrow stromal cells—athe dorsal surface of the spinal cord), and Schwann mixed cell population that generates bone, cartilage,cells, which (like oligodendrocytes in the CNS) make fat, fibrous connective tissue, and the reticular net-myelin in the PNS. The non-neural tissues that arise work that supports blood cell formation—werefrom the neural crest are diverse. They populate described shortly after the discovery of HSCs [30, 32,certain hormone-secreting glands—including the 73]. The mesenchymal stem cells of the boneadrenal medulla and Type I cells in the carotid marrow also give rise to these tissues, and maybody—pigment cells of the skin (melanocytes), constitute the same population of cells as the bonecartilage and bone in the face and skull, and marrow stromal cells [78]. Recently, a population ofconnective tissue in many parts of the body [76]. progenitor cells that differentiates into endothelial cells, a type of cell that lines the blood vessels, wasThus, neural crest cells migrate far more extensively isolated from circulating blood [8] and identified asthan other fetal neural stem cells during develop- originating in bone marrow [89]. Whether thesement, form mesenchymal tissues, most of which endothelial progenitor cells, which resemble thedevelop from embryonic mesoderm as well as the angioblasts that give rise to blood vessels duringcomponents of the CNS and PNS which arises from embryonic development, represent a bona fideembryonic ectoderm. This close link, in neural crest population of adult bone marrow stem cells remainsdevelopment, between ectodermally derived tissues uncertain. Thus, the bone marrow appears to containand mesodermally derived tissues accounts in part three stem cell populations—hematopoietic stemfor the interest in neural crest cells as a kind of stem cells, stromal cells, and (possibly) endothelialcell. In fact, neural crest cells meet several criteria of progenitor cells (see Figure 4.3. Hematopoietic andstem cells. They can self-renew (at least in the fetus) Stromal Stem Cell Differentiation).and can differentiate into multiple cells types, whichinclude cells derived from two of the three embryonic Two more apparent stem cell types have beengerm layers [76]. reported in circulating blood, but have not been shown to originate from the bone marrow. OneRecent studies indicate that neural crest cells persist population, called pericytes, may be closely relatedlate into gestation and can be isolated from E14.5 rat to bone marrow stromal cells, although their originsciatic nerve, a peripheral nerve in the hindlimb. The remains elusive [12]. The second population of blood-cells incorporate BrdU, indicating that they are divid- born stem cells, which occur in four species ofing in vivo. When transplanted into chick embryos, the 32
  • The Adult Stem Cell Natural killer (NK) cell Neutrophil Bone T lymphocytes Basophil Lymphoid progenitor cell Eosinophil B lymphocyte Hematopoietic stem cell Monocyte/macrophage Multipotential Myeloid stem cell progenitor cell Platelets Red blood cells Hematopoietic Marrow Bone (or cartilage) Bone Stromal supportive stroma adipocyte matrix cell Osteoblast Stromal Lining cell stem cell© 2001 Terese Winslow, Lydia Kibiuk Blood vessel Osteocyte Pericyte Pre-osteoblast Skeletal muscle stem cell? Hematopoietic stem cell Adipocyte Hepatocyte stem cell? Osteoclast Figure 4.3. Hematopoietic and Stromal Stem Cell Differentiation. animals tested—guinea pigs, mice, rabbits, and become a standard by which other candidate stem humans—resemble stromal cells in that they can cells are measured because it shows, without generate bone and fat [53]. question, that HSCs can regenerate an entire tissue system—in this case, the blood [9, 99]. HSCs were first Hematopoietic Stem Cells. Of all the cell types in the proven to be blood-forming stem cells in a series of body, those that survive for the shortest period of time experiments in mice; similar blood-forming stem cells are blood cells and certain kinds of epithelial cells. occur in humans. HSCs are defined by their ability to For example, red blood cells (erythrocytes), which self-renew and to give rise to all the kinds of blood lack a nucleus, live for approximately 120 days in the cells in the body. This means that a single HSC is bloodstream. The life of an animal literally depends capable of regenerating the entire hematopoietic on the ability of these and other blood cells to be system, although this has been demonstrated only a replenished continuously. This replenishment process few times in mice [72]. occurs largely in the bone marrow, where HSCs reside, divide, and differentiate into all the blood cell Over the years, many combinations of surface mark- types. Both HSCs and differentiated blood cells cycle ers have been used to identify, isolate, and purify from the bone marrow to the blood and back again, HSCs derived from bone marrow and blood. under the influence of a barrage of secreted factors Undifferentiated HSCs and hematopoietic progenitor that regulate cell proliferation, differentiation, and cells express c-kit, CD34, and H-2K. These cells usually migration (see Chapter 5. Hematopoietic Stem Cells). lack the lineage marker Lin, or express it at very low levels (Lin–/low). And for transplant purposes, cells that HSCs can reconstitute the hematopoietic system of are CD34+ Thy1+ Lin– are most likely to contain stem mice that have been subjected to lethal doses of cells and result in engraftment. radiation to destroy their own hematopoietic systems. This test, the rescue of lethally irradiated mice, has 33
  • The Adult Stem CellTwo kinds of HSCs have been defined. Long-term Also important to HSC proliferation and differentiationHSCs proliferate for the lifetime of an animal. In are interactions of the cells with adhesion moleculesyoung adult mice, an estimated 8 to 10 % of in the extracellular matrix of the bone marrow stromalong-term HSCs enter the cell cycle and divide each [83, 101, 110].day. Short-term HSCs proliferate for a limited time, Bone Marrow Stromal Cells. Bone marrow (BM) stromalpossibly a few months. Long-term HSCs have high cells have long been recognized for playing anlevels of telomerase activity. Telomerase is an enzyme important role in the differentiation of mature bloodthat helps maintain the length of the ends of chromo- cells from HSCs (see Figure 4.3. Hematopoietic andsomes, called telomeres, by adding on nucleotides. Stromal Stem Cell Differentiation). But stromal cellsActive telomerase is a characteristic of undifferentiat- also have other important functions [30, 31]. In addi-ed, dividing cells and cancer cells. Differentiated, tion to providing the physical environment in whichhuman somatic cells do not show telomerase activity. HSCs differentiate, BM stromal cells generate carti-In adult humans, HSCs occur in the bone marrow, lage, bone, and fat. Whether stromal cells are bestblood, liver, and spleen, but are extremely rare in any classified as stem cells or progenitor cells for these tis-of these tissues. In mice, only 1 in 10,000 to 15,000 sues is still in question. There is also a question as tobone marrow cells is a long-term HSC [105]. whether BM stromal cells and so-called mesenchy-Short-term HSCs differentiate into lymphoid and mal stem cells are the same population [78].myeloid precursors, the two classes of precursors for BM stromal cells have many features that distinguishthe two major lineages of blood cells. Lymphoid pre- them from HSCs. The two cell types are easy to sepa-cursors differentiate into T cells, B cells, and natural rate in vitro. When bone marrow is dissociated, andkiller cells. The mechanisms and pathways that lead the mixture of cells it contains is plated at low density,to their differentiation are still being investigated [1, 2]. the stromal cells adhere to the surface of the cultureMyeloid precursors differentiate into monocytes and dish, and the HSCs do not. Given specific in vitro con-macrophages, neutrophils, eosinophils, basophils, ditions, BM stromal cells form colonies from a singlemegakaryocytes, and erythrocytes [3]. In vivo, bone cell called the colony forming unit-F (CFU-F). Thesemarrow HSCs differentiate into mature, specialized colonies may then differentiate as adipocytes orblood cells that cycle constantly from the bone myelosupportive stroma, a clonal assay that indicatesmarrow to the blood, and back to the bone marrow the stem cell-like nature of stromal cells. Unlike HSCs,[26]. A recent study showed that short-term HSCs are which do not divide in vitro (or proliferate only to aa heterogeneous population that differ significantly in limited extent), BM stromal cells can proliferate for upterms of their ability to self-renew and repopulate the to 35 population doublings in vitro [16]. They growhematopoietic system [42]. rapidly under the influence of such mitogens asAttempts to induce HSC to proliferate in vitro—on platelet-derived growth factor (PDGF), epidermalmany substrates, including those intended to mimic growth factor (EGF), basic fibroblast growth factorconditions in the stroma—have frustrated scientists for (bFGF), and insulin-like growth factor-1 (IGF-1) [12].many years. Although HSCs proliferate readily in vivo, To date, it has not been possible to isolate a popula-they usually differentiate or die in vitro [26]. Thus, tion of pure stromal cells from bone marrow. Panels ofmuch of the research on HSCs has been focused onunderstanding the factors, cell-cell interactions, and markers used to identify the cells include receptors forcell-matrix interactions that control their proliferation certain cytokines (interleukin-1, 3, 4, 6, and 7) recep-and differentiation in vivo, with the hope that similar tors for proteins in the extracellular matrix, (ICAM-1conditions could be replicated in vitro. Many of the and 2, VCAM-1, the alpha-1, 2, and 3 integrins, andsoluble factors that regulate HSC differentiation in vivo the beta-1, 2, 3 and 4 integrins), etc. [64]. Despite theare cytokines, which are made by different cell types use of these markers and another stromal cell markerand are then concentrated in the bone marrow by called Stro-1, the origin and specific identity of stro-the extracellular matrix of stromal cells—the sites of mal cells have remained elusive. Like HSCs, BMblood formation [45, 107]. Two of the most-studied stromal cells arise from embryonic mesoderm duringcytokines are granulocyte-macrophage colony-stimu- development, although no specific precursor or stemlating factor (GM-CSF) and interleukin-3 (IL-3) [40, 81]. cell for stromal cells has been isolated and identified. 34
  • The Adult Stem CellOne theory about their origin is that a common kind new blood vessels in the embryo is called vasculoge-of progenitor cell—perhaps a primordial endothelial nesis. In the adult, the process of forming blood ves-cell that lines embryonic blood vessels—gives rise to sels from pre-existing blood vessels is called angio-both HSCs and to mesodermal precursors. The latter genesis [50].may then differentiate into myogenic precursors (the Evidence that hemangioblasts do exist comes fromsatellite cells that are thought to function as stem studies of mouse embryonic stem cells that arecells in skeletal muscle), and the BM stromal cells directed to differentiate in vitro. These studies have[10]. shown that a precursor cell derived from mouse ESIn vivo, the differentiation of stromal cells into fat and cells that express Flk-1 [the receptor for vascularbone is not straightforward. Bone marrow adipocytes endothelial growth factor (VEGF) in mice] can giveand myelosupportive stromal cells—both of which rise to both blood cells and blood vessel cells [88,are derived from BM stromal cells—may be regarded 109]. Both VEGF and fibroblast growth factor-2 (FGF-2)as interchangeable phenotypes [10, 11]. Adipocytes play critical roles in endothelial cell differentiationdo not develop until postnatal life, as the bones in vivo [79].enlarge and the marrow space increases to accom- Several recent reports indicate that the bone marrowmodate enhanced hematopoiesis. When the skele- contains cells that can give rise to new blood vesselston stops growing, and the mass of HSCs decreases in tissues that are ischemic (damaged due to thein a normal, age-dependent fashion, BM stromal cells deprivation of blood and oxygen) [8, 29, 49, 94]. Butdifferentiate into adipocytes, which fill the extra it is unclear from these studies what cell type(s) in thespace. New bone formation is obviously greater bone marrow induced angiogenesis. In a study whichduring skeletal growth, although bone “turns over” sought to address that question, researchers foundthroughout life. Bone forming cells are osteoblasts, that adult human bone marrow contains cells thatbut their relationship to BM stromal cells is not clear. resemble embryonic hemangioblasts, and mayNew trabecular bone, which is the inner region of therefore be called endothelial stem cells.bone next to the marrow, could logically developfrom the action of BM stromal cells. But the outside In more recent experiments, human bone marrow-surface of bone also turns over, as does bone next to derived cells were injected into the tail veins of ratsthe Haversian system (small canals that form concen- with induced cardiac ischemia. The human cellstric rings within bone). And neither of these surfaces is migrated to the rat heart where they generated newin contact with BM stromal cells [10, 11]. blood vessels in the infarcted muscle (a process akin to vasculogenesis), and also induced angiogenesis.Adult Stem Cells in Other Tissues The candidate endothelial stem cells are CD34+ (aIt is often difficult—if not impossible—to distinguish marker for HSCs), and they express the transcriptionadult, tissue-specific stem cells from progenitor cells. factor GATA-2 [51]. A similar study using transgenicWith that caveat in mind, the following summary mice that express the gene for enhanced green fluo-identifies reports of stem cells in various adult tissues. rescent protein (which allows the cells to be tracked),Endothelial Progenitor Cells. Endothelial cells line the showed that bone-marrow-derived cells could repopu-inner surfaces of blood vessels throughout the body, late an area of infarcted heart muscle in mice, andand it has been difficult to identify specific endothe- generate not only blood vessels, but also cardiomy-lial stem cells in either the embryonic or the adult ocytes that integrated into the host tissue [71] (seemammal. During embryonic development, just after Chapter 9. Can Stem Cells Repair a Damaged Heart?).gastrulation, a kind of cell called the hemangioblast, And, in a series of experiments in adult mammals,which is derived from mesoderm, is presumed to be progenitor endothelial cells were isolated fromthe precursor of both the hematopoietic and peripheral blood (of mice and humans) by using anti-endothelial cell lineages. The embryonic vasculature bodies against CD34 and Flk-1, the receptor for VEGF.formed at this stage is transient and consists of blood The cells were mononuclear blood cells (meaningislands in the yolk sac. But hemangioblasts, per se, they have a nucleus) and are referred to as MBCD34+have not been isolated from the embryo and their cells and MBFlk1+ cells. When plated in tissue-cultureexistence remains in question. The process of forming 35
  • The Adult Stem Celldishes, the cells attached to the substrate, became muscle may be present in muscle and bone marrow.spindle-shaped, and formed tube-like structures that SP stands for a side population of cells that can beresemble blood vessels. When transplanted into mice separated by fluorescence-activated cell sortingof the same species (autologous transplants) with analysis. Intravenously injecting these muscle-derivedinduced ischemia in one limb, the MBCD34+ cells stem cells restored the expression of dystrophin inpromoted the formation of new blood vessels [8]. mdx mice. Dystrophin is the protein that is defectiveAlthough the adult MBCD34+ and MBFlk1+ cells function in people with Duchenne’s muscular dystrophy; mdxin some ways like stem cells, they are usually mice provide a model for the human disease.regarded as progenitor cells. Dystrophin expression in the SP cell-treated mice was lower than would be needed for clinical benefit.Skeletal Muscle Stem Cells. Skeletal muscle, like the Injection of bone marrow- or muscle-derived SP cellscardiac muscle of the heart and the smooth muscle into the dystrophic muscle of the mice yieldedin the walls of blood vessels, the digestive system, equivocal results that the transplanted cells had inte-and the respiratory system, is derived from embryonic grated into the host tissue. The authors conclude thatmesoderm. To date, at least three populations of a similar population of SP stem cells can be derivedskeletal muscle stem cells have been identified: from either adult mouse bone marrow or skeletalsatellite cells, cells in the wall of the dorsal aorta, and muscle, and suggest “there may be some directso-called “side population” cells. relationship between bone marrow-derived stem cellsSatellite cells in skeletal muscle were identified 40 and other tissue- or organ-specific cells” [43]. Thus,years ago in frogs by electron microscopy [62], and stem cell or progenitor cell types from variousthereafter in mammals [84]. Satellite cells occur on mesodermally-derived tissues may be able to gen-the surface of the basal lamina of a mature muscle erate skeletal muscle.cell, or myofiber. In adult mammals, satellite cells Epithelial Cell Precursors in the Skin and Digestivemediate muscle growth [85]. Although satellite cells System. Epithelial cells, which constitute 60 percent ofare normally non-dividing, they can be triggered to the differentiated cells in the body are responsible forproliferate as a result of injury, or weight-bearing covering the internal and external surfaces of theexercise. Under either of these circumstances, muscle body, including the lining of vessels and other cavi-satellite cells give rise to myogenic precursor cells, ties. The epithelial cells in skin and the digestive tractwhich then differentiate into the myofibrils that typify are replaced constantly. Other epithelial cell popula-skeletal muscle. A group of transcription factors tions—in the ducts of the liver or pancreas, forcalled myogenic regulatory factors (MRFs) play example—turn over more slowly. The cell populationimportant roles in these differentiation events. The so- that renews the epithelium of the small intestinecalled primary MRFs, MyoD and Myf5, help regulate occurs in the intestinal crypts, deep invaginations inmyoblast formation during embryogenesis. The sec- the lining of the gut. The crypt cells are oftenondary MRFs, myogenin and MRF4, regulate the ter- regarded as stem cells; one of them can give riseminal differentiation of myofibrils [86]. to an organized cluster of cells called a structural-With regard to satellite cells, scientists have been proliferative unit [93].addressing two questions. Are skeletal muscle satellite The skin of mammals contains at least three popula-cells true adult stem cells or are they instead precur- tions of epithelial cells: epidermal cells, hair folliclesor cells? Are satellite cells the only cell type that can cells, and glandular epithelial cells, such as those thatregenerate skeletal muscle. For example, a recent make up the sweat glands. The replacement patternsreport indicates that muscle stem cells may also for epithelial cells in these three compartments differ,occur in the dorsal aorta of mouse embryos, and and in all the compartments, a stem cell populationconstitute a cell type that gives rise both to muscle has been postulated. For example, stem cells in thesatellite cells and endothelial cells. Whether the dorsal bulge region of the hair follicle appear to give rise toaorta cells meet the criteria of a self-renewing muscle multiple cell types. Their progeny can migrate downstem cell is a matter of debate [21]. to the base of the follicle where they become matrixAnother report indicates that a different kind of stem cells, which may then give rise to different cell typescell, called an SP cell, can also regenerate skeletal in the hair follicle, of which there are seven [39]. The 36
  • The Adult Stem Cellbulge stem cells of the follicle may also give rise to into hepatocytes (usually derived from endoderm)the epidermis of the skin [95]. [54, 77, 97]. But the question remains as to whether cells from the bone marrow normally generate hepa-Another population of stem cells in skin occurs in the tocytes in vivo. It is not known whether this kind ofbasal layer of the epidermis. These stem cells prolifer- plasticity occurs without severe damage to the liver orate in the basal region, and then differentiate as they whether HSCs from the bone marrow generate ovalmove toward the outer surface of the skin. The ker- cells of the liver [18]. Although hepatic oval cells existatinocytes in the outermost layer lack nuclei and act in the liver, it is not clear whether they actually gener-as a protective barrier. A dividing skin stem cell can ate new hepatocytes [87, 98]. Oval cells may arisedivide asymmetrically to produce two kinds of daugh- from the portal tracts in liver and may give rise toter cells. One is another self-renewing stem cell. The either hepatocytes [19, 55] and to the epithelium ofsecond kind of daughter cell is an intermediate pre- the bile ducts [37, 92]. Indeed, hepatocytes them-cursor cell which is then committed to replicate a few selves, may be responsible for the well-know regen-times before differentiating into keratinocytes. Self- erative capacity of liver.renewing stem cells can be distinguished from thisintermediate precusor cell by their higher level of ȋ1integrin expression, which signals keratinocytes to pro- SUMMARYliferate via a mitogen-activated protein (MAP) kinase What Do We Know About Adult Stem Cells?[112]. Other signaling pathways include that triggered • Adult stem cells can proliferate without differenti-by ȋ-catenin, which helps maintain the stem-cell ating for a long period (a characteristic referredstate [111], and the pathway regulated by the onco- to as long-term self-renewal), and they can giveprotein c-Myc, which triggers stem cells to give rise to rise to mature cell types that have characteristictransit amplifying cells [36]. shapes and specialized functions.Stem Cells in the Pancreas and Liver. The status of • Some adult stem cells have the capability tostem cells in the adult pancreas and liver is unclear. differentiate into tissues other than the ones fromDuring embryonic development, both tissues arise which they originated; this is referred to asfrom endoderm. A recent study indicates that a plasticity.single precursor cell derived from embryonic endo- • Adult stem cells are rare. Often they are difficultderm may generate both the ventral pancreas and to identify and their origins are not known.the liver [23]. In adult mammals, however, both the Current methods for characterizing adult stempancreas and the liver contain multiple kinds of cells are dependent on determining cell surfacedifferentiated cells that may be repopulated or markers and observations about their differentia-regenerated by multiple types of stem cells. In the tion patterns in test tubes and culture dishes.pancreas, endocrine (hormone-producing) cellsoccur in the islets of Langerhans. They include the • To date, published scientific literature indicatesbeta cells (which produce insulin), the alpha cells that adult stem cells have been derived from(which secrete glucagon), and cells that release the brain, bone marrow, peripheral blood, dentalpeptide hormones somatostatin and pancreatic pulp, spinal cord, blood vessels, skeletal muscle,polypeptide. Stem cells in the adult pancreas are epithelia of the skin and digestive system,postulated to occur in the pancreatic ducts or in the cornea, retina, liver, and pancreas; thus, adultislets themselves. Several recent reports indicate that stem cells have been found in tissues thatstem cells that express nestin—which is usually develop from all three embryonic germ layers.regarded as a marker of neural stem cells—can • Hematopoietic stem cells from bone marrow aregenerate all of the cell types in the islets [60, 113] the most studied and used for clinical applica-(see Chapter 7. Stem Cells and Diabetes). tions in restoring various blood and immune components to the bone marrow via transplan-The identity of stem cells that can repopulate the liver tation. There are at least two other populations ofof adult mammals is also in question. Recent studies adult stem cells that have been identified fromin rodents indicate that HSCs (derived from bone marrow and blood.mesoderm) may be able to home to liver after it isdamaged, and demonstrate plasticity in becoming 37
  • The Adult Stem Cell • Several populations of adult stem cells have adult stem cell may be one that circulates in been identified in the brain, particularly the the blood stream, can escape from the blood, hippocampus. Their function is unknown. and populate various adult tissues. In more than Proliferation and differentiation of brain stem cells one experimental system, researchers have are influenced by various growth factors. noted that dividing cells in adult tissues often • There are now several reports of adult stem cells appear near a blood vessel, such as candidate in other tissues (muscle, blood, and fat) that stem cells in the hippocampus, a region of the demonstrate plasticity. Very few published brain [75]. research reports on plasticity of adult stem cells • Do adult stem cells exhibit plasticity as a normal have, however, included clonality studies. That is, event in vivo? If so, is this true of all adult stem there is limited evidence that a single adult stem cells? What are the signals that regulate the cell or genetically identical line of adult stem proliferation and differentiation of stem cells that cells demonstrates plasticity. demonstrate plasticity? • Rarely have experiments that claim plasticity demonstrated that the adult stem cells have REFERENCES generated mature, fully functional cells or that 1. Akashi, K., Traver, D., Kondo, M., and Weissman, I.L. (1999). the cells have restored lost function in vivo. Lymphoid development from hematopoietic stem cells. Int. J. Hematol. 69, 217-226.What Do We Need to Know About Adult Stem Cells? 2. Akashi, K., Kondo, M., Cheshier, S., Shizuru, J., Gandy, K., • What are the sources of adult stem cells in the Domen, J., Mebius, R., Traver, D., and Weissman, I.L. (1999). body? Are they “leftover” embryonic stem cells, Lymphoid development from stem cells and the common lymphocyte progenitors. Cold Spring Harb. Symp. Quant. or do they arise in some other way? And if the Biol. 64, 1-12. latter is true—which seems to be the case— exactly how do adult stem cells arise, and 3. Akashi, K., Traver, D., Miyamoto, T., and Weissman, I.L. (2000). A clonogenic common myeloid progenitor that why do they remain in an undifferentiated gives rise to all myeloid lineages. Nature. 404, 193-197. state, when all the cells around them have 4. Alison, M.R., Poulsom, R., Jeffery, R., Dhillon, A.P Quaglia, ., differentiated? A., Jacob, J., Novelli, M., Prentice, G., Williamson, J., and • Is it possible to manipulate adult stem cells to Wright, N.A. (2000). Hepatocytes from non-hepatic adult stem cells. Nature. 406, 257. increase their ability to proliferate in vitro, so that adult stem cells can be used as a sufficient 5. Altman, J. and Das, G.D. (1965). Autoradiographic and his- source of tissue for transplants? tological evidence of postnatal hippocampal neurogene- sis in rats. J. Comp. Neurol. 124, 319-335. • How many kinds of adult stem cells exist, and in 6. Altman, J. (1969). Autoradiographic and histological studies which tissues do they exist? Evidence is accu- of postnatal neurogenesis. IV. Cell proliferation and migra- mulating that, although they occur in small tion in the anterior forebrain, with special reference to per- numbers, adult stem cells are present in many sisting neurogenesis in the olfactory bulb. J. Comp. Neurol. differentiated tissues. 137, 433-457. • What is the best evidence that adult stem cells 7. Anderson, D.J., Gage, F.H., and Weissman, I.L. (2001). Can stem cells cross lineage boundaries? Nat. Med. 7, 393-395. show plasticity and generate cell types of other tissues? 8. Asahara, T., Murohara, T., Sullivan, A., Silver, M., van der Zee R., Li, T., Witzenbichler, B., Schatteman, G., and Isner, J.M. • Is it possible to manipulate adult stem cells to (1997). Isolation of putative progenitor endothelial cells for increase their ability to proliferate in vitro so that angiogenesis. Science. 275, 964-967. adult stem cells can be used as a sufficient 9. Becker, A.J., McCullough, E.A., and Till, J.E. (1963). source of tissue for transplants? Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. • Is there a universal stem cell? An emerging Nature. 197, 452-454. concept is that, in adult mammals, there may be a population of “universal” stem cells. 10. Bianco, P and Cossu, G. (1999). Uno, nessuno e centomila: . searching for the identity of mesodermal progenitors. Exp. Although largely theoretical, the concept has Cell Res. 251, 257-263. some experimental basis. A candidate, universal 38
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  • The Adult Stem Cell102. Vescovi, A.L., Reynolds, B.A., Fraser, D.D., and Weiss, S. 109. Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., (1993). bFGF regulates the proliferative fate of unipotent Nishikawa, S., Yurugi, T., Naito, M., Nakao, K., and (neuronal) and bipotent (neuronal/astroglial) EGF- Nishikawa, S. (2000). Flk1-positive cells derived from generated CNS progenitor cells. Neuron. 11, 951-966. embryonic stem cells serve as vascular progenitors. Nature. 408, 92-96.103. Vescovi, A.L., Gritti, A., Galli, R., and Parati, E.A. (1999). Isolation and intracerebral grafting of nontransformed 110. Zandstra, P .W., Lauffenburger, D.A., and Eaves, C.J. (2000). multipotential embryonic human CNS stem cells. J. A ligand-receptor signaling threshold model of stem cell Neurotrauma. 16, 689-693. differentiation control: a biologically conserved mecha- nism applicable to hematopoiesis. Blood. 96, 1215-1222.104. Weiss, S. and van der Kooy D. (1998). CNS stem cells: where’s the biology (a.k.a. beef)? J. Neurobiol. 36, 111. Zhu, A.J. and Watt, F.M. (1999). beta-catenin signalling 307-314. modulates proliferative potential of human epidermal ker- atinocytes independently of intercellular adhesion.105. Weissman, I.L. (2000). Stem cells: units of development, Development. 126, 2285-2298. units of regeneration, and units in evolution. Cell. 100, 157-168. 112. Zhu, A.J., Haase, I., and Watt, F.M. (1999). Signaling via beta1 integrins and mitogen-activated protein kinase106. White, P .M., Morrison, S.J., Orimoto, K., Kubu, C.J., Verdi, determines human epidermal stem cell fate in vitro. Proc. J.M., and Anderson, D.J. (2001). Neural crest stem cells Natl. Acad. Sci. U. S. A. 96, 6728-6733. undergo cell-intrinsic developmental changes in sensitivity to instructive differentiation signals. Neuron. 29, 57-71. 113. Zulewski, H., Abraham, E.J., Gerlach, M.J., Daniel, P .B., Moritz, W., Muller, B., Vallejo, M., Thomas, M.K., and107. Whitlock, C.A., Tidmarsh, G.F., Muller-Sieburg, C., and Habener, J.F. (2001). Multipotential nestin-positive stem Weissman, I.L. (1987). Bone marrow stromal cell lines with cells isolated from adult pancreatic islets differentiate ex lymphopoietic activity express high levels of a pre-B neo- vivo into pancreatic endocrine, exocrine, and hepatic plasia-associated molecule. Cell. 48, 1009-1021. phenotypes. Diabetes. 50, 521-533.108. Williams, B.P Read, J., and Price, J. (1991). The generation ., of neurons and oligodendrocytes from a common pre- cursor cell. Neuron. 7, 685-693. 42
  • 5. HEMATOPOIETIC STEM CELLSWith more than 50 years of experience studying ultimately responsible for the constant renewal ofblood-forming stem cells called hematopoietic stem blood—the production of billions of new blood cellscells, scientists have developed sufficient understand- each day. Physicians and basic researchers haveing to actually use them as a therapy. Currently, no known and capitalized on this fact for more than 50other type of stem cell, adult, fetal or embryonic, years in treating many diseases. The first evidencehas attained such status. Hematopoietic stem cell and definition of blood-forming stem cells cametransplants are now routinely used to treat patients from studies of people exposed to lethal doses ofwith cancers and other disorders of the blood and radiation in 1945.immune systems. Recently, researchers have Basic research soon followed. After duplicatingobserved in animal studies that hematopoietic stem radiation sickness in mice, scientists found they couldcells appear to be able to form other kinds of cells, rescue the mice from death with bone marrowsuch as muscle, blood vessels, and bone. If this can transplants from healthy donor animals. In the earlybe applied to human cells, it may eventually be 1960s, Till and McCulloch began analyzing the bonepossible to use hematopoietic stem cells to replace marrow to find out which components were respon-a wider array of cells and tissues than once thought. sible for regenerating blood [56]. They defined whatDespite the vast experience with hematopoietic stem remain the two hallmarks of an HSC: it can renewcells, scientists face major roadblocks in expanding itself and it can produce cells that give rise to all thetheir use beyond the replacement of blood and different types of blood cells (see Chapter 4. Theimmune cells. First, hematopoietic stem cells are Adult Stem Cell).unable to proliferate (replicate themselves) anddifferentiate (become specialized to other cell types) in WHAT IS A HEMATOPOIETICvitro (in the test tube or culture dish). Second, scientists STEM CELL?do not yet have an accurate method to distinguishstem cells from other cells recovered from the blood A hematopoietic stem cell is a cell isolated from theor bone marrow. Until scientists overcome these blood or bone marrow that can renew itself, cantechnical barriers, they believe it is unlikely that differentiate to a variety of specialized cells, canhematopoietic stem cells will be applied as cell mobilize out of the bone marrow into circulatingreplacement therapy in diseases such as diabetes, blood, and can undergo programmed cell death,Parkinson’s Disease, spinal cord injury, and many others. called apoptosis—a process by which cells that are detrimental or unneeded self-destruct.INTRODUCTION A major thrust of basic HSC research since the 1960sBlood cells are responsible for constant maintenance has been identifying and characterizing these stemand immune protection of every cell type of the cells. Because HSCs look and behave in culture likebody. This relentless and brutal work requires that ordinary white blood cells, this has been a difficultblood cells, along with skin cells, have the greatest challenge and this makes them difficult to identify bypowers of self-renewal of any adult tissue. morphology (size and shape). Even today, scientists must rely on cell surface proteins, which serve, onlyThe stem cells that form blood and immune cells are roughly, as markers of white blood cells.known as hematopoietic stem cells (HSCs). They are 43
  • Hematopoietic Stem Cells Identifying and characterizing properties of HSCs hematopoietic system over some months, they are began with studies in mice, which laid the ground- considered to be long-term stem cells that are work for human studies. The challenge is formidable capable of self-renewal. Other cells from bone as about 1 in every 10,000 to 15,000 bone marrow marrow can immediately regenerate all the different cells is thought to be a stem cell. In the blood stream types of blood cells, but under normal circumstances the proportion falls to 1 in 100,000 blood cells. To this cannot renew themselves over the long term, and end, scientists began to develop tests for proving the these are referred to as short-term progenitor or self-renewal and the plasticity of HSCs. precursor cells. Progenitor or precursor cells are rela- tively immature cells that are precursors to a fully The “gold standard” for proving that a cell derived differentiated cell of the same tissue type. They are from mouse bone marrow is indeed an HSC is still capable of proliferating, but they have a limited based on the same proof described above and capacity to differentiate into more than one cell type used in mice many years ago. That is, the cells are as HSCs do. For example, a blood progenitor cell injected into a mouse that has received a dose of may only be able to make a red blood cell (see irradiation sufficient to kill its own blood-producing Figure 5.1. Hematopoietic and Stromal Stem Cell cells. If the mouse recovers and all types of blood Differentiation). cells reappear (bearing a genetic marker from the donor animal), the transplanted cells are deemed to Harrison et al. write that short-term blood-progenitor have included stem cells. cells in a mouse may restore hematopoiesis for three to four months [36]. The longevity of short-term stem These studies have revealed that there appear to be cells for humans is not firmly established. A true stem two kinds of HSCs. If bone marrow cells from the cell, capable of self-renewal, must be able to renew transplanted mouse can, in turn, be transplanted to itself for the entire lifespan of an organism. It is these another lethally irradiated mouse and restore its Natural killer (NK) cell Neutrophil Bone T lymphocytes Basophil Lymphoid progenitor cell Eosinophil B lymphocyte Hematopoietic stem cell Monocyte/macrophage Multipotential Myeloid stem cell progenitor cell Platelets Red blood cells Hematopoietic Marrow Bone (or cartilage) Bone Stromal supportive stroma adipocyte matrix cell Osteoblast Stromal Lining cell stem cell© 2001 Terese Winslow, Lydia Kibiuk Blood vessel Osteocyte Pericyte Pre-osteoblast Skeletal muscle stem cell? Hematopoietic stem cell Adipocyte Hepatocyte stem cell? Osteoclast Figure 5.1. Hematopoietic and Stromal Stem Cell Differentiation. 44
  • Hematopoietic Stem Cellslong-term replicating HSCs that are most important cell was a long-term HSC [50]. Four years later, thefor developing HSC-based cell therapies. Unfortu- laboratory proposed a comparable set of markers fornately, to date, researchers cannot distinguish the the human stem cell [3]. Weissman proposes thelong-term from the short-term cells when they are markers shown in Table 5.1 as the closest markers forremoved from the bloodstream or bone marrow. mouse and human HSCs [62].The central problem of the assays used to identify Table 5.1. Proposed cell-surface markers oflong-term stem cells and short-term progenitor cells is undifferentiated hematopoietic stem cells.that they are difficult, expensive, and time-consuming Listed here are cell surface markers found on mouseand cannot be done in humans. A few assays are and human hematopoietic stem cells as they exist innow available that test cells in culture for their ability their undifferentiated state in vivo and in vitro. As these cells begin to develop as distinct cell lineages the cellto form primitive and long-lasting colonies of cells, surface markers are no longer identified.but these tests are not accepted as proof that a cellis a long-term stem cell. Some genetically altered Mouse Humanmice can receive transplanted human HSCs to test CD34 low/- CD 34+the cells’ self-renewal and hematopoietic capabilities SCA-1+ CD59+*during the life of a mouse, but the relevance of this Thy1+/low Thy1+test for the cells in humans—who may live fordecades—is open to question. CD38+ CD38 low/- C-kit+ C-kit -/lowThe difficulty of HSC assays has contributed to two lin -* lin -**mutually confounding research problems: definitivelyidentifying the HSC and getting it to proliferate, or * Only one of a family of CD59 markers has thus far been evaluated.increase its numbers, in a culture dish. More rapid ** Lin- cells lack 13 to 14 different mature blood-lineageresearch progress on characterizing and using HSCs markers.would be possible if they could be readily grown inthe laboratory. Conversely, progress in identifyinggrowth conditions suitable for HSCs and getting the Such cell markers can be tagged with monoclonalcells to multiply would move more quickly if scientists antibodies bearing a fluorescent label and culled outcould reliably and readily identify true HSCs. of bone marrow with fluorescence-activated cell sorting (FACS).CAN CELL MARKERS BE USED The groups of cells thus sorted by surface markers areTO IDENTIFY HEMATOPOIETIC heterogeneous and include some cells that are true,STEM CELLS? long-term self-renewing stem cells, some shorter-term progenitors, and some non-stem cells. Weissman’sHSCs have an identity problem. First, the ones with group showed that as few as five genetically taggedlong-term replicating ability are rare. Second, there cells, injected along with larger doses of stem cellsare multiple types of stem cells. And, third, the stem into lethally irradiated mice, could establish them-cells look like many other blood or bone marrow cells. selves and produce marked donor cells in all bloodSo how do researchers find the desired cell popula- cell lineages for the lifetime of the mouse. A singletions? The most common approach is through tagged cell could produce all lineages for as manymarkers that appear on the surface of cells. (For a as seven weeks, and 30 purified cells were sufficientmore detailed discussion, see Appendix E.i. Markers: to rescue mice and fully repopulate the bone mar-How Do Researchers Use Them to Identify Stem Cells?) row without extra doses of backup cells to rescue theThese are useful, but not perfect tools for the mice [49]. Despite these efforts, researchers remainresearch laboratory. divided on the most consistently expressed set of HSC markers [27, 32]. Connie Eaves of the University ofIn 1988, in an effort to develop a reliable means of British Columbia says none of the markers are tied toidentifying these cells, Irving Weissman and his unique stem cell functions or truly define the stemcollaborators focused attention on a set of protein cell [14]. “Almost every marker I am aware of hasmarkers on the surface of mouse blood cells that been shown to be fickle,” she says.were associated with increased likelihood that the 45
  • Hematopoietic Stem CellsMore recently, Diane Krause and her colleagues at will be true HSCs. Thus, when medical researchersYale University, New York University, and Johns Hopkins commonly refer to peripherally harvested “stemUniversity, used a new technique to home in on a sin- cells,” this is something of a misnomer. As is true forgle cell capable of reconstituting all blood cell line- bone marrow, the CD34+ cells are a mixture of stemages of an irradiated mouse [27]. After marking bone cells, progenitors, and white blood cells of variousmarrow cells from donor male mice with a nontoxic degrees of maturity.dye, they injected the cells into female recipient In the past three years, the majority of autologousmice that had been given a lethal dose of radiation. (where the donor and recipient are the same person)Over the next two days, some of the injected cells and allogeneic (where the donor and recipient aremigrated, or homed, to the bone marrow of the different individuals) “bone marrow” transplants haverecipients and did not divide; when transplanted into actually been white blood cells drawn from peripherala second set of irradiated female mice, they eventu- circulation, not bone marrow. Richard Childs, an intra-ally proved to be a concentrated pool of self-renew- mural investigator at the NIH, says peripheral harvesting stem cells. The cells also reconstituted blood pro- of cells is easier on the donor—with minimal pain, noduction. The scientists estimate that their technique anesthesia, and no hospital stay—but also yieldsconcentrated the long-term stem cells 500 to 1,000- better cells for transplants [6]. Childs points to evi-fold compared with bone marrow. dence that patients receiving peripherally harvested cells have higher survival rates than bone marrowWHAT ARE THE SOURCES OF recipients do. The peripherally harvested cells containHEMATOPOIETIC STEM CELLS? twice as many HSCs as stem cells taken from boneBone Marrow marrow and engraft more quickly. This means patients may recover white blood cells, platelets, andThe classic source of hematopoietic stem cells (HSCs) their immune and clotting protection several daysis bone marrow. For more than 40 years, doctors faster than they would with a bone marrow graft.performed bone marrow transplants by anesthetizing Scientists at Stanford report that highly purified,the stem cell donor, puncturing a bone—typically a mobilized peripheral cells that have CD34+ andhipbone—and drawing out the bone marrow cells Thy-1+ surface markers engraft swiftly and withoutwith a syringe. About 1 in every 100,000 cells in the complication in breast cancer patients receiving anmarrow is a long-term, blood-forming stem cell; other autologous transplant of the cells after intensivecells present include stromal cells, stromal stem cells, chemotherapy [41].blood progenitor cells, and mature and maturingwhite and red blood cells. Umbilical Cord BloodPeripheral Blood In the late 1980s and early 1990s, physicians began to recognize that blood from the human umbilicalAs a source of HSCs for medical treatments, bone cord and placenta was a rich source of HSCs. Thismarrow retrieval directly from bone is quickly fading tissue supports the developing fetus during pregnancy,into history. For clinical transplantation of human is delivered along with the baby, and, is usually dis-HSCs, doctors now prefer to harvest donor cells from carded. Since the first successful umbilical cordperipheral, circulating blood. It has been known for blood transplants in children with Fanconi anemia,decades that a small number of stem and progenitor the collection and therapeutic use of these cells hascells circulate in the bloodstream, but in the past 10 grown quickly. The New York Blood Center’s Placentalyears, researchers have found that they can coax the Blood Program, supported by NIH, is the largest U.S.cells to migrate from marrow to blood in greater public umbilical cord blood bank and now hasnumbers by injecting the donor with a cytokine, such 13,000 donations available for transplantation intoas granulocyte-colony stimulating factor (GCSF). The small patients who need HSCs. Since it began col-donor is injected with GCSF a few days before the lecting umbilical cord blood in 1992, the center hascell harvest. To collect the cells, doctors insert an provided thousands of cord blood units to patients.intravenous tube into the donor’s vein and pass his Umbilical cord blood recipients—typically children—blood through a filtering system that pulls out CD34+ have now lived in excess of eight years, relying on thewhite blood cells and returns the red blood cells to HSCs from an umbilical cord blood transplant [31, 57].the donor. Of the cells collected, just 5 to 20 percent 46
  • Hematopoietic Stem CellsThere is a substantial amount of research being con- embryonic HSCs. Scientists in Europe, includingducted on umbilical cord blood to search for ways to Coulombel, Peault, and colleagues, first describedexpand the number of HSCs and compare and con- hematopoietic precursors in human embryos only atrast the biological properties of cord blood with adult few years ago [20, 53]. Most recently, Gallacher andbone marrow stem cells. There have been sugges- others reported finding HSCs circulating in the bloodtions that umbilical cord blood contains stem cells of 12- to 18-week aborted human fetuses [16, 28, 54]that have the capability of developing cells of multi- that was rich in HSCs. These circulating cells hadple germ layers (multipotent) or even all germ layers, different markers than did cells from fetal liver, fetale.g., endoderm, ectoderm, and mesoderm bone marrow, or umbilical cord blood.(pluripotent). To date, there is no published scientific Embryonic Stem Cells and Embryonic Germ Cellsevidence to support this claim. While umbilical cordblood represents a valuable resource for HSCs, In 1985, it was shown that it is possible to obtainresearch data have not conclusively shown qualita- precursors to many different blood cells from mousetive differences in the differentiated cells produced embryonic stem cells [9]. Perkins was able to obtainbetween this source of HSCs and peripheral blood all the major lineages of progenitor cells from mouseand bone marrow. embryoid bodies, even without adding hemato- poietic growth factors [45].Fetal Hematopoietic System Mouse embryonic stem cells in culture, given the rightAn important source of HSCs in research, but not in growth factors, can generate most, if not all, theclinical use, is the developing blood-producing tissues different blood cell types [19], but no one has yetof fetal animals. Hematopoietic cells appear early in achieved the “gold standard” of proof that they canthe development of all vertebrates. Most extensively produce long-term HSCs from these sources—namelystudied in the mouse, HSC production sweeps by obtaining cells that can be transplanted intothrough the developing embryo and fetus in waves. lethally irradiated mice to reconstitute long-termBeginning at about day 7 in the life of the mouse hematopoiesis [32].embryo, the earliest hematopoietic activity is indicat-ed by the appearance of blood islands in the yolk The picture for human embryonic stem and germsac (see Appendix A. Early Development). The point is cells is even less clear. Scientists from Jamesdisputed, but some scientists contend that yolk sac Thomson’s laboratory reported in 1999 that they wereblood production is transient and will generate some able to direct human embryonic stem cells—whichblood cells for the embryo, but probably not the bulk can now be cultured in the lab—to produce bloodof the HSCs for the adult animal [12, 26, 44]. progenitor cells [23]. Israeli scientists reported thatAccording to this proposed scenario, most stem cells they had induced human ES cells to producethat will be found in the adult bone marrow and hematopoietic cells, as evidenced by their produc-circulation are derived from cells that appear slightly tion of a blood protein, gamma-globin [21]. Cell lineslater and in a different location. This other wave of derived from human embryonic germ cells (culturedhematopoietic stem cell production occurs in the cells derived originally from cells in the embryo thatAGM—the region where the aorta, gonads, and fetal would ultimately give rise to eggs or sperm) that arekidney (mesonephros) begin to develop. The cells cultured under certain conditions will produce CD34+that give rise to the HSCs in the AGM may also give cells [47]. The blood-producing cells derived fromrise to endothelial cells that line blood vessels. [13]. human ES and embryonic germ (EG) cells have notThese HSCs arise at around days 10 to 11 in the been rigorously tested for long-term self-renewal ormouse embryo (weeks 4 to 6 in human gestation), the ability to give rise to all the different blood cells.divide, and within a couple of days, migrate to theliver [11]. The HSCs in the liver continue to divide and As sketchy as data may be on the hematopoieticmigrate, spreading to the spleen, thymus, and—near powers of human ES and EG cells, blood experts arethe time of birth—to the bone marrow. intrigued by their clinical potential and their potential to answer basic questions on renewal and differen-Whereas an increasing body of fetal HSC research is tiation of HSCs [19]. Connie Eaves, who has madeemerging from mice and other animals, there is comparisons of HSCs from fetal liver, cord blood,much less information about human fetal and 47
  • Hematopoietic Stem Cells The Stem Cell Database http://stemcell.princeton.edu Ihor Lemischka and colleagues at Princeton University related database, constructed in collaboration with and the Computational Biology and Informatics Kateri A. Moore, also at Princeton University, will docu- Laboratory at the University of Pennsylvania are colla- ment all genes active in stromal cells, which provide the borating to record all the findings about hematopoietic microenvironment in which stem cells are maintained. stem cell (HSC) genes and markers in the Stem Cell The combined power of the two databases, along with Database. new tools and methods for studying molecular biology, will help researchers put together a complete portrait of The collaborators started the database five years ago. the hematopoietic stem cell and how it works. The Its goal is listing and annotating all the genes that are databases will continue to grow and take advantage differentially expressed in mouse liver HSCs and their of other efforts, such as those to complete the gene cellular progeny. The database is growing to include sequences of mammals. Data will be publicly available human HSCs from different blood sources, and a to researchers around the world.and adult bone marrow, expects cells derived from The authors believe these changes reflect prepara-embryonic tissues to have some interesting traits. She tions for the cells to relocate—from homing in fetalsays actively dividing blood-producing cells from ES liver to homing in bone marrow [52].cell culture—if they are like other dividing cells—will The point is controversial, but a paper by Chen et al.not themselves engraft or rescue hematopoiesis in an provides evidence that at least in some strains ofanimal whose bone marrow has been destroyed. mice, HSCs from old mice are less able to repopu-However, they may play a critical role in developing late bone marrow after transplantation than are cellsan abundant supply of HSCs grown in the lab. from young adult mice [5]. Cells from fetal mice wereIndications are that the dividing cells will also more 50 to 100 percent better at repopulating marrowreadily lend themselves to gene manipulations than than were cells from young adult mice were. Thedo adult HSCs. Eaves anticipates that HSCs derived specific potential for repopulating marrow appears tofrom early embryo sources will be developmentally be strain-specific, but the scientists found this poten-more “plastic” than later HSCs, and more capable of tial declined with age for both strains. Other scientistsself-renewal [14]. find no decreases or sometimes increases in num- bers of HSCs with age [51]. Because of the difficultyHOW DO HSCs FROM VARYING in identifying a long-term stem cell, it remains difficultSOURCES DIFFER? to quantify changes in numbers of HSCs as aScientists in the laboratory and clinic are beginning to person ages.measure the differences among HSCs from different Effectiveness of Transplants of Adult versus Umbilicalsources. In general, they find that HSCs taken from Cord Blood Stem Cellstissues at earlier developmental stages have a A practical and important difference between HSCsgreater ability to self-replicate, show different homing collected from adult human donors and from um-and surface characteristics, and are less likely to be bilical cord blood is simply quantitative. Doctors arerejected by the immune system—making them rarely able to extract more than a few million HSCspotentially more useful for therapeutic transplantation. from a placenta and umbilical cord—too few to useStem cell populations of the bone marrow in a transplant for an adult, who would ideally get 7 toWhen do HSCs move from the early locations in the 10 million CD34+ cells per kilogram body weight, butdeveloping fetus to their adult “home” in the bone often adequate for a transplant for a child [33, 48].marrow? European scientists have found that the rela- Leonard Zon says that HSCs from cord blood are lesstive number of CD34+ cells in the collections of cord likely to cause a transplantation complication calledblood declined with gestational age, but expression graft-versus-host disease, in which white blood cellsof cell-adhesion molecules on these cells increased. 48
  • Hematopoietic Stem Cellsfrom a donor attack tissues of the recipient [65]. In a differentiation of cells to progenitor cells that can giverecent review of umbilical cord blood transplantation, rise to different lineages of blood cells but cannotLaughlin cites evidence that cord blood causes less renew themselves. True stem cells divide and replacegraft-versus-host disease [31]. Laughlin writes that it is themselves slowly in adult bone marrow.yet to be determined whether umbilical cord blood New tools for gene-expression analysis will now allowHSCs are, in fact, longer lived in a transplant recipient. scientists to study developmental changes in telo-In lab and mouse-model tests comparing CD34+ merase activity and telomeres. Telomeres are regionscells from human cord with CD34+ cells derived from of DNA found at the end of chromosomes that areadult bone marrow, researchers found cord blood extended by the enzyme telomerase. Telomerasehad greater proliferation capacity [24]. White blood activity is necessary for cells to proliferate and activitycells from cord blood engrafted better in a mouse decreases with age leading to shortened telomeres.model, which was genetically altered to tolerate the Scientists hypothesize that declines in stem cellhuman cells, than did their adult counterparts. renewal will be associated with declines in telomere length and telomerase activity. Telomerase activity inEffectiveness in Transplants of Peripheral Versus hematopoietic cells is associated with self-renewalBone Marrow Stem Cells potential [40].In addition to being far easier to collect, peripherallyharvested white blood cells have other advantages Because self-renewal divisions are rare, hard toover bone marrow. Cutler and Antin’s review says that induce in culture, and difficult to prove, scientists doperipherally harvested cells engraft more quickly, but not have a definitive answer to the burning question:are more likely to cause graft-versus-host disease [8]. what puts—or perhaps keeps—HSCs in a self-renewalProspecting for the most receptive HSCs for gene division mode? HSCs injected into an anemic patienttherapy, Orlic and colleagues found that mouse HSCs or mouse—or one whose HSCs have otherwise beenmobilized with cytokines were more likely to take up suppressed or killed—will home to the bone marrowgenes from a viral vector than were non-mobilized and undergo active division to both replenish all thebone marrow HSCs [43]. different types of blood cells and yield additional self- renewing HSCs. But exactly how this happens remainsWHAT DO HEMATOPOIETIC STEM a mystery that scientists are struggling to solve by manipulating cultures of HSCs in the laboratory.CELLS DO AND WHAT FACTORS AREINVOLVED IN THESE ACTIVITIES? Two recent examples of progress in the culturing studies of mouse HSCs are by Ema and coworkersAs stated earlier, an HSC in the bone marrow has four and Audet and colleagues [2, 15]. Ema et al. foundactions in its repertoire: 1) it can renew itself, 2) it can that two cytokines—stem cell factor and thrombo-differentiate, 3) it can mobilize out of the bone poietin—efficiently induced an unequal first cellmarrow into circulation (or the reverse), or 4) it can division in which one daughter cell gave rise toundergo programmed cell death, or apoptosis. repopulating cells with self-renewal potential. AudetUnderstanding the how, when, where, which, and why et al. found that activation of the signaling moleculeof this simple repertoire will allow researchers to gp130 is critical to survival and proliferation of mousemanipulate and use HSCs for tissue and organ repair. HSCs in culture.Self-renewal of Hematopoietic Stem Cells Work with specific cytokines and signaling moleculesScientists have had a tough time trying to grow— builds on several earlier studies demonstrating mod-or even maintain—true stem cells in culture. This is an est increases in the numbers of stem cells that couldimportant goal because cultures of HSCs that could be induced briefly in culture. For example, Van Zantmaintain their characteristic properties of self-renewal and colleagues used continuous-perfusion cultureand lack of differentiation could provide an unlimited and bioreactors in an attempt to boost human HSCsource of cells for therapeutic transplantation and numbers in single cord blood samples incubated forstudy. When bone marrow or blood cells are observed one to two weeks [58]. They obtained a 20-foldin culture, one often observes large increases in the increase in “long-term culture initiating cells.”number of cells. This usually reflects an increase in 49
  • Hematopoietic Stem CellsMore clues on how to increase numbers of stem cells Differentiation of HSCs into Components of themay come from looking at other animals and various Blood and Immune Systemdevelopmental stages. During early developmental Producing differentiated white and red blood cells isstages—in the fetal liver, for example—HSCs may the real work of HSCs and progenitor cells. M.C.undergo more active cell division to increase their MacKey calculates that in the course of producingnumbers, but later in life, they divide far less often [30, a mature, circulating blood cell, the original42]. Culturing HSCs from 10- and 11-day-old mouse hematopoietic stem cell will undergo between 17embryos, Elaine Dzierzak at Erasmus University in the and 19.5 divisions, “giving a net amplification ofNetherlands finds she can get a 15-fold increase in between ~170,000 and ~720,000” [35].HSCs within the first 2 or 3 days after she removes theAGM from the embryos [38]. Dzierzak recognizes that Through a series of careful studies of cultured cells—this is dramatically different from anything seen with often cells with mutations found in leukemia patientsadult stem cells and suggests it is a difference with or cells that have been genetically altered—practical importance. She suspects that the increase investigators have discovered many key growthis not so much a response to what is going on in the factors and cytokines that induce progenitor cells toculture but rather, it represents the developmental make different types of blood cells. These factorsmomentum of this specific embryonic tissue. That is, it interact with one another in complex ways to createis the inevitable consequence of divisions that were a system of exquisite genetic control and coordina-cued by that specific embryonic microenvironment. tion of blood cell production.After five days, the number of HSCs plateaus and can Migration of Hematopoietic Stem Cells Into andbe maintained for up to a month. Dzierzak says that Out of Marrow and Tissuesthe key to understanding how adult-derived HSCs canbe expanded and manipulated for clinical purposes Scientists know that much of the time, HSCs live inmay very well be found by defining the cellular com- intimate connection with the stroma of bone marrowposition and complex molecular signals in the AGM in adults (see Chapter 4. The Adult Stem Cell). Butregion during development [13]. HSCs may also be found in the spleen, in peripheral blood circulation, and other tissues. Connection toIn another approach, Lemischka and coworkers have the interstices of bone marrow is important to bothbeen able to maintain mouse HSCs for four to seven the engraftment of transplanted cells and to theweeks when they are grown on a clonal line of cells maintenance of stem cells as a self-renewing popu-(AFT024) derived from the stroma, the other major lation. Connection to stroma is also important to thecellular constituent of bone marrow [39]. No one orderly proliferation, differentiation, and maturation ofknows which specific factors secreted by the stromal blood cells [63].cells maintain the stem cells. He says ongoing genecloning is rapidly zeroing in on novel molecules from Weissman says HSCs appear to make brief forays outthe stromal cells that may “talk” to the stem cells and of the marrow into tissues, then duck back into mar-persuade them to remain stem cells—that is, row [62]. At this time, scientists do not understand whycontinue to divide and not differentiate. or how HSCs leave bone marrow or return to it [59]. Scientists find that HSCs that have been mobilizedIf stromal factors provide the key to stem cell self- into peripheral circulation are mostly non-dividingrenewal, research on maintaining stromal cells may cells [64]. They report that adhesion molecules on thebe an important prerequisite. In 1999, researchers at stroma, play a role in mobilization, in attachment toOsiris Therapeutics and Johns Hopkins University the stroma, and in transmitting signals that regulatereported culturing and expanding the numbers of HSC self-renewal and progenitor differentiation [61].mesenchymal stem cells, which produce the stromalenvironment [46]. Whereas cultured HSCs rush to Apoptosis and Regulation of Hematopoietic Stemdifferentiate and fail to retain primitive, self-renewing Cell Populationscells, the mesenchymal stem cells could be The number of blood cells in the bone marrow andincreased in numbers and still retained their powers to blood is regulated by genetic and moleculargenerate the full repertoire of descendant lineages. mechanisms. How do hematopoietic stem cells know 50
  • Hematopoietic Stem Cellswhen to stop proliferating? Apoptosis is the process Thomas and Clift describe the history of treatment forof programmed cell death that leads cells to self- chronic myeloid leukemia as it moved from largelydestruct when they are unneeded or detrimental. If ineffective chemotherapy to modestly successfulthere are too few HSCs in the body, more cells divide use of a cytokine, interferon, to bone marrow trans-and boost the numbers. If excess stem cells were plants—first in identical twins, then in HLA-matchedinjected into an animal, they simply wouldn’t divide siblings [55]. Although there was significant risk ofor would undergo apoptosis and be eliminated [62]. patient death soon after the transplant either fromExcess numbers of stem cells in an HSC transplant infection or from graft-versus-host disease, for the firstactually seem to improve the likelihood and speed of time, many patients survived this immediate chal-engraftment, though there seems to be no rigorous lenge and had survival times measured in years oridentification of a mechanism for this empirical even decades, rather than months. The authors write,observation. “In the space of 20 years, marrow transplantation has contributed to the transformation of [chronicThe particular signals that trigger apoptosis in HSCs myelogenous leukemia] CML from a fatal diseaseare as yet unknown. One possible signal for apoptosis to one that is frequently curable. At the same time,might be the absence of life-sustaining signals from experience acquired in this setting has improved ourbone marrow stroma. Michael Wang and others understanding of many transplant-related problems.found that when they used antibodies to disrupt the It is now clear that morbidity and mortality are notadhesion of HSCs to the stroma via VLA-4/VCAM-1, inevitable consequences of allogeneic transplanta-the cells were predisposed to apoptosis [61]. tion, [and] that an allogeneic effect can add to theUnderstanding the forces at play in HSC apoptosis is anti-leukemic power of conditioning regimens…”important to maintaining or increasing their numbers In a recent development, CML researchers havein culture. For example, without growth factors, sup- taken their knowledge of hematopoietic regulationplied in the medium or through serum or other feeder one step farther. On May 10, 2001, the Food andlayers of cells, HSCs undergo apoptosis. Domen and Drug Administration approved Gleevec™ (imatinibWeissman found that stem cells need to get two mesylate), a new, rationally designed oral drug forgrowth factor signals to continue life and avoid treatment of CML. The new drug specifically targets aapoptosis: one via a protein called BCL-2, the other mutant protein, produced in CML cancer cells, thatfrom steel factor, which, by itself, induces HSCs to sabotages the cell signals controlling orderly divisionproduce progenitor cells but not to self-renew [10]. of progenitor cells. By silencing this protein, the new drug turns off cancerous overproduction of whiteWHAT ARE THE CLINICAL USES OF blood cells, so doctors do not have to resort to boneHEMATOPOIETIC STEM CELLS? marrow transplantation. At this time, it is unknown whether the new drug will provide sustained remissionLeukemia and Lymphoma or will prolong life for CML patients.Among the first clinical uses of HSCs were thetreatment of cancers of the blood—leukemia and Inherited Blood Disorderslymphoma, which result from the uncontrolled Another use of allogeneic bone marrow transplants isproliferation of white blood cells. In these applica- in the treatment of hereditary blood disorders, suchtions, the patient’s own cancerous hematopoietic as different types of inherited anemia (failure to pro-cells were destroyed via radiation or chemotherapy, duce blood cells), and inborn errors of metabolismthen replaced with a bone marrow transplant, or, as is (genetic disorders characterized by defects in keydone now, with a transplant of HSCs collected from enzymes need to produce essential body compo-the peripheral circulation of a matched donor. A nents or degrade chemical byproducts). The bloodmatched donor is typically a sister or brother of the disorders include aplastic anemia, beta-thalassemia,patient who has inherited similar human leukocyte Blackfan-Diamond syndrome, globoid cell leuko-antigens (HLAs) on the surface of their cells. Cancers dystrophy, sickle-cell anemia, severe combinedof the blood include acute lymphoblastic leukemia, immunodeficiency, X-linked lymphoproliferativeacute myeloblastic leukemia, chronic myelogenous syndrome, and Wiskott-Aldrich syndrome. Inborn errorsleukemia (CML), Hodgkin’s disease, multiple of metabolism that are treated with bone marrowmyeloma, and non-Hodgkin’s lymphoma. 51
  • Hematopoietic Stem Cells The National Marrow Donor Program http://www.marrow.org Launched in 1987, the National Marrow Donor Program recruit potential donors. As of February 28, 2001, the (NMDP) was created to connect patients who need NMDP listed 4,291,434 potential donors. Since its start, blood-forming stem cells or bone marrow with potential the Minneapolis-based group has facilitated almost nonrelated donors. About 70 percent of patients who 12,000 transplants—75 percent of them for leukemia. need a life-saving HSC transplant cannot find a match Major recruiting efforts have led to substantial increases in their own family. in the number of donations from minorities, but the chance that African Americans, Native Americans, The NMDP is made up of an international network of Asian/Pacific Islanders, or Hispanics will find a match is centers and banks that collect cord blood, bone still lower than it is for Caucasians. marrow, and peripherally harvested stem cells and thattransplants include: Hunter’s syndrome, Hurler’s syn- far have had their tumors reduced. The researchdrome, Lesch Nyhan syndrome, and osteopetrosis. protocol is now expanding to treatment of other solidBecause bone marrow transplantation has carried a tumors that resist standard therapy, including cancersignificant risk of death, this is usually a treatment of of the lung, prostate, ovary, colon, esophagus, liver,last resort for otherwise fatal diseases. and pancreas.Hematopoietic Stem Cell Rescue in Cancer This experimental treatment relies on an allogeneicChemotherapy stem cell transplant from an HLA-matched siblingChemotherapy aimed at rapidly dividing cancer cells whose HSCs are collected peripherally. The patient’sinevitably hits another target—rapidly dividing own immune system is suppressed, but not totallyhematopoietic cells. Doctors may give cancer destroyed. The donor’s cells are transfused into thepatients an autologous stem cell transplant to patient, and for the next three months, doctorsreplace the cells destroyed by chemotherapy. They closely monitor the patient’s immune cells, using DNAdo this by mobilizing HSCs and collecting them from fingerprinting to follow the engraftment of the donor’speripheral blood. The cells are stored while the cells and regrowth of the patient’s own blood cells.patient undergoes intensive chemotherapy or radio- They must also judiciously suppress the patient’stherapy to destroy the cancer cells. Once the drugs immune system as needed to deter his/her T cellshave washed out of a patient’s body, the patient from attacking the graft and to reduce graft-versus-receives a transfusion of his or her stored HSCs. host disease.Because patients get their own cells back, there is no A study by Joshi et al. shows that umbilical cordchance of immune mismatch or graft-versus-host blood and peripherally harvested human HSCs showdisease. One problem with the use of autologous antitumor activity in the test tube against leukemiaHSC transplants in cancer therapy has been that cells and breast cancer cells [22]. Grafted into acancer cells are sometimes inadvertently collected mouse model that tolerates human cells, HSCs attackand reinfused back into the patient along with the human leukemia and breast cancer cells. Althoughstem cells. One team of investigators finds that they untreated cord blood lacks natural killer (NK) lympho-can prevent reintroducing cancer cells by purifying cytes capable of killing tumor cells, researchers havethe cells and preserving only the cells that are found that at least in the test tube and in mice, theyCD34+, Thy-1+ [41]. can greatly enhance the activity and numbers ofGraft-Versus-Tumor Treatment of Cancer these cells with cytokines IL-15 [22, 34].One of the most exciting new uses of HSC Other Applications of Hematopoietic Stem Cellstransplantation puts the cells to work attacking other- Substantial basic and limited clinical research explor-wise untreatable tumors. A group of researchers in ing the experimental uses of HSCs for other diseasesNIH’s intramural research program recently described is underway. Among the primary applications arethis approach to treating metastatic kidney cancer autoimmune diseases, such as diabetes, rheumatoid[7]. Just under half of the 38 patients treated so 52
  • Hematopoietic Stem Cellsarthritis, and system lupus erythematosis. Here, the mice were on the liver-protective drug. The drug wasbody’s immune system turns to destroying body then removed, launching deterioration of the liver—tissues. Experimental approaches similar to those and a test to see whether cells from the transplantapplied above for cancer therapies are being con- would be recruited and rescue the liver. The scientistsducted to see if the immune system can be found that transplants of as few as 50 cells led toreconstituted or reprogrammed. More detailed dis- abundant growth of marked, donor-derived liver cellscussion on this application is provided in Chapter 6. in the female mice.Autoimmune Diseases and the Promise of Stem Cell- Recently, Krause has shown in mice that a singleBased Therapies. The use of HSCs as a means to selected donor hematopoietic stem cell could dodeliver genes to repair damaged cells is another more than just repopulate the marrow andapplication being explored. The use of HSCs for gene hematopoietic system of the recipient [27]. Thesetherapies is discussed in detail in Chapter 11. Use of investigators also found epithelial cells derived fromGenetically Modified Stem Cells in Experimental the donors in the lungs, gut, and skin of the recipientGene Therapies. mice. This suggests that HSCs may have grown in the other tissues in response to infection or damage fromPLASTICITY OF HEMATOPOIETIC the irradiation the mice received.STEM CELLS In humans, observations of male liver cells in femaleA few recent reports indicate that scientists have patients who have received bone marrow grafts frombeen able to induce bone marrow or HSCs to males, and in male patients who have received liverdifferentiate into other types of tissue, such as brain, transplants from female donors, also suggest themuscle, and liver cells. These concepts and the possibility that some cells in bone marrow have theexperimental evidence supporting this concept are capacity to integrate into the liver and formdiscussed in Chapter 4. The Adult Stem Cell. hepatocytes [1].Research in a mouse model indicates that cells fromgrafts of bone marrow or selected HSCs may home WHAT ARE THE BARRIERS TO THEto damaged skeletal and cardiac muscle or liver and DEVELOPMENT OF NEW ANDregenerate those tissues [4, 29]. One recent advance IMPROVED TREATMENTS USINGhas been in the study of muscular dystrophy, a HEMATOPOIETIC STEM CELLS?genetic disease that occurs in young people andleads to progressive weakness of the skeletal muscles. Boosting the Numbers of Hematopoietic Stem CellsBittner and colleagues used mdx mice, a genetically Clinical investigators share the same fundamentalmodified mouse with muscle cell defects similar to problem as basic investigators—limited ability to growthose in human muscular dystrophy. Bone marrow and expand the numbers of human HSCs. Cliniciansfrom non-mdx male mice was transplanted into repeatedly see that larger numbers of cells in stemfemale mdx mice with chronic muscle damage; cell grafts have a better chance of survival in aafter 70 days, researchers found that nuclei from the patient than do smaller numbers of cells. The limitedmales had taken up residence in skeletal and cardiac number of cells available from a placenta andmuscle cells. umbilical cord blood transplant currently means that cord blood banks are useful to pediatric but not adultLagasse and colleagues’ demonstration of liver repair patients. Investigators believe that the main cause ofby purified HSCs is a similarly encouraging sign that failure of HSCs to engraft is host-versus-graft disease,HSCs may have the potential to integrate into andgrow in some non-blood tissues. These scientists and larger grafts permit at least some donor cellslethally irradiated female mice that had an unusual to escape initial waves of attack from a patient’sgenetic liver disease that could be halted with a residual or suppressed immune system [6]. Ability todrug. The mice were given transplants of genetically expand numbers of human HSCs in vivo or in vitro would clearly be an enormous boost to all currentmarked, purified HSCs from male mice that did not and future medical uses of HSC transplantation.have the liver disease. The transplants were given achance to engraft for a couple of months while the 53
  • Hematopoietic Stem CellsOnce stem cells and their progeny can be multiplied cancerous transformation. Glimm et al. [17] highlightin culture, gene therapists and blood experts could some of these problems, for example, with theircombine their talents to grow limitless quantities of confirmation that human stem cells lose their ability“universal donor” stem cells, as well as progenitors to repopulate the bone marrow as they enter andand specific types of red and white blood cells. If the progress through the cell cycleælike mouse stemcells were engineered to be free of markers that cells that have been stimulated to divide lose theirprovoke rejection, these could be transfused to any transplantability [18]. Observations on the inverserecipient to treat any of the diseases that are now relationship between progenitor cell division rate andaddressed with marrow, peripheral, cord, or other longevity in strains of mice raise an additional con-transfused blood. If gene therapy and studies of the cern that culture tricks or selection of cells thatplasticity of HSCs succeed, the cells could also be expand rapidly may doom the cells to a short life.grown to repair other tissues and treat non-blood- Pragmatically, some scientists say it may not berelated disorders [32]. necessary to be able to induce the true, long-termSeveral research groups in the United States, Canada, HSC to divide in the lab. If they can manipulateand abroad have been striving to find the key factor progenitors and coax them into division on com-or factors for boosting HSC production. Typical mand, gene uptake, and differentiation into keyapproaches include comparing genes expressed in blood cells and other tissues, that may be sufficientprimitive HSCs versus progenitor cells; comparing to accomplish clinical goals. It might be sufficient togenes in actively dividing fetal HSCs versus adult boost HSCs or subpopulations of hematopoietic cellsHSCs; genetic screening of hematopoietically within the body by chemically prodding the bonemutated zebrafish; studying dysregulated genes in marrow to supply the as-yet-elusive factors tocancerous hematopoietic cells; analyzing stromal or rejuvenate cell division.feeder-layer factors that appear to boost HSC Outfoxing the Immune System in Host, Graft, anddivision; and analyzing factors promoting homing Pathogen Attacksand attachment to the stroma. Promising candidatefactors have been tried singly and in combination, Currently, the risks of bone marrow transplants—graftand researchers claim they can now increase the rejection, host-versus-graft disease, and infectionnumber of long-term stem cells 20-fold, albeit briefly, during the period before HSCs have engrafted andin culture. resumed full blood cell production—restrict their use to patients with serious or fatal illnesses. AllogeneicThe specific assays researchers use to prove that their grafts must come from donors with a close HLAexpanded cells are stem cells vary, which makes it match to the patient (see Chapter 6. Autoimmunedifficult to compare the claims of different research Diseases and the Promise of Stem Cell-Basedgroups. To date, there is only a modest ability to Therapies). If doctors could precisely manipulateexpand true, long-term, self-renewing human HSCs. immune reactions and protect patients fromNumbers of progenitor cells are, however, more pathogens before their transplants begin to function,readily increased. Kobari et al., for example, can HSC transplants could be extended to less ill patientsincrease progenitor cells for granulocytes and and patients for whom the HLA match was not asmacrophages 278-fold in culture [25]. close as it must now be. Physicians might use transplants with greater impunity in gene therapy,Some investigators are now evaluating whether these autoimmune disease, HIV/AIDS treatment, and thecomparatively modest increases in HSCs are clinically preconditioning of patients to accept a major organuseful. At this time, the increases in cell numbers are transplant.not sustainable over periods beyond a few months,and the yield is far too low for mass production. In Scientists are zeroing in on subpopulations of T cellsaddition, the cells produced are often not rigorously that may cause or suppress potentially lethal host-characterized. A host of other questions remain— versus-graft rejection and graft-versus-host disease infrom how well the multiplied cells can be altered for allogeneic-transplant recipients. T cells in a graft aregene therapy to their potential longevity, immuno- a two-edged sword. They fight infections and helpgenicity, ability to home correctly, and susceptibility to 54
  • Hematopoietic Stem Cellsthe graft become established, but they also can Could there be embryological underpinnings to thecause graft-versus-host disease. Identifying sub- apparent plasticity of adult cells? Researchers havepopulations of T cells responsible for deleterious and suggested that a lot of the tissues that are showingbeneficial effects—in the graft, but also in residual plasticity are adjacent to one another after gastru-cells surviving or returning in the host—could allow lation in the sheet of mesodermal tissue that will goclinicians to make grafts safer and to ratchet up on to form blood—muscle, blood vessels, kidney,graft-versus-tumor effects [48]. Understanding the mesenchyme, and notochord. Plasticity may reflectpresentation of antigens to the immune system and derivation from the mesoderm, rather than being athe immune system’s healthy and unhealthy fixed trait of hematopoietic cells. One lab is nowresponses to these antigens and maturation and studying the adjacency of embryonic cells and howprogrammed cell death of T cells is crucial. the developing embryo makes the decision to make one tissue instead of another—and whether theThe approach taken by investigators at Stanford— decision is reversible [65].purifying peripheral blood—may also help eliminatethe cells causing graft-versus-host disease. Transplants In vivo studies of the plasticity of bone marrow orin mouse models support the idea that purified HSCs, purified stem cells injected into mice are in theircleansed of mature lymphocytes, engraft readily and infancy. Even if follow-up studies confirm and moreavoid graft-versus-host disease [60]. precisely characterize and quantify plasticity potential of HSCs in mice, there is no guarantee that it willKnowledge of the key cellular actors in autoimmune occur or can be induced in humans.disease, immune grafting, and graft rejection couldalso permit scientists to design gentler “minitrans-plants.” Rather than obliterating and replacing the SUMMARYpatient’s entire hematopoietic system, they could Grounded in half a century of research, the study ofreplace just the faulty components with a selection of hematopoietic stem cells is one of the most excitingcells custom tailored to the patient’s needs. Clinicians and rapidly advancing disciplines in biomedicineare currently experimenting with deletion of T cells today. Breakthrough discoveries in both the laboratoryfrom transplants in some diseases, for example, and clinic have sharply expanded the use andthereby reducing graft-versus-host disease. supply of life-saving stem cells. Yet even more promising applications are on the horizon andResearchers are also experimenting with the possibility scientists’ current inability to grow HSCs outside theof knocking down the patient’s immune system—but body could delay or thwart progress with these newnot knocking it out. A blow that is sublethal to the therapies. New treatments include graft-versus-tumorpatient’s hematopoietic cells given before an allo- therapy for currently incurable cancers, autologousgeneic transplant can be enough to give the graft a transplants for autoimmune diseases, and genechance to take up residence in the bone marrow. therapy and tissue repair for a host of other problems.The cells replace some or all of the patient’s original The techniques, cells, and knowledge thatstem cells, often making their blood a mix of donor researchers have now are inadequate to realize theand original cells. For some patients, this mix of cells full promise of HSC-based therapy.will be enough to accomplish treatment objectivesbut without subjecting them to the vicious side Key issues for tapping the potential of hematopoieticeffects and infection hazards of the most powerful stem cells will be finding ways to safely and efficientlytreatments used for total destruction of their hemato- expand the numbers of transplantable human HSCspoietic systems [37]. in vitro or in vivo. It will also be important to gain a better understanding of the fundamentals of howUnderstanding the Differentiating Environment and immune cells work—in fighting infections, in causingDevelopmental Plasticity transplant rejection, and in graft-versus-host disease asAt some point in embryonic development, all cells well as master the basics of HSC differentiation.are plastic, or developmentally flexible enough to Concomitant advances in gene therapy techniquesgrow into a variety of different tissues. Exactly what is and the understanding of cellular plasticity could makeit about the cell or the embryonic environment that HSCs one of the most powerful tools for healing.instructs cells to grow into one organ and not another? 55
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  • 6. AUTOIMMUNE DISEASES AND THE PROMISE OF STEM CELL-BASED THERAPIESOne of the more perplexing questions in biomedical different symptoms, they share the unfortunate realityresearch is—why does the body’s protective shield that, for some reason, the body’s immune system hasagainst infections, the immune system, attack its own turned against itself (see Box 6.1. Immune Systemvital cells, organs, and tissues? The answer to this Components: Common Terms and Definitions).question is central to understanding an array ofautoimmune diseases, such as rheumatoid arthritis, HOW DOES THE IMMUNE SYSTEMtype 1 diabetes, systemic lupus erythematosus, and NORMALLY KEEP US HEALTHY?Sjogren’s syndrome. When some of the bodys cellularproteins are recognized as “foreign” by immune cells The “soldiers” of the immune system are white bloodcalled T lymphocytes, a destructive cascade of cells, including T and B lymphocytes, which originateinflammation is set in place. Current therapies to in the bone marrow from hematopoietic stem cells.combat these cases of cellular mistaken identity Every day the body comes into contact with manydampen the body’s immune response and leave organisms such as bacteria, viruses, and parasites.patients vulnerable to life-threatening infections. Unopposed, these organisms have the potential toResearch on stem cells is now providing new cause serious infections, such as pneumonia or AIDS.approaches to strategically remove the misguided When a healthy individual is infected, the bodyimmune cells and restore normal immune cells to responds by activating a variety of immune cells.the body. Presented here are some of the basic Initially, invading bacteria or viruses are engulfed byresearch investigations that are being guided by an antigen presenting cell (APC), and their compo-adult and embryonic stem cell discoveries. nent proteins (antigens) are cut into pieces and dis- played on the cell’s surface. Pieces of the foreignINTRODUCTION protein (antigen) bind to the major histocompatibility complex (MHC) proteins, also known as human leuko-The body’s main line of defense against invasion by cyte antigen (HLA) molecules, on the surface of theinfectious organisms is the immune system. To suc- APCs (see Figure 6.1 Immune Response to Self orceed, an immune system must distinguish the many Foreign Antigens). This complex, formed by a foreigncellular components of its own body (self) from the protein and an MHC protein, then binds to a T cellcells or components of invading organisms (nonself). receptor on the surface of another type of immune“Nonself” should be attacked while “self” should not. cell, the CD4 helper T cell. They are so namedTherefore, two general types of errors can be made because they “help” immune responses proceedby the immune system. If the immune system fails to and have a protein called CD4 on their surface. Thisquickly detect and destroy an invading organism, an complex enables these T cells to focus the immuneinfection will result. However, if the immune system response to a specific invading organism. The anti-fails to recognize self cells or components and gen-specific CD4 helper T cells divide and multiplymistakenly attacks them, the result is known as an while secreting substances called cytokines, whichautoimmune disease. Common autoimmune cause inflammation and help activate other immunediseases include rheumatoid arthritis, systemic lupus cells. The particular cytokines secreted by the CD4erythematosis (lupus), type 1 diabetes, multiple helper T cells act on cells known as the CD8 “cytotox-sclerosis, Sjogren’s syndrome and inflammatory bowel ic” T cells (because they can kill the cells that aredisease. Although each of these diseases has infected by the invading organism and have the CD8 59
  • Autoimmune Diseases and the Promise of Stem Cell-Based Therapies Free pathogen clearance by specific antibody Antibodies Production of pathogenic self-reactive antibodies B Cell Cytokines CD8 Whole self Foreign (or self) cytotoxic antigen antigen T cell CD4 helper T cell Antigen Presenting Infected cells (displays Cell (APC) foreign T cell epitope Antigen-specific T cell receptor on its surface) or self Cell death (i.e., loss of self tolerance) CD4 helper T cell© 2001 Terese Winslow MHC/antigen complex Figure 6.1. Immune Response to Self or Foreign Antigens. protein on their surface). The helper T cells can also marrow. Upon their departure from the bone marrow, activate antigen-specific B cells to produce antibod- immature T cells undergo a final maturation process ies, which can neutralize and help eliminate bacteria in the thymus, a small organ located in the upper and viruses from the body. Some of the antigen-specif- chest, before being dispersed to the body with the ic T and B cells that are activated to rid the body of rest of the immune cells (e.g., B cells). Within the infectious organisms become long-lived “memory” thymus, T cells undergo an important process that cells. Memory cells have the capacity to act quickly “educates” them to distinguish between self (the when confronted with the same infectious organism at proteins of their own body) and nonself (the invading later times. It is the memory cells that cause us to organism’s) antigens. Here, the T cells are selected for become “immune” from later reinfections with the their ability to bind to the particular MHC proteins same organism. expressed by the individual. The particular array of MHCs varies slightly between individuals, and this HOW DO THE IMMUNE CELLS OF variation is the basis of the immune response when a THE BODY KNOW WHAT TO ATTACK transplanted organ is rejected. MHCs and other less easily characterized molecules called minor histo- AND WHAT NOT TO? compatibility antigens are genetically determined All immune and blood cells develop from multipotent and this is the reason why donor organs from relatives hematopoietic stem cells that originate in the bone of the recipient are preferred over unrelated donors. 60
  • Autoimmune Diseases and the Promise of Stem Cell-Based Therapies Box 6.1 Immune System Components: Common Terms and Definitions Antibody — A Y-shaped protein secreted by B cells in Immune system cells — White blood cells or leukocytes response to an antigen. An antibody binds specifically that originate from the bone marrow. They include to the antigen that induced its production. Antibodies antigen presenting cells, such as dendritic cells, T and B directed against antigens on the surface of infectious lymphocytes, and neutrophils, among many others. organisms help eliminate those organisms from the Lymphatic system — A network of lymph vessels and body. nodes that drain and filter antigens from tissue fluids Antigen — A substance (often a protein) that induces before returning lymphocytes to the blood. the formation of an antibody. Antigens are commonly Memory cells — A subset of antigen-specific T or B found on the surface of infectious organisms, transfused cells that “recall” prior exposure to an antigen and blood cells, and organ transplants. respond quickly without the need to be activated Antigen presenting cells (APC) — One of a variety of again by CD4 helper T cells. cells within the body that can process antigens and Major histocompatibility complex (MHC) — A group display them on their surface in a form recognizable of genes that code for cell-surface histocompatibility by T cells. antigens. These antigens are the primary reason why Autoantibody — An antibody that reacts with antigens organ and tissue transplants from incompatible found on the cells and tissues of an individual’s own donors fail. body. Autoantibodies can cause autoimmune diseases. T cells — Also known as T lymphocytes. There are two Autoimmune disease — A condition that results from primary subsets of T cells. CD4 helper T cells (identified the formation of antibodies that attack the cells or by the presence of the CD4 protein on their surfaces) tissues of an individual’s own body. are instrumental in initiating an immune response by B cells — Also known as B lymphocytes. Each B cell supplying special cytokines. CD8 cytotoxic (killer) T cells is capable of making one specific antibody. When (identified by the presence of the CD8 protein on their stimulated by antigen and helper T cells, B cells mature surfaces), after being activated by the CD4 helper cells, into plasma cells that secrete large amounts of their are capable of killing infected cells in the body. CD4 specific antibody helper T cells are destroyed by the HIV virus in AIDS patients, resulting in an ineffective immune system. Bone marrow — The soft, living tissue that fills most bone cavities and contains hematopoietic stem cells, Thymus — A lymphoid organ located in the upper from which all red and white blood cells evolve. The chest cavity. Maturing T cells leave the bone marrow bone marrow also contains mesenchymal stem cells and go directly to the thymus, where they are educated that a number of cell types come from, including to discriminate between self and nonself proteins. chondrocytes, which produce cartilage. (See tolerance.) Cytokines — A generic term for a large variety of Tolerance — A state of specific immunologic regulatory proteins produced and secreted by cells unresponsiveness. Individuals should normally be and used to communicate with other cells. One class tolerant of the cells and tissues that make up our own of cytokines is the interleukins, which act as intercellular bodies. Should tolerance fail, an autoimmune disease mediators during the generation of an immune response. may result.In the bone marrow, a highly diverse and random Tolerance usually ensures that T cells do not attackarray of T cells is produced. Collectively, these T cells the “autoantigens” (self-proteins) of the body. Givenare capable of recognizing an almost unlimited the importance of this task, it is not surprising thatnumber of antigens. Because the process of there are multiple checkpoints for destroying orgenerating a T cell’s antigen specificity is a random inactivating T cells that might react to auto-antigens.one, many immature T cells have the potential toreact with the body’s own (self) proteins. To avoid this Autoimmune diseases arise when this intricate systempotential disaster, the thymus provides an environ- for the induction and maintenance of immunement where T cells that recognize self-antigens tolerance fails. These diseases result in cell and tissue(autoreactive or self-reactive T cells) are deleted or destruction by antigen-specific CD8 cytotoxic T cells or autoantibodies (antibodies to self-proteins) and theinactivated in a process called tolerance induction. 61
  • Autoimmune Diseases and the Promise of Stem Cell-Based Therapiesaccompanying inflammatory process. These disproportionately afflicts African-American andmechanisms can lead to the destruction of the joints Hispanic women [11]. A major obstacle in the treat-in rheumatoid arthritis, the destruction of the insulin- ment of non-organ-specific autoimmune diseasesproducing beta cells of the pancreas in type 1 such as lupus is the lack of a single specific target fordiabetes, or damage to the kidneys in lupus. The rea- the application of therapy.sons for the failure to induce or maintain tolerance The objective of hematopoietic stem cell therapy forare enigmatic. However, genetic factors, along with lupus is to destroy the mature, long-lived, and auto-environmental and hormonal influences and certain reactive immune cells and to generate a new,infections, may contribute to tolerance and the properly functioning immune system. In most of thesedevelopment of autoimmune disease [4, 7]. trials, the patient’s own stem cells have been used in a procedure known as autologous (from “one’s self”)HEMATOPOIETIC STEM CELL hematopoietic stem cell transplantation. First, patientsTHERAPY FOR AUTOIMMUNE receive injections of a growth factor, which coaxesDISEASES large numbers of hematopoietic stem cells to be released from the bone marrow into the blood stream.The current treatments for many autoimmune diseases These cells are harvested from the blood, purifiedinclude the systemic use of anti-inflammatory drugs away from mature immune cells, and stored. Afterand potent immunosuppressive and immunomodu- sufficient quantities of these cells are obtained, thelatory agents (i.e., steroids and inhibitor proteins that patient undergoes a regimen of cytotoxic (cell-killing)block the action of inflammatory cytokines). However, drug and/or radiation therapy, which eliminates thedespite their profound effect on immune responses, mature immune cells. Then, the hematopoietic stemthese therapies are unable to induce clinically signifi- cells are returned to the patient via a blood transfusioncant remissions in certain patients. In recent years, into the circulation where they migrate to the boneresearchers have contemplated the use of stem cells marrow and begin to differentiate to become matureto treat autoimmune disorders. Discussed here is immune cells. The body’s immune system is thensome of the rationale for this approach, with a focus restored. Nonetheless, the recovery phase, until theon experimental stem cell therapies for lupus, immune system is reconstituted represents a period ofrheumatoid arthritis, and type 1 diabetes. dramatically increased susceptibility to bacterial, fun-The immune-mediated injury in autoimmune diseases gal, and viral infection, making this a high-risk therapy.can be organ-specific, such as type 1 diabetes Recent reports suggest that this replacement therapywhich is the consequence of the destruction of the may fundamentally alter the patient’s immune sys-pancreatic beta islet cells or multiple sclerosis which tem. Richard Burt and his colleagues [18] conductedresults from the breakdown of the myelin covering of a long-term follow-up (one to three years) of sevennerves. These autoimmune diseases are amenable lupus patients who underwent this procedure andto treatments involving the repair or replacement of found that they remained free from active lupus anddamaged or destroyed cells or tissue (see Chapter 7. improved continuously after transplantation, withoutStem Cells and Diabetes and Chapter 11. Use of the need for immunosuppressive medications. OneGenetically Modified Stem Cells in Experimental of the hallmarks of lupus is that during the natural pro-Gene Therapies). In contrast, non-organ-specific gression of disease, the normally diverse repertoire ofautoimmune diseases, such as lupus, are character- T cells become limited in the number of different anti-ized by widespread injury due to immune reactions gens they recognize, suggesting that an increasingagainst many different organs and tissues. proportion of the patient’s T cells are autoreactive.One approach is being evaluated in early clinical Burt and colleagues found that following hemato-trials of patients with poorly responsive, life-threatening poietic stem cell transplantation, levels of T celllupus. This is a severe disease affecting multiple diversity were restored to those of healthy individuals.organs in the body including muscles, skin, joints, and This finding provides evidence that stem cell replace-kidneys as well as the brain and nerves. Over 239,000 ment may be beneficial in reestablishing tolerance inAmericans, of which more than 90 percent are T cells, thereby decreasing the likelihood of diseasewomen, suffer from lupus. In addition, lupus reoccurrence. 62
  • Autoimmune Diseases and the Promise of Stem Cell-Based TherapiesDEVELOPMENT OF HEMATOPOIETIC One risk of using nonself hematopoietic stem cells isSTEM CELL LINES FOR of immune rejection of the transplanted cells. Immune rejection is caused by MHC protein differ-TRANSPLANTATION ences between the donor and the patient (recipient).The ability to generate and propagate unlimited In this scenario, the transplanted hematopoietic stemnumbers of hematopoietic stem cells outside the cells and their progeny are rejected by the patient’sbody—whether from adult, umbilical cord blood, own T cells, which are originating from the patient’sfetal, or embryonic sources—would have a major surviving bone marrow hematopoietic stem cells. Inimpact on the safety, cost, and availability of stem this regard, embryonic stem cell-derived hemato-cells for transplantation. The current approach of poietic stem cells may offer distinct advantages overisolating hematopoietic stem cells from a patient’s cord blood and bone marrow hematopoietic stemown peripheral blood places the patient at risk for a cell lines in avoiding rejection of the transplant.flare-up of their autoimmune disease. This is a poten- Theoretically, banks of embryonic stem cells express-tial consequence of repeated administration of the ing various combinations of the three most criticalstem cell growth factors needed to mobilize MHC proteins could be generated to allow closehematopoietic stem cells from the bone marrow to matching to the recipient’s MHC composition.the blood stream in numbers sufficient for trans- Additionally, there is evidence that embryonic stemplantation. In addition, contamination of the purified cells are considerably more receptive to genetichematopoietic stem cells with the patient’s mature manipulation than are hematopoietic stem cells (seeautoreactive T and B cells could affect the success of Chapter 11. Use of Genetically Modified Stem Cells inthe treatment in some patients. Propagation of pure Experimental Gene Therapies).cell lines in the laboratory would avoid these potentialdrawbacks and increase the numbers of stem cells This characteristic means that embryonic stem cellsavailable to each patient, thus shortening the at-risk could be useful in strategies that could prevent theirinterval before full immune reconstitution. recognition by the patient’s surviving immune cells. For example, it may be possible to introduce theWhether embryonic stem cells will provide advan- recipient’s MHC proteins into embryonic stem cellstages over stem cells derived from cord blood or through targeted gene transfer. Alternatively, it isadult bone marrow hematopoietic stem cells remains theoretically possible to generate a universal donorto be determined. However, hematopoietic stem embryonic stem cell line by genetic alteration orcells, whether from umbilical cord blood or bone removal of the MHC proteins. Researchers havemarrow, have a more limited potential for self- accomplished this by genetically altering a mouserenewal than do pluripotent embryonic stem cells. so that it has little or no surface expression of MHCAlthough new information will be needed to direct molecules on any of the cells or tissues. There is nothe differentiation of embryonic stem cells into rejection of pancreatic beta islet cells from thesehematopoietic stem cells, hematopoietic cells are genetically altered mice when the cells are trans-present in differentiated cultures from human embry- planted into completely MHC-mismatched mice [13].onic stem cells [9] and from human fetal-derived Additional research will be needed to determine theembryonic germ stem cells [17]. feasibility of these alternative strategies for preventionOne potential advantage of using hematopoietic of graft rejection in humans [6].stem cell lines for transplantation in patients with Jon Odorico and colleagues have shown that expres-autoimmune diseases is that these cells could be sion of MHC proteins on mouse embryonic stem cellsgenerated from unaffected individuals or, as pre- and differentiated embryonic stem cell progeny isdisposing genetic factors are defined, from embryonic either absent or greatly decreased compared withstem cells lacking these genetic influences. In addition, MHC expression on adult cells [8]. These preliminaryuse of genetically selected or genetically engineered findings raise the intriguing possibility that lines derivedcell types may further limit the possibility of disease from embryonic stem cells may be inherently lessprogression or reemergence. susceptible to rejection by the recipient’s immune 63
  • Autoimmune Diseases and the Promise of Stem Cell-Based Therapiessystem than lines derived from adult cells. This could studies have advanced understanding of the diseasehave important implications for the transplantation of processes and the particular inflammatory cytokinescells other than hematopoietic stem cells. involved in disease progression [15, 19].Another potential advantage of using pure popula- Serious obstacles to the development of effectivetions of donor hematopoietic stem cells achieved gene therapies for humans remain, however.through stem cell technologies would be a lower Foremost among these are the difficulty of reliablyincidence and severity of graft-versus-host disease, a transferring genetic material into adult and slowlypotentially fatal complication of bone marrow trans- dividing cells (including hematopoietic stem cells)plantation. Graft-versus-host disease results from the and of producing long-lasting expression of theimmune-mediated injury to recipient tissues that intended protein at levels that can be tightly con-occurs when mature organ-donor T cells remain with- trolled in response to disease activity. Importantly,in the organ at the time of transplant. Such mature embryonic stem cells are substantially more permis-donor alloreactive T cells would be absent from pure sive to gene transfer compared with adult cells, andpopulations of multipotent hematopoietic stem cells, embryonic cells sustain protein expression duringand under ideal conditions of immune tolerance extensive self-renewal. Whether adult-derived steminduction in the recipient’s thymus, the donor-derived cells, other than hematopoietic stem cells, aremature T cell population would be tolerant to the host. similarly amenable to gene transfer has not yet been determined.GENE THERAPY AND STEM CELL Ultimately, stem cell gene therapy should allow theAPPROACHES FOR THE TREATMENT development of novel methods for immune modula-OF AUTOIMMUNE DISEASES tion in autoimmune diseases. One example is the genetic modification of hematopoietic stem cells orGene therapy is the genetic modification of cells to differentiated tissue cells with a “decoy” receptor forproduce a therapeutic effect (see Chapter 11. Use of the inflammatory cytokine interferon gamma to treatGenetically Modified Stem Cells in Experimental lupus. For example, in a lupus mouse model, geneGene Therapies). In most investigational protocols, transfer of the decoy receptor, via DNA injection,DNA containing the therapeutic gene is transferred arrested disease progression [12]. Other investigatorsinto cultured cells, and these cells are subsequently have used a related but distinct approach in aadministered to the animal or patient. DNA can also mouse model of type 1 diabetes. Interleukin-12be injected directly, entering cells at the site of the (IL-12), an inflammatory cytokine, plays a prominentinjection or in the circulation. Under ideal conditions, role in the development of diabetes in these mice.cells take up the DNA and produce the therapeutic The investigators transferred the gene for a modifiedprotein encoded by the gene. form of IL-12, which blocks the activity of the naturalCurrently, there is an extensive amount of gene IL-12, into pancreatic beta islet cells (the target oftherapy research being conducted in animal models autoimmune injury in type 1 diabetes). The islet cellof autoimmune disease. The goal is to modify the gene therapy prevented the onset of diabetes inaberrant, inflammatory immune response that is these mice [20]. Theoretically, embryonic stem cellscharacteristic of autoimmune diseases [15, 19]. or adult stem cells could be genetically modifiedResearchers most often use one of two general before or during differentiation into pancreatic betastrategies to modulate the immune system. The first islet cells to be used for transplantation. The resultingstrategy is to block the actions of an inflammatory immune-modulating islet cells might diminish thecytokine (secreted by certain activated immune cells occurrence of ongoing autoimmunity, increase theand inflamed tissues) by transferring a gene into cells likelihood of long-term function of the transplantedthat encodes a “decoy” receptor for that cytokine. cells, and eliminate the need for immunosuppressiveAlternatively, a gene is transferred that encodes an therapy following transplantation.anti-inflammatory cytokine, redirecting the auto- Researchers are exploring similar genetic approachesinflammatory immune response to a more “tolerant” to prevent progressive joint destruction and loss ofstate. In many animal studies, promising results have cartilage and to repair damaged joints in animalbeen achieved by using these approaches, and the 64
  • Autoimmune Diseases and the Promise of Stem Cell-Based Therapiesmodels of rheumatoid arthritis. Rheumatoid arthritis is derived from human bone marrow [14]. Little is knowna debilitating autoimmune disease characterized by about the intermediate cells that ultimately differ-acute and chronic inflammation, in which the entiate into chondrocytes. In addition to adult boneimmune system primarily attacks the joints of the marrow as a source for stromal stem cells, humanbody. In a recent study, investigators genetically embryonic stem cells can differentiate into precursortransferred an anti-inflammatory cytokine, interleukin-4 cells believed to lead ultimately to the stromal stem(IL-4), into a specialized, highly efficient antigen- cells [16]. However, extensive research is needed topresenting cell called a dendritic cell, and then reliably achieve the directed derivation of the stromalinjected these IL-4-secreting cells into mice that can stem cells from embryonic stem cells and, subse-be induced to develop a form of arthritis similar to quently, the differentiation of chondrocytes fromrheumatoid arthritis in humans. These IL-4-secreting these stromal stem cells.dendritic cells are presumed to act on the CD4 The ideal cell for optimum cartilage repair may be ahelper T cells to reintroduce tolerance to self-proteins. more primitive cell than the chondrocyte, such as theTreated mice showed complete suppression of their stromal cell, or an intermediate cell in the pathwaydisease and, in addition to its immune-modulatory (e.g., a connective tissue precursor) leading to theproperties, IL-4 blocked bone resorption (a serious chondrocyte. Stromal stem cells can generate newcomplication of rheumatoid arthritis), making it a chondrocytes and facilitate cartilage repair in a rab-particularly attractive cytokine for this therapy [10]. bit model [3]. Such cells may also prove to be idealHowever, one obstacle to this approach is that human targets for the delivery of immune-modulatory genedendritic cells are difficult to isolate in large numbers. therapy. Like hematopoietic stem cells, stromal stemInvestigators have also directed the differentiation of cells have been used in animal models for delivery ofdendritic cells from mouse embryonic stem cells, gene therapy [1]. For example, a recent studyindicating that a stem cell-based approach might demonstrated that genetically engineered chondro-work in patients with rheumatoid arthritis [5]. Longer- cytes, expressing a growth factor, can enhance theterm follow-up and further characterization will be function of transplanted chondrocytes [2].needed in animal models before researchers Two obstacles to the use of adult stromal stem cellsproceed with the development of such an approach or chondrocytes are the limited numbers of thesein humans. In similar studies, using other inhibitors of cells that can be harvested and the difficulties ininflammatory cytokines such as a decoy receptor for propagating them in the laboratory. Embryonic stemtumor necrosis factor–Ȋ (a prominent inflammatory cells, genetically modified and expanded beforecytokine in inflamed joints), an inhibitor of nuclear directed differentiation to a connective tissue stemfactor–Ȕdz (a protein within cells that turns on the cell, may be an attractive alternative.production of many inflammatory cytokines), andinterleukin-13 (an anti-inflammatory cytokine), Collectively, these results illustrate the tremendousresearchers have shown promising results in animal potential these cells may offer for the treatment ofmodels of rheumatoid arthritis [19]. Because of the rheumatoid arthritis and other autoimmune diseases.complexity and redundancy of immune systemsignaling networks, it is likely that a multifaceted CONCLUSIONapproach involving inhibitors of several differentinflammatory cytokines will be successful, whereas Stem cell-based therapies offer many excitingapproaches targeting single cytokines might fail or possibilities for the development of novel treatments,produce only short-lived responses. In addition, other and perhaps even cures, for autoimmune diseases.cell types may prove to be even better vehicles for A challenging research effort remains to fully realizethe delivery of gene therapy in this disease. this potential and to address the many remaining questions, which include how best to direct the differ-Chondrocytes, cells that build cartilage in joints, may entiation of specific cell types and determine whichprovide another avenue for stem cell-based treat- particular type of stem cell will be optimum for eachment of rheumatoid arthritis. These cells have been therapeutic approach. Gene therapy with cytokinesderived from human bone marrow stromal stem cells or their inhibitors is still in its infancy, but stem cells or 65
  • Autoimmune Diseases and the Promise of Stem Cell-Based Therapiestheir progeny may provide one of the better avenues 11. Lawrence, R.C., Helmick, C.G., Arnett, F.C., Deyo, R.A.,for future delivery of immune-based therapies. Felson, D.T., Giannini, E.H., Heyse, S.P Hirsch, R., Hochberg, ., M.C., Hunder, G.G., Liang, M.H., Pillemer, S.R., Steen, V.D.,Ultimately, the potential to alleviate these devastating and Wolfe, F. (1998). Estimates of the prevalence of arthritischronic diseases with the use of stem cell-based and selected musculoskeletal disorders in the United States.technologies is enormous. Arthritis Rheum. 41, 778-799. 12. Lawson, B.R., Prud’homme, G.J., Chang, Y., Gardner, H.A.,REFERENCES Kuan, J., Kono, D.H., and Theofilopoulos, A.N. (2000). Treatment of murine lupus with cDNA encoding IFN- 1. Allay, J.A., Dennis, J.E., Haynesworth, S.E., Majumdar, M.K., gammaR/Fc. J. Clin. Invest. 106, 207-215. Clapp, D.W., Shultz, L.D., Caplan, A.I., and Gerson, S.L. (1997). LacZ and interleukin-3 expression in vivo after retro- 13. Osorio, R.W., Ascher, N.L., Jaenisch, R., Freise, C.E., Roberts, viral transduction of marrow-derived human osteogenic J.P and Stock, P (1993). Major histocompatibility com- ., .G. mesenchymal progenitors. Hum. Gene Ther. 8, 1417-1427. plex class I deficiency prolongs islet allograft survival. Diabetes. 42, 1520-1527. 2. Brower-Toland, B.D., Saxer, R.A., Goodrich, L.R., Mi, Z., Robbins, P.D., Evans, C.H., and Nixon, A.J. (2001). Direct 14. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., adenovirus-mediated insulin-like growth factor I gene Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., transfer enhances transplant chondrocyte function. Hum. Craig, S., and Marshak, D.R. (1999). Multilineage potential Gene Ther. 12, 117-129. of adult human mesenchymal stem cells. Science. 284, 143-147. 3. Caplan, A.I., Elyaderani, M., Mochizuki, Y., Wakitani, S., and Goldberg, V.M. (1997). Principles of cartilage repair and 15. Prud’homme, G.J. (2000). Gene therapy of autoimmune regeneration. Clin. Orthop. 342, 254-269. diseases with vectors encoding regulatory cytokines or inflammatory cytokine inhibitors. J. Gene. Med. 2, 222-232. 4. Cooper, G.S., Dooley, M.A., Treadwell, E.L., St Clair, E.W., Parks, C.G., and Gilkeson, G.S. (1998). Hormonal, environ- 16. Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D., and mental, and infectious risk factors for developing systemic Benvenisty, N. (2000). Effects of eight growth factors on the lupus erythematosus. Arthritis Rheum. 41, 1714-1724. differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 97, 11307-11312. 5. Fairchild, P Brook, F.A., Gardner, R.L., Graca, L., Strong, V., .J., Tone, Y., Tone, M., Nolan, K.F., and Waldmann, H. (2000). 17. Shamblott, M.J., Axelman, J., Littlefield, J.W., Blumenthal, Directed differentiation of dendritic cells from mouse P.D., Huggins, G.R., Cui, Y., Cheng, L., and Gearhart, J.D. embryonic stem cells. Curr. Biol. 10, 1515-1518. (2000). Human embryonic germ cell derivatives express a broad range of develpmentally distinct markers and prolif- 6. Gearhart, J. (1998). New potential for human embryonic erate extensively in vitro. Proc. Natl. Acad. Sci. U. S. A. 98, stem cells. Science. 282, 1061-1062. 113-118. 7. Grossman, J.M. and Tsao, B.P (2000). Genetics and sys- . 18. Traynor, A.E., Schroeder, J., Rosa, R.M., Cheng, D., Stefka, J., temic lupus erythematosus. Curr. Rheumatol. Rep. 2, 13-18. Mujais, S., Baker, S., and Burt, R.K. (2000). Treatment of severe systemic lupus erythematosus with high-dose 8. Harley, C.B., Gearhart, J., Jaenisch, R., Rossant, J., and chemotherapy and haemopoietic stem-cell transplanta- Thomson, J. (2001). Keystone Symposia. Pluripotent stem tion: a phase I study. Lancet. 356, 701-707. cells: biology and applications. Durango, CO. 19. Tsokos, G.C. and Nepom, G.T. (2000). Gene therapy in the 9. Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., treatment of autoimmune diseases. J. Clin. Invest. 106, Yanuka, O., Amit, M., Soreq, H., and Benvenisty, N. (2000). 181-183. Differentiation of human embryonic stem cells into embry- oid bodies comprising the three embryonic germ layers. 20. Yasuda, H., Nagata, M., Arisawa, K., Yoshida, R., Fujihira, K., Mol. Med. 6, 88-95. Okamoto, N., Moriyama, H., Miki, M., Saito, I., Hamada, H., Yokono, K., and Kasuga, M. (1998). Local expression of10. Kim, S.H., Kim, S., Evans, C.H., Ghivizzani, S.C., Oligino, T., immunoregulatory IL-12p40 gene prolonged syngeneic islet and Robbins, P (2001). Effective treatment of established .D. graft survival in diabetic NOD mice. J. Clin. Invest. 102, murine collagen-induced arthritis by systemic administra- 1807-1814. tion of dendritic cells genetically modified to express IL-4. J. Immunol. 166, 3499-3505. 66
  • 7. STEM CELLS AND DIABETESDiabetes exacts its toll on many Americans, young the islet cells of the pancreas, which normallyand old. For years, researchers have painstakingly produce insulin, are destroyed. In the absence ofdissected this complicated disease caused by the insulin, glucose cannot enter the cell and glucosedestruction of insulin producing islet cells of the accumulates in the blood. Type 2 diabetes, alsopancreas. Despite progress in understanding the called adult-onset diabetes, tends to affect older,underlying disease mechanisms for diabetes, there is sedentary, and overweight individuals with a familystill a paucity of effective therapies. For years investi- history of diabetes. Type 2 diabetes occurs when thegators have been making slow, but steady, progress body cannot use insulin effectively. This is calledon experimental strategies for pancreatic transplan- insulin resistance and the result is the same as withtation and islet cell replacement. Now, researchers type 1 diabetes—a build up of glucose in the blood.have turned their attention to adult stem cells thatappear to be precursors to islet cells and embryonic There is currently no cure for diabetes. People withstem cells that produce insulin. type 1 diabetes must take insulin several times a day and test their blood glucose concentration three to four times a day throughout their entire lives. FrequentINTRODUCTION monitoring is important because patients who keepFor decades, diabetes researchers have been their blood glucose concentrations as close tosearching for ways to replace the insulin-producing normal as possible can significantly reduce many ofcells of the pancreas that are destroyed by a the complications of diabetes, such as retinopathypatient’s own immune system. Now it appears that (a disease of the small blood vessels of the eyethis may be possible. Each year, diabetes affects which can lead to blindness) and heart disease,more people and causes more deaths than breast that tend to develop over time. People with type 2cancer and AIDS combined. Diabetes is the seventh diabetes can often control their blood glucoseleading cause of death in the United States today, concentrations through a combination of diet,with nearly 200,000 deaths reported each year. The exercise, and oral medication. Type 2 diabetesAmerican Diabetes Association estimates that nearly often progresses to the point where only insulin16 million people, or 5.9 percent of the United States therapy will control blood glucose concentrations.population, currently have diabetes. Each year, approximately 1,300 people with type 1Diabetes is actually a group of diseases character- diabetes receive whole-organ pancreas transplants.ized by abnormally high levels of the sugar glucose in After a year, 83 percent of these patients, onthe bloodstream. This excess glucose is responsible average, have no symptoms of diabetes and dofor most of the complications of diabetes, which not have to take insulin to maintain normal glucoseinclude blindness, kidney failure, heart disease, stroke, concentrations in the blood. However, the demandneuropathy, and amputations. Type 1 diabetes, also for transplantable pancreases outweighs theirknown as juvenile-onset diabetes, typically affects availability. To prevent the body from rejecting thechildren and young adults. Diabetes develops when transplanted pancreas, patients must take powerfulthe body’s immune system sees its own cells as drugs that suppress the immune system for their entireforeign and attacks and destroys them. As a result, lives, a regimen that makes them susceptible to a 67
  • Stem Cells and Diabeteshost of other diseases. Many hospitals will not perform DEVELOPMENT OF THE PANCREASa pancreas transplant unless the patient also needs akidney transplant. That is because the risk of infection Before discussing cell-based therapies for diabetes,due to immunosuppressant therapy can be a greater it is important to understand how the pancreashealth threat than the diabetes itself. But if a patient is develops. In mammals, the pancreas contains threealso receiving a new kidney and will require immuno- classes of cell types: the ductal cells, the acinar cells,suppressant drugs anyway, many hospitals will and the endocrine cells. The endocrine cells produceperform the pancreas transplant. the hormones glucagon, somatostatin, pancreatic polypeptide (PP), and insulin, which are secreted intoOver the past several years, doctors have attempted the blood stream and help the body regulate sugarto cure diabetes by injecting patients with pancreatic metabolism. The acinar cells are part of the exocrineislet cells—the cells of the pancreas that secrete system, which manufactures digestive enzymes, andinsulin and other hormones. However, the requirement ductal cells from the pancreatic ducts, whichfor steroid immunosuppressant therapy to prevent connect the acinar cells to digestive organs.rejection of the cells increases the metabolicdemand on insulin-producing cells and eventually In humans, the pancreas develops as an outgrowththey may exhaust their capacity to produce insulin. of the duodenum, a part of the small intestine. TheThe deleterious effect of steroids is greater for islet cells of both the exocrine system—the acinar cells—cell transplants than for whole-organ transplants. As and of the endocrine system—the islet cells—seema result, less than 8 percent of islet cell transplants to originate from the ductal cells during develop-performed before last year had been successful. ment. During development these endocrine cells emerge from the pancreatic ducts and formMore recently, James Shapiro and his colleagues in aggregates that eventually form what is known asEdmonton, Alberta, Canada, have developed an Islets of Langerhans. In humans, there are four typesexperimental protocol for transplanting islet cells that of islet cells: the insulin-producing beta cells; theinvolves using a much larger amount of islet cells and alpha cells, which produce glucagon; the delta cells,a different type of immunosuppressant therapy. In a which secrete somatostatin; and the PP-cells, whichrecent study, they report that [17], seven of seven produce pancreatic polypeptide. The hormonespatients who received islet cell transplants no longer released from each type of islet cell have a role inneeded to take insulin, and their blood glucose regulating hormones released from other islet cells. Inconcentrations were normal a year after surgery. the human pancreas, 65 to 90 percent of islet cellsThe success of the Edmonton protocol is now being are beta cells, 15 to 20 percent are alpha-cells, 3 totested at 10 centers around the world. 10 percent are delta cells, and one percent is PP cells. Acinar cells form small lobules contiguous withIf the success of the Edmonton protocol can be the ducts (see Figure 7.1. Insulin Production in theduplicated, many hurdles still remain in using this Human Pancreas). The resulting pancreas is aapproach on a wide scale to treat diabetes. First, combination of a lobulated, branched acinar glanddonor tissue is not readily available. Islet cells used in that forms the exocrine pancreas, and, embeddedtransplants are obtained from cadavers, and the pro- in the acinar gland, the Islets of Langerhans, whichcedure requires at least two cadavers per transplant. constitute the endocrine pancreas.The islet cells must be immunologically compatible,and the tissue must be freshly obtained—within eight During fetal development, new endocrine cellshours of death. Because of the shortage of organ appear to arise from progenitor cells in the pancre-donors, these requirements are difficult to meet and atic ducts. Many researchers maintain that somethe waiting list is expected to far exceed available sort of islet stem cell can be found intermingled withtissue, especially if the procedure becomes widely ductal cells during fetal development and that theseaccepted and available. Further, islet cell transplant stem cells give rise to new endocrine cells as therecipients face a lifetime of immunosuppressant fetus develops. Ductal cells can be distinguishedtherapy, which makes them susceptible to other from endocrine cells by their structure and by theserious infections and diseases. genes they express. For example, ductal cells typically express a gene known as cytokeratin-9 68
  • Stem Cells and Diabetes Human pancreas Islet of Langerhans Beta cell Muscle fiber Blood vessel Insulin© 2001 Terese Winslow, Lydia Kibiuk Glucose Figure 7.1. Insulin Production in the Human Pancreas. The pancreas is located in the abdomen, adjacent to the duodenum (the first portion of the small intestine). A cross-section of the pancreas shows the islet of Langerhans which is the functional unit of the endocrine pancreas. Encircled is the beta cell that synthesizes and secretes insulin. Beta cells are located adjacent to blood vessels and can easily respond to changes in blood glucose concentration by adjusting insulin production. Insulin facilitates uptake of glucose, the main fuel source, into cells of tissues such as muscle. 69
  • Stem Cells and Diabetes(CK-9), which encodes a structural protein. Beta islet Isolated beta cells, as well as islet clusters withcells, on the other hand, express a gene called lower-than-normal amounts of non-beta cells, do notPDX-1, which encodes a protein that initiates release insulin in this biphasic manner. Instead insulintranscription from the insulin gene. These genes, is released in an all-or-nothing manner, with nocalled cell markers, are useful in identifying particular fine-tuning for intermediate concentrations of glucosecell types. in the blood [5, 18]. Therefore, many researchers believe that it will be preferable to develop a systemFollowing birth and into adulthood, the source of new in which stem or precursor cell types can beislet cells is not clear, and some controversy exists cultured to produce all the cells of the islet cluster inover whether adult stem cells exist in the pancreas. order to generate a population of cells that will beSome researchers believe that islet stem cell-like cells able to coordinate the release of the appropriatecan be found in the pancreatic ducts and even in amount of insulin to the physiologically relevantthe islets themselves. Others maintain that the ductal concentrations of glucose in the blood.cells can differentiate into islet precursor cells, whileothers hold that new islet cells arise from stem cells inthe blood. Researchers are using several approaches FETAL TISSUE AS SOURCE FORfor isolating and cultivating stem cells or islet pre- ISLET CELLScursor cells from fetal and adult pancreatic tissue. In Several groups of researchers are investigating theaddition, several new promising studies indicate that use of fetal tissue as a potential source of islet pro-insulin-producing cells can be cultivated from embry- genitor cells. For example, using mice, researchersonic stem cell lines. have compared the insulin content of implants from several sources of stem cells—fresh human fetalDEVELOPMENT OF CELL-BASED pancreatic tissue, purified human islets, and culturedTHERAPIES FOR DIABETES islet tissue [2]. They found that insulin content was initially higher in the fresh tissue and purified islets.In developing a potential therapy for patients with However, with time, insulin concentration decreaseddiabetes, researchers hope to develop a system that in the whole tissue grafts, while it remained the samemeets several criteria. Ideally, stem cells should be in the purified islet grafts. When cultured islets wereable to multiply in culture and reproduce themselves implanted, however, their insulin content increasedexactly. That is, the cells should be self-renewing. over the course of three months. The researchersStem cells should also be able to differentiate in concluded that precursor cells within the culturedvivo to produce the desired kind of cell. For diabetes islets were able to proliferate (continue to replicate)therapy, it is not clear whether it will be desirable to and differentiate (specialize) into functioning isletproduce only beta cells—the islet cells that manufac- tissue, but that the purified islet cells (alreadyture insulin—or whether other types of pancreatic islet differentiated) could not further proliferate whencells are also necessary. Studies by Bernat Soria and grafted. Importantly, the researchers found, however,colleagues, for example, indicate that isolated beta that it was also difficult to expand cultures of fetal isletcells—those cultured in the absence of the other progenitor cells in culture [7].types of islet cells—are less responsive to changes inglucose concentration than intact islet clusters madeup of all islet cell types. Islet cell clusters typically ADULT TISSUE AS SOURCE FORrespond to higher-than-normal concentrations of ISLET CELLSglucose by releasing insulin in two phases: a quick Many researchers have focused on culturing islet cellsrelease of high concentrations of insulin and a slower from human adult cadavers for use in developingrelease of lower concentrations of insulin. In this transplantable material. Although differentiated betamanner the beta cells can fine-tune their response cells are difficult to proliferate and culture, someto glucose. Extremely high concentrations of glucose researchers have had success in engineering suchmay require that more insulin be released quickly, cells to do this. For example, Fred Levine and hiswhile intermediate concentrations of glucose can be colleagues at the University of California, San Diego,handled by a balance of quickly and slowly have engineered islet cells isolated from humanreleased insulin. cadavers by adding to the cells’ DNA special genes 70
  • Stem Cells and Diabetesthat stimulate cell proliferation. However, because established cells lines that can grow indefinitely.once such cell lines that can proliferate in culture are However the cells can be expanded. According toestablished, they no longer produce insulin. The cell the researchers, it might be possible in principle to dolines are further engineered to express the beta islet a biopsy and remove duct cells from a patient andcell gene, PDX-1, which stimulates the expression of then proliferate the cells in culture and give thethe insulin gene. Such cell lines have been shown to patient back his or her own islets. This would work withpropagate in culture and can be induced to patients who have type 1 diabetes and who lackdifferentiate to cells, which produce insulin. When functioning beta cells, but their duct cells remaintransplanted into immune-deficient mice, the cells intact. However, the autoimmune destruction wouldsecrete insulin in response to glucose. The researchers still be a problem and potentially lead to destructionare currently investigating whether these cells will of these transplanted cells [3]. Type 2 diabetesreverse diabetes in an experimental diabetes patients might benefit from the transplantation ofmodel in mice [6, 8]. cells expanded from their own duct cells since they would not need any immunosuppression. However,These investigators report that these cells do not many researchers believe that if there is a geneticproduce as much insulin as normal islets, but it is component to the death of beta cells, then betawithin an order of magnitude. The major problem in cells derived from ductal cells of the same individualdealing with these cells is maintaining the delicate would also be susceptible to autoimmune attack.balance between growth and differentiation. Cellsthat proliferate well do not produce insulin efficiently, Some researchers question whether the ductal cellsand those that do produce insulin do not proliferate are indeed undergoing a dedifferentiation or whetherwell. According to the researchers, the major issue is a subset of stem-like or islet progenitors populate thedeveloping the technology to be able to grow large pancreatic ducts and may be co-cultured alongnumbers of these cells that will reproducibly produce with the ductal cells. If ductal cells die off but isletnormal amounts of insulin [9]. precursors proliferate, it is possible that the islet precursor cells may overtake the ductal cells inAnother promising source of islet progenitor cells lies culture and make it appear that the ductal cellsin the cells that line the pancreatic ducts. Some are dedifferentiating into stem cells. According toresearchers believe that multipotent (capable of Bonner-Weir, both dedifferentiated ductal cells andforming cells from more than one germ layer) stem islet progenitor cells may occur in pancreatic ducts.cells are intermingled with mature, differentiated ductcells, while others believe that the duct cells Ammon Peck of the University of Florida, Vijayakumarthemselves can undergo a differentiation, or a Ramiya of Ixion Biotechnology in Alachua, FL, andreversal to a less mature type of cell, which can then their colleagues [13, 14] have also cultured cells fromdifferentiate into an insulin-producing islet cell. the pancreatic ducts from both humans and mice. Last year, they reported that pancreatic ductalSusan Bonner-Weir and her colleagues reported last epithelial cells from adult mice could be cultured toyear that when ductal cells isolated from adult yield islet-like structures similar to the cluster of cellshuman pancreatic tissue were cultured, they could found by Bonner-Weir. Using a host of islet-cell markersbe induced to differentiate into clusters that they identified cells that produced insulin, glucagon,contained both ductal and endocrine cells. Over somatostatin, and pancreatic polypeptide. When thethe course of three to four weeks in culture, the cells cells were implanted into diabetic mice, the diabetessecreted low amounts of insulin when exposed to was reversed.low concentrations of glucose, and higher amountsof insulin when exposed to higher glucose Joel Habener has also looked for islet-like stem cellsconcentrations. The researchers have determined from adult pancreatic tissue. He and his colleaguesby immunochemistry and ultrastructural analysis that have discovered a population of stem-like cells withinthese clusters contain all of the endocrine cells of both the adult pancreas islets and pancreatic ducts.the islet [4]. These cells do not express the marker typical of ductal cells, so they are unlikely to be ductal cells,Bonner-Weir and her colleagues are working with according to Habener. Instead, they express a markerprimary cell cultures from duct cells and have not called nestin, which is typically found in developing 71
  • Stem Cells and Diabetesneural cells. The nestin-positive cells do not express into insulin-producing cells [19]. Bernat Soria and hismarkers typically found in mature islet cells. However, colleagues at the Universidad Miguel Hernandez independing upon the growth factors added, the cells San Juan, Alicante, Spain, added DNA containingcan differentiate into different types of cells, including part of the insulin gene to embryonic cells from mice.liver, neural, exocrine pancreas, and endocrine pan- The insulin gene was linked to another gene thatcreas, judged by the markers they express, and can rendered the mice resistant to an antibiotic drug. Bybe maintained in culture for up to eight months [20]. growing the cells in the presence of an antibiotic, only those cells that were activating the insulinEMBRYONIC STEM CELLS promoter were able to survive. The cells were cloned and then cultured under varying conditions. CellsThe discovery of methods to isolate and grow human cultured in the presence of low concentrations ofembryonic stem cells in 1998 renewed the hopes of glucose differentiated and were able to respond todoctors, researchers, and diabetes patients and their changes in glucose concentration by increasingfamilies that a cure for type 1 diabetes, and perhaps insulin secretion nearly sevenfold. The researcherstype 2 diabetes as well, may be within striking then implanted the cells into the spleens ofdistance. In theory, embryonic stem cells could be diabetic mice and found that symptoms ofcultivated and coaxed into developing into the diabetes were reversed.insulin-producing islet cells of the pancreas. With aready supply of cultured stem cells at hand, the Manfred Ruediger of Cardion, Inc., in Erkrath,theory is that a line of embryonic stem cells could Germany, is using the approach developed by Soriabe grown up as needed for anyone requiring a and his colleagues to develop insulin-producingtransplant. The cells could be engineered to avoid human cells derived from embryonic stem cells. Byimmune rejection. Before transplantation, they could using this method, the non-insulin-producing cells willbe placed into nonimmunogenic material so that be killed off and only insulin-producing cells shouldthey would not be rejected and the patient would survive. This is important in ensuring that undifferenti-avoid the devastating effects of immunosuppressant ated cells are not implanted that could give rise todrugs. There is also some evidence that differentiated tumors [15]. However, some researchers believe thatcells derived from embryonic stem cells might be it will be important to engineer systems in which allless likely to cause immune rejection (see Chapter the components of a functioning pancreatic islet are10. Assessing Human Stem Cell Safety). Although allowed to develop.having a replenishable supply of insulin-producing Recently Ron McKay and his colleagues described acells for transplant into humans may be a long way series of experiments in which they induced mouseoff, researchers have been making remarkable embryonic cells to differentiate into insulin-secretingprogress in their quest for it. While some researchers structures that resembled pancreatic islets [10].have pursued the research on embryonic stem cells, McKay and his colleagues started with embryonicother researchers have focused on insulin-producing stem cells and let them form embryoid bodies—anprecursor cells that occur naturally in adult and aggregate of cells containing all three embryonicfetal tissues. germ layers. They then selected a population of cellsSince their discovery three years ago, several teams from the embryoid bodies that expressed the neuralof researchers have been investigating the possibility marker nestin (see Appendix B. Mouse Embryonicthat human embryonic stem cells could be Stem Cells). Using a sophisticated five-stage culturingdeveloped as a therapy for treating diabetes. technique, the researchers were able to induce theRecent studies in mice show that embryonic stem cells to form islet-like clusters that resembled thosecells can be coaxed into differentiating into found in native pancreatic islets. The cells respondedinsulin-producing beta cells, and new reports indicate to normal glucose concentrations by secreting insulin,that this strategy may be possible using human although insulin amounts were lower than thoseembryonic cells as well. secreted by normal islet cells (see Figure 7.2. Development of Insulin-Secreting Pancreatic-LikeLast year, researchers in Spain reported using mouse Cells From Mouse Embryonic Stem Cells). Whenembryonic stem cells that were engineered to allow the cells were injected into diabetic mice, theyresearchers to select for cells that were differentiating 72
  • Stem Cells and Diabetes Mouse blastocyst Inner cell mass Mouse embryonic cells Pancreatic islet-like cells secrete Differentiation insulin Diabetic mouse Pancreatic islet-like cells© 2001 Terese Winslow, Lydia Kibiuk Insulin secreted Figure 7.2. Development of Insulin-Secreting Pancreatic-Like Cells From Mouse Embryonic Stem Cells. Mouse embryonic stem cells were derived from the inner cell mass of the early embryo (blastocyst) and cultured under specific conditions. The embryonic stem cells (in blue) were then expanded and differentiated. Cells with markers consistent with islet cells were selected for further differentiation and characterization. When these cells (in purple) were grown in culture, they spontaneously formed three-dimentional clusters similar in structure to normal pancreatic islets. The cells produced and secreted insulin. As depicted in the chart, the pancreatic islet-like cells showed an increase in release of insulin as the glucose concentration of the culture media was increased. When the pancreatic islet-like cells were implanted in the shoulder of diabetic mice, the cells became vascularized, synthesized insulin, and maintained physical characteristics similar to pancreatic islets. 73
  • Stem Cells and Diabetessurvived, although they did not reverse the symptoms spontaneously form embryoid bodies contained aof diabetes. significant percentage of cells that express insulin. Based on the binding of antibodies to the insulinAccording to McKay, this system is unique in that the protein, Itskovitz-Eldor estimates that 1 to 3 percentembryonic cells form a functioning pancreatic islet, of the cells in embryoid bodies are insulin-producingcomplete with all the major cell types. The cells beta-islet cells. The researchers also found that cellsassemble into islet-like structures that contain another in the embryoid bodies express glut-2 and islet-specif-layer, which contains neurons and is similar to intact ic glucokinase, genes important for beta cell functionislets from the pancreas [11]. Several research groups and insulin secretion. Although the researchers didare trying to apply McKay’s results with mice to not measure a time-dependent response to glucose,induce human embryonic stem cells to differentiate they did find that cells cultured in the presence ofinto insulin-producing islets. glucose secrete insulin into the culture medium. TheRecent research has also provided more evidence researchers concluded that embryoid bodies containthat human embryonic cells can develop into cells a subset of cells that appear to function as beta cellsthat can and do produce insulin. Last year, Melton, and that the refining of culture conditions may soonNissim Benvinisty of the Hebrew University in yield a viable method for inducing the differentiationJerusalem, and Josef Itskovitz-Eldor of the Technion in of beta cells and, possibly, pancreatic islets.Haifa, Israel, reported that human embryonic stem Taken together, these results indicate that the devel-cells could be manipulated in culture to express the opment of a human embryonic stem cell system thatPDX-1 gene, a gene that controls insulin transcription can be coaxed into differentiating into functioning[16]. In these experiments, researchers cultured insulin-producing islets may soon be possible.human embryonic stem cells and allowed them tospontaneously form embryoid bodies (clumps ofembryonic stem cells composed of many types of FUTURE DIRECTIONScells from all three germ layers). The embryoid bodies Ultimately, type 1 diabetes may prove to bewere then treated with various growth factors, includ- especially difficult to cure, because the cells areing nerve growth factor. The researchers found that destroyed when the body’s own immune systemboth untreated embryoid bodies and those treated attacks and destroys them. This autoimmunity mustwith nerve growth factor expressed PDX-1. Embryonic be overcome if researchers hope to use transplantedstem cells prior to formation of the aggregated cells to replace the damaged ones. Manyembryoid bodies did not express PDX-1. Because researchers believe that at least initially, immunosup-expression of the PDX-1 gene is associated with the pressive therapy similar to that used in the Edmontonformation of beta islet cells, these results suggest that protocol will be beneficial. A potential advantage ofbeta islet cells may be one of the cell types that embryonic cells is that, in theory, they could bespontaneously differentiate in the embryoid bodies. engineered to express the appropriate genes thatThe researchers now think that nerve growth would allow them to escape or reduce detection byfactor may be one of the key signals for inducing the immune system. Others have suggested that athe differentiation of beta islet cells and can be technology should be developed to encapsulate orexploited to direct differentiation in the laboratory. embed islet cells derived from islet stem or progenitorComplementing these findings is work done by Jon cells in a material that would allow small moleculesOdorico of the University of Wisconsin in Madison such as insulin to pass through freely, but would notusing human embryonic cells of the same source. allow interactions between the islet cells and cells ofIn preliminary findings, he has shown that human the immune system. Such encapsulated cells couldembryonic stem cells can differentiate and express secrete insulin into the blood stream, but remainthe insulin gene [12]. inaccessible to the immune system.More recently, Itskovitz-Eldor and his Technion Before any cell-based therapy to treat diabetescolleagues further characterized insulin-producing makes it to the clinic, many safety issues must becells in embryoid bodies [1]. The researchers found addressed (see Chapter 10. Assessing Human Stemthat embryonic stem cells that were allowed to Cell Safety). A major consideration is whether any 74
  • Stem Cells and Diabetesprecursor or stem-like cells transplanted into the body 7. Hayek, A., personal communication.might revert to a more pluripotent state and induce 8. Itkin-Ansari, P Demeterco, C., Bossie, S., Dufayet de la Tour, .,the formation of tumors. These risks would seemingly D., Beattie, G.M., Movassat, J., Mally, M.I., Hayek, A., andbe lessened if fully differentiated cells are used in Levine, F. (2001). PDX-1 and cell-cell contact act in synergy to promote d-cell development in a human pancreatictransplantation. endocrine precursor cell line. Mol. Endocrinol. 14, 814-822.But before any kind of human islet-precursor cells 9. Levine, F., personal communication.can be used therapeutically, a renewable source of 10. Lumelsky, N., Blondel, O., Laeng, P Velasco, I., Ravin, R., .,human stem cells must be developed. Although and McKay, R. (2001). Differentiation of Embryonic Stemmany progenitor cells have been identified in adult Cells to Insulin-Secreting Structures Similiar to Pancreatictissue, few of these cells can be cultured for multiple Islets. Science. 292, 1389-1394.generations. Embryonic stem cells show the greatest 11. McKay, R., personal communicationpromise for generating cell lines that will be free of 12. Odorico, J. S., personal communication.contaminants and that can self renew. However,most researchers agree that until a therapeutically 13. Peck, A., personal communication.useful source of human islet cells is developed, all 14. Ramiya, V. K., personal communication.avenues of research should be exhaustivelyinvestigated, including both adult and embryonic 15. Ruediger, M., personal communication.sources of tissue. 16. Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D., and Benvenisty, N. (2000). Effects of eight growth factors on the differentiation of cells derived from human embryonic stemREFERENCES cells. Proc. Natl. Acad. Sci. U. S. A. 97, 11307-11312. 1. Assady, S., Maor, G., Amit, M., Itskovitz-Eldor, J., Skorecki, 17. Shapiro, J., Lakey, J.R.T., Ryan, E.A., Korbutt, G.S., Toth, E., K.L., and Tzukerman, M. (2001). Insulin production by Warnock, G.L., Kneteman, N.M., and Rajotte, R.V. (2000). human embryonic stem cells. Diabetes. 50. Islet transplantation in seven patients with type 1 diabetes http://www.diabetes.org/Diabetes_Rapids/Suheir_Assady_ mellitus using a glucocorticoid-free immunosuppressive 06282001.pdf regimen. N. Engl. J. Med. 343, 230-238. 2. Beattie, G.M., Otonkoski, T., Lopez, A.D., and Hayek, A. 18. Soria, B., Martin, F., Andreu, E., Sanchez-Andrés, J.V., (1997). Functional beta-cell mass after transplantation of Nacher, V., and Montana, E. (1996). Diminished fraction of human fetal pancreatic cells: differentiation or prolifera- blockable ATP-sensitive K+ channels in islets transplanted tion? Diabetes. 46, 244-248. into diabetic mice. Diabetes. 45, 1755-1760. 3. Bonner-Weir, S., personal communication. 19. Soria, B., Roche, E., Berná, G., Leon-Quinto, T., Reig, J.A., 4. Bonner-Weir, S., Taneja, M., Weir, G.C., Tatarkiewicz, K., Song, and Martin, F. (2000). Insulin-secreting cells derived from K.H., Sharma, A., and ONeil, J.J. (2000). In vitro cultivation embryonic stem cells normalize glycemia in streptozotocin- of human islets from expanded ductal tissue. Proc. Natl. induced diabetic mice. Diabetes. 49, 157-162. Acad. Sci. U. S. A. 97, 7999-8004. 20. Zulewski, H., Abraham, E.J., Gerlach, M.J., Daniel, P .B., 5. Bosco, D. and Meda, P (1997). Reconstructing islet function . Moritz, W., Muller, B., Vallejo, M., Thomas, M.K., and in vitro. Adv. Exp. Med. Biol. 426, 285-298. Habener, J.F. (2001). Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo 6. Dufayet de la Tour, D., Halvorsen, T., Demeterco, C., into pancreatic endocrine, exocrine, and hepatic pheno- Tyrberg, B., Itkin-Ansari, P Loy, M., Yoo, S.J., Hao, S., Bossie, ., types. Diabetes. 50, 521-533. S., and Levine, F. (2001). b-cell differentiation from a human pancreatic cell line in vitro and in vivo. Mol. Endocrinol. 15, 476-483. 75
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  • 8. REBUILDING THE NERVOUS SYSTEM WITH STEM CELLSToday, most treatments for damage to the brain or The discovery of a regenerative capacity in the adultspinal cord aim to relieve symptoms and limit further central nervous system holds out the promise that itdamage. But recent research into the regeneration may eventually be possible to repair damage frommechanisms of the central nervous system, including terrible degenerative diseases such as Parkinson’sthe discovery of stem cells in the adult brain that can Disease and amyotrophic lateral sclerosis (ALS, alsogive rise to new neurons and neural support cells, known as Lou Gehrig’s disease), as well as from brainhas raised hopes that researchers can find ways to and spinal cord injuries resulting from stroke or traumaactually repair central nervous system damage. (see Box 8.1. Early Research Shows Stem Cells CanResearch on stem cells in nervous system disorders is Improve Movement in Paralyzed Mice).one of the few areas in which there is evidence thatcell-replacement therapy can restore lost function. Researchers are pursuing two fundamental strategies to exploit this discovery. One is to grow differentiated cells in a laboratory dish that are suitable for implan-STEM CELLS BRING NEW tation into a patient by starting with undifferentiatedSTRATEGIES FOR DEVELOPING neural cells. The idea is either to treat the cells in cul-REPLACEMENT NEURONS ture to nudge them toward the desired differentiated neuronal cell type before implantation, or to implantJust a decade ago, neuroscience textbooks held them directly and rely on signals inside the body tothat neurons in the adult human brain and spinal direct their maturation into the right kind of brain cell.cord could not regenerate. Once dead, it was A variety of stem cells might be used for this task,thought, central nervous system neurons were gone including so-called “neural precursor cells” that arefor good. Because rebuilding nervous tissue seemed inwardly committed to differentiating into a particularout of the question, research focused almost entirely cell type but are outwardly not yet changed oron therapeutic approaches to limiting further damage. pluripotent embryonic stem cells—cells derived fromThat dogma that brain tissue could not be regenerated a very early stage human embryo that retain theis history. In the mid-1990s, neuroscientists learned that capacity to become any cell type in the body andsome parts of the adult human brain do, in fact, gener- that can be maintained in culture for a very longate new neurons, at least under certain circumstances. time without differentiating.Moreover, they found that the new neurons arise from The other repair strategy relies on finding growth"neural stem cells” in the fetal as well as the adult brain hormones and other “trophic factors”—growth fac-(see Chapter 4. The Adult Stem Cell). These undifferenti- tors, hormones, and other signaling molecules thatated cells resemble cells in a developing fetus that give help cells survive and grow—that can fire up arise to the brain and spinal cord. The researchers also patient’s own stem cells and endogenous repairfound that these neural stem cells could generate mechanisms, to allow the body to cope with dam-many, if not all, types of cells found in the brain. This age from disease or injury. Researchers are vigorouslyincludes neurons—the main message carriers in the pursuing both strategies to find therapies for centralnervous system, which use long, thin projections called nervous system disorders that involve cell death, butaxons to transmit signals over long distances—as well a great deal more basic research must be carriedas crucial neural-support cells called oligodendrocytes out before effective new therapies emerge.and astrocytes (see Figure 8.1. The Neuron). 77
  • Rebuilding the Nervous System with Stem Cells Dendrites Cell Body Axon Oligodendrocyte Myelin sheath Synaptic terminal Receptor Postsynaptic membrane Neurotransmitter Synapse release© 2001 Terese Winslow Figure 8.1. The Neuron. When sufficient neurotransmitters cross synapses and bind receptors on the neuronal cell body and dendrites, the neuron sends an electrical signal down its axon to synaptic terminals, which in turn release neurotransmitters into the synapse that affects the following neuron. The brain neurons that die in Parkinsons Disease release the transmitter dopamine. Oligodendrocytes supply the axon with an insulating myelin sheath. 78
  • Rebuilding the Nervous System with Stem Cells Box 8.1 Early Research Shows Stem Cells Can Improve Movement in Paralyzed Mice Researchers at Johns Hopkins University recently reported To test this idea, the researchers selected from laborato- preliminary evidence that cells derived from embryonic ry culture dishes barely differentiated embryonic germs stem cells can restore movement in an animal model cells that displayed the molecular markers of neural of amyotrophic lateral sclerosis (ALS) [1]. This degenera- stem cells, including the proteins nestin and neuron spe- tive disorder, also called as Lou Gehrig’s disease, pro- cific enolase. They grew these cells in large quantities gressively destroys special nerves found in the spinal and injected them into the fluid surrounding the spinal cord, known as motor neurons, that control movement. cords of partially paralyzed, Sindbis-virus-treated rats. Patients with ALS develop increasing muscle weakness The response was impressive. Three months after the over months to years, which ultimately leads to paralysis injections, many of the treated rats were able to move and death. The cause is largely unknown, and there are their hind limbs and walk, albeit clumsily, while the rats no effective treatments. that did not receive cell injections remained paralyzed. In this new study, the researchers used a rat model of Moreover, at autopsy the researchers found that cells ALS to test for possible nerve cell- restoring properties of derived from human embryonic germ cells had migrat- stem cells. The rats were exposed to Sindbis virus, which ed throughout the spinal fluid and continued to devel- infects the central nervous system and destroys the op, displaying both the shape and molecular markers motor neurons in the spinal cord. Rats that survive are characteristic of mature motor neurons. The researchers left with paralyzed muscles in their hindquarters and are quick to caution that their results are preliminary, weakened back limbs. Scientists assess the degree of and that they do not know for certain whether the treat- impairment by measuring the rats’ movement, quantify- ment helped the paralyzed rats because new neurons ing electrical activity in the nerves serving the back took the place of the old, or because trophic factors limbs, and visually judging the extent of nerve damage from the injected cells facilitated the recovery of the through a microscope. rats’ remaining nerve cells and helped the rats improve in their ability to use their hind limbs. Nor do they know The researchers wanted to see whether stem cells could how well this strategy will translate into a therapy for restore nerves and improve mobility in rats. Because sci- human neurodegenerative diseases like ALS. And they entists have had difficulty sustaining stem cell lines emphasize that there are many hurdles to cross before derived from rat embryos, the investigators conducted the use of stem cells to repair damaged motor neurons their experiments with embryonic germ cells that John in patients can be considered. Nevertheless, researchers Gearhart and colleagues isolated from human fetal tis- are excited about these results, which, if confirmed, sue in 1998. These cells can produce unchanged would represent a major step toward using specialized copies of themselves when maintained in culture, and stem cells from embryonic and fetal tissue sources to they form into clumps called embryoid bodies. Under restore nervous system function. certain conditions, research has shown that the cells in the embryoid bodies begin to look and function like REFERENCES neurons when subjected to specific laboratory condi- 1. Kerr, D.A., Llado, J., Shamblott, M., Maragakis, N., Irani, D.N., tions [2]. The researchers had an idea that these embry- Dike, S., Sappington, A., Gearhart, J., and Rothstein, J. (2001). oid body cells in their nonspecialized state might Human embryonic germ cell derivatives facillitate motor become specialized as replacement neurons if placed recovery of rats with diffuse motor neuron injury. into the area of the damaged spinal cord. So they 2. Shamblott, M.J., Axelman, J., Wang, S., Bugg, E.M., Littlefield, carefully prepared cells from the embryoid bodies and J.W., Donovan, P Blumenthal, P .J., .D., Huggins, G.R., and injected them into the fluid surrounding the spinal cord Gearhart, J.D. (1998). Derivation of pluripotent stem cells of the paralyzed rats that had their motor neurons from cultured human primordial germ cells. Proc. Natl. Acad. Sci. U. S. A. 95, 13726-13731. destroyed by the Sindbis virus.MULTIPLE APPROACHES FOR USING tem. As is the case with other disorders, both the cell-STEM CELLS IN PARKINSON’S implantation and the trophic-factor strategies are under active development. Both approaches areDISEASE RESEARCH promising. This is especially true of cell implantation,Efforts to develop stem cell based therapies for which involves using primary tissue transplantedParkinson’s Disease provide a good example of directly form developing fetal brain tissue. Parkinson’sresearch aimed at rebuilding the central nervous sys- is a progressive movement disorder that usually strikes 79
  • Rebuilding the Nervous System with Stem Cells after age 50. Symptoms often begin with an uncon- (see Figure 8.2. Neuronal Pathways that Degenerate trollable hand tremor, followed by increasing rigidity, in Parkinson’s Disease). These “nigro-striatal” neurons difficulty walking, and trouble initiating voluntary release the chemical transmitter dopamine onto their movement. The symptoms result from the death of a target neurons in the striatum. One of dopamine’s particular set of neurons deep in the brain. major roles is to regulate the nerves that control body movement. As these cells die, there is less dopamine The neurons that die in Parkinson’s Disease connect a produced, leading to the movement difficulties char- structure in the brain called the substantia nigra to acteristic of Parkinson’s. At this point, no one knows for another structure called the striatum, composed certain why the neurons die. of the caudate nucleus and the putamen Lateral ventricles Striatum } Caudate nucleus Putamen Nigro-striatal neurons Substantia nigra© 2001 Terese Winslow, Lydia Kibiuk Figure 8.2. Neuronal Pathways that Degenerate in Parkinsons Disease. Signals that control body movements travel along neurons that project from the substantia nigra to the caudate nucleus and putamen (collectively called the striatum). These “nigro-striatal” neurons release dopamine at their stargets in the striatum. In Parkinsons patients, dopamine neurons in the nigro-striatal pathway degenerate for unknown reasons. 80
  • Rebuilding the Nervous System with Stem CellsMost patients suffering from Parkinson’s Disease are transplanted directly from the developing nigro-striataltreated with a drug called levodopa, which the brain pathways of embryonic mice into the anterior cham-converts into dopamine. It initially helps most patients, ber of an adult rat’s eye continues to mature into fullybut unfortunately, side effects of the drug increase developed dopamine neurons [3]. By the early 1980sover time and its effectiveness wanes. This leaves Anders Bjorkland and others had shown that trans-Parkinson’s patients and their doctors fighting a long, plantation of fetal tissue into the damaged areas ofuphill battle to balance medication with side effects the brains of rats and monkeys used as models of theto maintain function. In the end, many patients are disease could reverse their Parkinson’s-like symptoms.utterly helpless. Subsequently, researchers refined their surgical tech- niques and showed that functional recovery dependsFETAL TISSUE TRANSPLANTS IN on the implanted neurons growing and making func-PARKINSON’S DISEASE RESEARCH tional connections at the appropriate brain loca- tions—essentially finishing their maturation by integrat-The idea of growing dopamine cells in the laboratory ing into the adult host brain [3].to treat Parkinson’s is the most recent step in the longhistory of cell or tissue transplantation to reverse this The promising animal results led to human trials indevastating disease. The concept was, and still is, several centers worldwide, starting in the mid-1980s.straightforward: implant cells into the brain that can Using tissue removed from a fetus electively abortedreplace the lost dopamine-releasing neurons. seven to nine weeks after conception, these earlyAlthough conceptually straightforward, this is not an human transplantation studies showed encouraging,easy task. Fully developed and differentiated but inconsistent, benefit to patients. Although not alldopamine neurons do not survive transplantation, so patients improved, in the best cases patients receiv-direct transplantation of fully developed brain tissue ing fetal tissue transplants showed a clear reductionfrom cadavers, for example, is not an option. in the severity of their symptoms. Also, researchersMoreover, full functional recovery depends on more could measure an increase in dopamine neuronthan cell survival and dopamine release; transplant- function in the striatum of these patients by using aed cells must also make appropriate connections brain-imaging method called positron emissionwith their normal target neurons in the striatum. tomography (PET) (see Figure 8.3. Positron Emission Tomography [PET] images from a Parkinson’s patientOne of the first attempts at using cell transplantation before and after fetal tissue transplantation). Also,in humans was tried in the 1980s. This surgical autopsies done on the few patients who died fromapproach involved the transplantation of dopamine- causes unrelated to either Parkinson’s or the surgeryproducing cells found in the adrenal glands, which sit revealed a robust survival of the grafted neurons.atop the kidneys in the abdomen. Neurosurgeons in Moreover, the grafted neurons sent outgrowths fromMexico reported that they had achieved dramatic the cell body that integrated well into the normalimprovement in Parkinson’s patients by transplanting target areas in the striatum.dopamine-producing chromaffin cells from severalpatients’ own adrenal glands to the nigro-striatal area A major weakness in these initial studies was that theyof their brains. Surgeons in the United States, however, were all done “open label,” meaning that bothobserved only very modest and inconsistent improve- researchers and patients knew which patientsment in their patients’ symptoms, and any gains dis- received the transplanted tissue. When appropriate,appeared within a year after surgery. Furthermore, it the best test of a new therapy is a placebo con-became clear that the risks associated with the pro- trolled, double-blind trial, in which neither researchercedure—which required both brain and abdominal nor patient knows who has received the experimentalsurgery on patients who are often frail and elderly— treatment. In the mid-1990s, NIH approved fundingoutweigh the benefits [13]. for two rigorous clinical trials of fetal tissue trans- plantation for Parkinson’s patients. Both studiesAnother strategy, based on transplanting developing provided for placebo control, in the form of shamdopamine neurons from fetal brain tissue, has surgery conducted on half the study patients, andfared better, however. Lars Olsen and his colleagues they were done double blind—neither theshowed in the early 1970s that fetal tissue 81
  • Rebuilding the Nervous System with Stem Cells Dopamine-Neuron Transplantation Before Surgery After Surgery Figure 8.3. Positron Emission Tomography (PET) images from a Parkinson’s patient before and after fetal tissue transplantation. The image taken before surgery (left) shows uptake of a radioactive form of dopamine (red) only in the caudate nucleus, indicating that dopamine neurons have degenerated. Twelve months after surgery, an image from the same patient (right) reveals increased dopamine function, especially in the putamen. (Reprinted with permission from N Eng J Med 2001;344 (10) p. 710.)researchers evaluating the effects of the surgery nor to limit rejection of the implanted tissue, and the teststhe patients themselves knew who got tissue implants. used to assess patient response—and are closer to those used in the most successful of the early open-The results of one of these trials, led by Curt Freed, label experiments.were published recently [5]. Compared with control,patients who received the fetal-tissue transplant Most Parkinson’s researchers are still hopeful that theshowed no significant benefit in a subjective assess- cell-implantation approach will one day lead to ament of the patient’s quality of life, which was the useful and widely used therapy for Parkinson’sstudy’s primary endpoint. Moreover, two years after Disease. At the same time, however, mostsurgery, 5 out of 33 treated patients developed per- researchers are also convinced they must find asistent dyskinesia—uncontrolled flailing movements— different source of cells for transplant. The logisticalthat had not been observed in the open-label work and technical problems involved in recoveringdescribed above. enough developing dopamine neurons from fetal tissue are very great. Moreover, it is virtually impossibleThe Freed study results, nonetheless, provide impor- to standardize the tissue collected from differenttant information about the ability of dopamine fetuses and to fully characterize the cells implanted.neurons to survive in humans. Moreover, PET-scanning This absence of tissue standardization makes it verydata from the treated patients, as well as autopsies difficult to determine the most important factors thatof two patients who died of unrelated causes several lead to a good patient response and may add riskmonths after the surgery, showed that many of the (see Chapter 10. Assessing Human Stem Cell Safety).dopamine neurons survived and grew. Researchersare now awaiting the results of the second NIH- One alternative to cell implantation with human fetalsponsored double-blind trial, led by Warren Olanow tissue is to use fetal cells and tissues from animals.[12]. The procedures used in this study differ substan- Researchers at Diacrin and Genzyme, twotially from those of Freed and his colleagues— biotechnology companies, recently announcedincluding the tissue-handling method, the number of preliminary results from a clinical trial in which 10cells implanted, the use of immunosuppressive drugs Parkinson’s patients received neural cells from the 82
  • Rebuilding the Nervous System with Stem Cellsbrains of fetal pigs. Eighteen months after the surgery, becoming not just nervous system cells but every celltreated patients did not improve enough to show a type in the body. If researchers want to be able tostatistically significant difference from eight control implant cells derived from undifferentiated embryon-patients who received a sham immunosuppression ic stem cells, they must take care that no cells in theregimen and underwent sham surgery. Autopsy of mix give rise to unwanted cell types, such as muscleone treated patient who died of a pulmonary or bone, within the nervous system. Stem cells fromembolism eight months after surgery revealed that a other tissues—including umbilical cord blood andsmall portion of the transplanted pig cells had sur- human bone marrow—can also be coaxed tovived [2], but PET studies looking for improvement in display many of the surface-protein “markers” char-dopamine uptake in all treated patients did not show acteristic of nervous system cells. It is not yet clear,clear improvement. The researchers are still analyzing however, whether these cells are capable of givingtheir data [15]. rise to fully functional neurons. A great deal of basic research remains to be done toRAISING NEURONS FOR find which of these cells provides the best way to getREPLACEMENT IN PATIENTS WITH a workable therapy for Parkinson’s Disease. For exam-PARKINSON’S DISEASE ple, although researchers have shown for certain that both primary human fetal cells and mouse embryon-What Parkinson’s researchers ultimately want is a ic stem cells can become fully functional dopaminerenewable source of cells that can differentiate into neurons, they do not yet know if adult neural stemfunctional dopamine neurons when placed in the cells have the same potential. Also, no one has yetstriatum. Laboratory-grown cells derived from a stem published evidence that cells from any renewablecell may be the best potential alternative source for source that are laboratory-directed to differentiatetransplantable material. One way to get these is to into dopamine neurons can eliminate symptoms infind the right combination of growth factors and cell- animal models of Parkinson’s when implanted.culture conditions to bring undifferentiated cells alongin a culture dish to a point where they are committed Researchers are making rapid progress, however. Forto becoming dopamine neurons, then implant them example, Ron McKay and his colleagues at NIHto finish growth and differentiation in the host brain. reported in 1998 that they were able to expand aAnother possibility is to put less-committed cells into a population of neurons from embryonic mouse braindamaged brain and rely on “environmental” signals in culture, and that these cells relieved Parkinson’s-likein the brain to guide them into becoming the right symptoms in a rat model [16]. And last year, McKay’skind of replacement cell. These developmental sig- lab also described a procedure for efficiently con-nals may be expressed in the brain transiently follow- verting mouse pluripotent embryonic stem cells intoing neural degeneration or acute damage. neurons that have all the characteristics of dopamine neurons, including the ability to form synapses [17].Whether the cells ultimately implanted are half- McKay and other researchers say they have encour-differentiated or completely immature, however, aging unpublished results that dopamine precursorsresearchers need a reliable source. To that end, they derived from mouse embryonic stem cells can elimi-have identified a whole host of different immature nate symptoms in rat models of Parkinson’s Diseasecells that may have the potential to become, [7, 10].among other things, dopamine neurons, and theyare now in the process of sorting out how best to Privately funded researchers are following an analo-make them do so. Neural stem cells isolated from gous path using pluripotent human embryonic stemanimals and humans cannot be grown efficiently in cells. Thomas Okarma of Geron Corporation confirmsthe lab without changing them in some way, such as that his company is testing the potential of humanby engineering them to express a gene normally embryonic stem cells in animal models of Parkinson’sturned on only early in development. Embryonic stem Disease, but the results are not yet complete [11]. Incells—derived from the inner cell mass of an embryo abstracts presented at a recent conference, Geronat the blastocyst stage, when only a few hundred reports having succeeded in directing humancells are present—can be kept in culture in a com- embryonic stem cells to become mature neural cellspletely undifferentiated state. They are still capable of 83
  • Rebuilding the Nervous System with Stem Cellsin laboratory culture, including cells that have the a toxin called 6-hydroxydopamine—a frequently usedstructural and chemical characteristics of dopamine animal model for Parkinson’s Disease—two thingsneurons [6]. happen. After several days of cell proliferation, Fallon observes what he calls a “wave of migration” of theTURNING ON THE BRAIN’S OWN stem cells to the damaged areas, where they differ-STEM CELLS AS A REPAIR entiate into dopamine neurons. Most importantly, the treated rats do not show the behavioral abnormalitiesMECHANISM associated with the loss of the neurons. Whether theParkinson’s researchers are also looking for ways to beneficial effect on symptoms is the result of thespark the repair mechanisms already in a patient’s newly formed cells or some other trophic effect is notbrain to fix damage that these mechanisms could yet entirely clear [4].not otherwise manage. This strategy is less developedthan cell implantation, but it also holds promise [1]. STEM CELLS’ FUTURE ROLE INIn the future, researchers may use stem cells from SPINAL CORD INJURY REPAIRembryonic or adult sources not to replace lost cellsdirectly, but rather to turn on the body’s own repair Parkinson’s Disease is only one of many nervousmechanisms. Alternatively, researchers may find system disorders that researchers are trying to solveeffective drug treatments that help a patient’s own by regenerating damaged tissue. But Parkinson’s,stem cells and repair mechanisms work more difficult as it is to reverse, is a relatively easy targeteffectively. because a regenerative therapy need only replace one particular cell type in one part of the brain.Stem cells in the adult primate brain occur in twolocations. One, the subventricular zone, is an area Therapies for other disorders face much biggerunder fluid-filled spaces called ventricles. The other is hurdles. Complete restoration after severe spinal cordthe dentate gyrus of the hippocampus. In primates, injury, for example, is probably far in the future, if itvery few new neurons normally appear in either can ever be done at all. Many cell types areplace, which is why the phenomenon escaped destroyed in these injuries, including neurons thatnotice until recently. Researchers showed in the mid- carry messages between the brain and the rest of1990s that when the brain is injured, stem cells in the body. Getting these neurons to grow past anthese two areas proliferate and migrate toward the injury site and connect appropriately with their targetssite of the damage. The researchers are now trying to is extraordinarily difficult. But spinal cord injury patientsdiscover how far this kind of response can go toward would benefit greatly from an even limited restorationameliorating certain kinds of damage. of lost functions—gaining partial use of a limb instead of none, or restoring bladder control, or being freedRecent research shows the direction that this may be from pain. Such limited restoration of part of aheading for Parkinson’s Disease. James Fallon and patient’s lost functions is, for some less severe types ofcolleagues studied the effects on rat brain of a pro- injury, perhaps a more achievable goal.tein called transforming growth factor alpha (TGFȊ)—a natural peptide found in the body from the very In many spinal injuries, the spinal cord is not actuallyearliest stages of embryonic development onward cut and at least some of the signal-carrying neuronalthat is important in activating normal repair processes axons are intact. But the surviving axons no longerin several organs, including liver and skin. Fallons carry messages because cells called oligodendro-studies suggest that the brain’s normal repair process cytes, which make the axons’ insulating myelinmay never be adequately triggered in a slowly devel- sheath, are lost. Researchers have recently made theoping degenerative disease like Parkinsons and that first steps in learning to replace these lost myelin-pro-providing more TGFȊ can turn it on. Specifically, ducing cells [14]. For example, researchers haveFallon found that TGFȊ injected into healthy rat brain shown that stem cells can aid remyelination incauses stem cells in the subventricular zone to prolif- rodents [8, 9]. Specifically, they found that injection oferate for several days, after which they disappear. But oligodendrocytes derived from mouse embryonicif the researchers make similar injections into rats in stem cells could remyelinate axons in chemicallywhich they first damage the nigro-striatal neurons with demyelinated rat spinal cord and that the treated 84
  • Rebuilding the Nervous System with Stem Cellsrats regained limited use of their hind limbs com- 6. Harley, C.B., Gearhart, J., Jaenisch, R., Rossant, J., andpared with the controls. They are not certain, however, Thomson, J. (2001). Pluripotent stem cells: biology and applications. Durango, CO.whether the limited increase in function theyobserved in rats is actually due to the remyelination 7. Isacson, O., personal communication.or an unidentified trophic effect of the treatment. 8. Liu, S., Qu, Y., Stewart, T.J., Howard, M.J., Chakrabortty, S., Holekamp, T.F., and McDonald, J.W. (2000). EmbryonicSpinal injury researchers emphasize that much more stem cells differentiate into oligodendrocytes and myeli-basic and preclinical research must be done before nate in culture and after spinal cord transplantation. Proc.attempting human trials using stem cell therapies to Natl. Acad. Sci. U. S. A. 97, 6126-6131.repair the damaged nervous system. Despite the fact 9. McDonald, J.W., Liu, X.Z., Qu, Y., Liu, S., Mickey, S.K., Turetsky,that there is much basic work left to do and many D., Gottlieb, D.I., and Choi, D.W. (1999). Transplanted embryonic stem cells survive, differentiate and promotefundamental questions still to be answered, recovery in injured rat spinal cord. Nat. Med. 5, 1410-1412.researchers are hopeful that effective repair for once-hopeless nervous system damage may eventually be 10. McKay, R., personal communication.achieved. Whether through developing replacement 11. Okarma, T., personal communication.cells or activating the body’s own stem cells in vivo, 12. Olanow, C. W., personal communication.research on the use of stem cells for nervous systemdisorders is a rapidly advancing field. This research 13. Quinn, N.P (1990). The clinical application of cell grafting .promises to answer key questions about how to repair techniques in patients with Parkinsons disease. Prog. Brain Res. 82, 619-625.nervous system damage and how to restore keybody functions damaged by disease or disability. 14. Raisman, G. (2001). Olfactory ensheathing cells—another miracle cure for spinal cord injury? Nat. Rev. Neurosci. 2, 369-374.REFERENCES 15. Schumacher, J.M., Ellias, S.A., Palmer, E.P Kott, H.S., .,1. Bjorklund, A. and Lindvall, O. (2000). Self-repair in the brain. Dinsmore, J., Dempsey, P Fischman, A.J., Thomas, C., .K., Nature. 405, 892-895. Feldman, R.G., Kassissieh, S., Raineri, R., Manhart, C., Penney, D., Fink, J.S., and Isacson, O. (2000).2. Deacon, T., Schumacher, J., Dinsmore, J., Thomas, C., Transplantation of embryonic porcine mesencephalic Palmer, P Kott, S., Edge, A., Penney, D., Kassissieh, S., ., tissue in patients with PD. Neurology. 54, 1042-1050. Dempsey, P and Isacson, O. (1997). Histological evidence ., of fetal pig neural cell survival after transplantation into a 16. Studer, L., Csete, M., Lee, S.H., Kabbani, N., Walikonis, J., patient with Parkinsons disease. Nat. Med. 3, 350-353. Wold, B., and McKay, R. (2000). Enhanced proliferation, survival, and dopaminergic differentiation of CNS precur-3. Dunnett, S.B., Bjorklund, A., and Lindvall, O. (2001). Cell thera- sors in lowered oxygen. J. Neurosci. 20, 7377-7383. py in Parkinsons disease—stop or go? Nat. Rev. Neurosci. 2, 365-369. 17. Studer, L., Tabar, V., and McKay, R.D. (1998). Transplantation of expanded mesencephalic precursors leads to recovery4. Fallon, J., Reid, S., Kinyamu, R., Opole, I., Opole, R., Baratta, in parkinsonian rats. Nat. Neurosci. 1, 290-295. J., Korc, M., Endo, T.L., Duong, A., Nguyen, G., Karkehabadhi, M., Twardzik, D., and Loughlin, S. (2000). In vivo induction of massive proliferation, directed migration, and differentiation of neural cells in the adult mammalian brain. Proc. Natl. Acad. Sci. U. S. A. 97, 14686-14691. 5. Freed, C.R., Greene, P Breeze, R.E., Tsai, W.Y., .E., DuMouchel, W., Kao, R., Dillon, S., Winfield, H., Culver, S., Trojanowski, J.Q., Eidelberg, D., and Fahn, S. (2001). Transplantation of embryonic dopamine neurons for severe Parkinsons disease. N. Engl. J. Med. 344, 710-719. 85
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  • 9. CAN STEM CELLS REPAIR A DAMAGED HEART?Heart attacks and congestive heart failure remain den closing of a blood vessel supplying oxygen toamong the Nation’s most prominent health the heart. Despite advances in surgical procedures,challenges despite many breakthroughs in cardio- mechanical assistance devices, drug therapy, andvascular medicine. In fact, despite successful organ transplantation, more than half of patients withapproaches to prevent or limit cardiovascular congestive heart failure die within five years of initialdisease, the restoration of function to the damaged diagnosis. Research has shown that therapies such asheart remains a formidable challenge. Recent clot-busting medications can reestablish blood flowresearch is providing early evidence that adult and to the damaged regions of the heart and limit theembryonic stem cells may be able to replace death of cardiomyocytes. Researchers are nowdamaged heart muscle cells and establish new exploring ways to save additional lives by usingblood vessels to supply them. Discussed here are replacement cells for dead or impaired cells sosome of the recent discoveries that feature stem cell that the weakened heart muscle can regain itsreplacement and muscle regeneration strategies for pumping power.repairing the damaged heart. How might stem cells play a part in repairing the heart? To answer this question, researchers are build-INTRODUCTION ing their knowledge base about how stem cells areFor those suffering from common, but deadly, heart directed to become specialized cells. One importantdiseases, stem cell biology represents a new medical type of cell that can be developed is the cardiomy-frontier. Researchers are working toward using stem ocyte, the heart muscle cell that contracts to ejectcells to replace damaged heart cells and literally the blood out of the heart’s main pumping chamberrestore cardiac function. (the ventricle). Two other cell types are important to a properly functioning heart are the vascular endothe-Today in the United States, congestive heart failure— lial cell, which forms the inner lining of new blood ves-the ineffective pumping of the heart caused by the sels, and the smooth muscle cell, which forms theloss or dysfunction of heart muscle cells—afflicts 4.8 wall of blood vessels. The heart has a large demandmillion people, with 400,000 new cases each year. for blood flow, and these specialized cells are impor-One of the major contributors to the development of tant for developing a new network of arteries to bringthis condition is a heart attack, known medically as a nutrients and oxygen to the cardiomyocytes after amyocardial infarction, which occurs in nearly 1.1 mil- heart has been damaged. The potential capability oflion Americans each year. It is easy to recognize that both embryonic and adult stem cells to develop intoimpairments of the heart and circulatory system rep- these cells types in the damaged heart is now beingresent a major cause of death and disability in the explored as part of a strategy to restore heart func-United States [5]. tion to people who have had heart attacks or haveWhat leads to these devastating effects? The destruc- congestive heart failure. It is important that work withtion of heart muscle cells, known as cardiomyocytes, stem cells is not confused with recent reports thatcan be the result of hypertension, chronic insufficien- human cardiac myocytes may undergo cell divisioncy in the blood supply to the heart muscle caused by after myocardial infarction [1]. This work suggests thatcoronary artery disease, or a heart attack, the sud- injured heart cells can shift from a quiescent state into active cell division. This is not different from the 87
  • Can Stem Cells Repair a Damaged Heart? Coronary artery ligated inducing an infarct Infarct© 2001 Terese Winslow, Lydia Kibiuk Figure 9.1. Rodent Model of Myocardial Infarction. ability of a host of other cells in the body that begin approach has immense advantages over heart to divide after injury. There is still no evidence that transplant, particularly in light of the paucity of donor there are true stem cells in the heart which can hearts available to meet current transplantation proliferate and differentiate. needs. Researchers now know that under highly specific What is the evidence that such an approach to growth conditions in laboratory culture dishes, stem restoring cardiac function might work? In the research cells can be coaxed into developing as new car- laboratory, investigators often use a mouse or rat diomyocytes and vascular endothelial cells. Scientists model of a heart attack to study new therapies are interested in exploiting this ability to provide (see Figure 9.1. Rodent Model of Myocardial replacement tissue for the damaged heart. This Infarction). To create a heart attack in a mouse or rat, 88
  • Can Stem Cells Repair a Damaged Heart?a ligature is placed around a major blood vessel induced cardiac injury, the survival rate was 26 per-serving the heart muscle, thereby depriving the car- cent. As with the study by Orlic et al., analysis of thediomyocytes of their oxygen and nutrient supplies. region surrounding the damaged tissue in survivingDuring the past year, researchers using such models mice showed the presence of donor-derived car-have made several key discoveries that kindled inter- diomyocytes and endothelial cells. Thus, the mouseest in the application of adult stem cells to heart hematopoietic stem cells transplanted into the bonemuscle repair in animal models of heart disease. marrow had responded to signals in the injured heart, migrated to the border region of the damaged area,Recently, Orlic and colleagues [9] reported on an and differentiated into several types of tissue neededexperimental application of hematopoietic stem cells for cardiac repair. This study suggests that mousefor the regeneration of the tissues in the heart. In this hematopoietic stem cells may be delivered to thestudy, a heart attack was induced in mice by tying off heart through bone marrow transplantation as well asa major blood vessel, the left main coronary artery. through direct injection into the cardiac tissue, thusThrough the identification of unique cellular surface providing another possible therapeutic strategy formarkers, the investigators then isolated a select group regenerating injured cardiac tissue.of adult primitive bone marrow cells with a highcapacity to develop into cells of multiple types. When More evidence for potential stem cell-based thera-injected into the damaged wall of the ventricle, these pies for heart disease is provided by a study thatcells led to the formation of new cardiomyocytes, showed that human adult stem cells taken from thevascular endothelium, and smooth muscle cells, thus bone marrow are capable of giving rise to vasculargenerating de novo myocardium, including coronary endothelial cells when transplanted into rats [6]. As inarteries, arterioles, and capillaries. The newly formed the Jackson study, these researchers induced a heartmyocardium occupied 68 percent of the damaged attack by tying off the LAD coronary artery. They tookportion of the ventricle nine days after the bone mar- great care to identify a population of humanrow cells were transplanted, in effect replacing the hematopoietic stem cells that give rise to new blooddead myocardium with living, functioning tissue. The vessels. These stem cells demonstrate plasticityresearchers found that mice that received the trans- meaning that they become cell types that theyplanted cells survived in greater numbers than mice would not normally be. The cells were used to formwith heart attacks that did not receive the mouse new blood vessels in the damaged area of the rats’stem cells. Follow-up experiments are now being hearts and to encourage proliferation of preexistingconducted to extend the posttransplantation analysis vasculature following the experimental heart attack.time to determine the longer-range effects of such Like the mouse stem cells, these human hematopoi-therapy [8]. The partial repair of the damaged heart etic stem cells can be induced under the appropri-muscle suggests that the transplanted mouse ate culture conditions to differentiate into numeroushematopoietic stem cells responded to signals in the tissue types, including cardiac muscle [10] (see Figureenvironment near the injured myocardium. The cells 9.2. Heart Muscle Repair with Adult Stem Cells). Whenmigrated to the damaged region of the ventricle, injected into the bloodstream leading to the dam-where they multiplied and became “specialized” aged rat heart, these cells prevented the death ofcells that appeared to be cardiomyocytes. hypertrophied or thickened but otherwise viableA second study, by Jackson et al. [3], demonstrated myocardial cells and reduced progressive formationthat cardiac tissue can be regenerated in the mouse of collagen fibers and scars. Control rats that under-heart attack model through the introduction of adult went surgery with an intact LAD coronary artery, asstem cells from mouse bone marrow. In this model, well as LAD-ligated rats injected with saline or controlinvestigators purified a “side population” of cells, did not demonstrate an increase in the numberhematopoietic stem cells from a genetically altered of blood vessels. Furthermore, the hematopoieticmouse strain. These cells were then transplanted into cells could be identified on the basis of highly specif-the marrow of lethally irradiated mice approximately ic cell markers that differentiate them from cardiomy-10 weeks before the recipient mice were subjected ocyte precursor cells, enabling the cells to be usedto heart attack via the tying off of a different major alone or in conjunction with myocyte-regenerationheart blood vessel, the left anterior descending (LAD) strategies or pharmacological therapies. (For morecoronary artery. At two to four weeks after the 89
  • Can Stem Cells Repair a Damaged Heart? Mouse adult stem cells are Human adult bone marrow injected into the muscle of stem cells are injected into the damaged left ventricular the tail vasculature of a rat. wall of the mouse heart. Mouse heart The stem cells induce new blood vessel Adult stem cells formation in the damaged heart muscle and proliferation of existing vasculature. Adult stem cells New blood Stem cells help vessels regenerate damaged heart muscle.© 2001 Terese Winslow, Lydia Kibiuk Damaged Damaged heart muscle heart muscle cells cells Figure 9.2. Heart Muscle Repair with Adult Stem Cells. about stem cell markers see Appendix E.i. How Do In a continuation of this early work, Kehat et al. [4] Researchers Use Markers to Identify Stem Cells?) displayed structural and functional properties of early stage cardiomyocytes in the cells that develop from Exciting new advances in cardiomyocyte regenera- the embryoid bodies. The cells that have sponta- tion are being made in human embryonic stem cell neously contracting activity are positively identified by research. Because of their ability to differentiate into using markers with antibodies to myosin heavy chain, any cell type in the adult body, embryonic stem cells alpha-actinin, desmin, antinaturietic protein, and car- are another possible source population for cardiac- diac troponin—all proteins found in heart tissue. These repair cells. The first step in this application was taken investigators have done genetic analysis of these by Itskovitz-Eldor et al. [2] who demonstrated that cells and found that the transcription-factor genes human embryonic stem cells can reproducibly differ- expressed are consistent with early stage cardio- entiate in culture into embryoid bodies made up of myocytes. Electrical recordings from these cells, cell types from the body’s three embryonic germ changes in calcium-ion movement within the cells, layers. Among the various cell types noted were cells and contractile responsiveness to catecholamine that had the physical appearance of cardiomy- hormone stimulation by the cells were similar to the ocytes, showed cellular markers consistent with heart recordings, changes, and responsiveness seen in cells, and demonstrated contractile activity similar to early cardiomyocytes observed during mammalian cardiomyocytes when observed under the microscope. 90
  • Can Stem Cells Repair a Damaged Heart?development. A next step in this research is to see the rodent research models accurately reflect humanwhether the experimental evidence of improvement heart conditions and transplantation responses? Doin outcome from heart attack in rodents can be these new replacement cardiomyocytes derivedreproduced using embryonic stem cells. from stem cells have the electrical-signal-conducting capabilities of native cardiac muscle cells?These breakthrough discoveries in rodent modelspresent new opportunities for using stem cells to Stem cells may well serve as the foundation uponrepair damaged heart muscle. The results of the which a future form of “cellular therapy” is construct-studies discussed above are growing evidence that ed. In the current animal models, the time betweenadult stem cells may develop into more cell types the injury to the heart and the application of stemthan first thought. In those studies, hematopoietic cells affects the degree to which regeneration takesstem cells appear to be able to develop not only into place, and this has real implications for the patientblood, but also into cardiac muscle and endothelial who is rushed unprepared to the emergency room intissue. This capacity of adult stem cells, increasingly the wake of a heart attack. In the future, could thereferred to as “plasticity,” may make such adult stem patient’s cells be harvested and expanded for use incells a viable candidate for heart repair. But this an efficient manner? Alternatively, can at-risk patientsevidence is not complete; the mouse hematopoietic donate their cells in advance, thus minimizing thestem cell populations that give rise to these replace- preparation necessary for the cells’ administration?ment cells are not homogenous. Rather, they are Moreover, can these stem cells be geneticallyenriched for the cells of interest through specific and “programmed” to migrate directly to the site of injuryselective stimulating factors that promote cell growth. and to synthesize immediately the heart proteinsThus, the originating cell population for these injected necessary for the regeneration process? Investigatorscells has not been identified, and the possibility exists are currently using stem cells from all sources tofor inclusion of other cell populations that could address these questions, thus providing a promisingcause the recipient to reject the transplanted cells. future for therapies for repairing or replacing theThis is a major issue to contend with in clinical appli- damaged heart and addressing the Nation’s leadingcations, but it is not as relevant in the experimental causes of death.models described here because the rodents havebeen bred to be genetically similar. REFERENCESWhat are the implications for extending the research 1. Beltrami, A.P Urbanek, K., Kajstura, J., Yan, S.M., Finato, N., .,on differentiated growth of replacement tissues for Bussani, R., Nadal-Ginard, B., Silvestri, F., Leri, A., Beltrami, C.A., and Anversa, P (2001). Evidence that human cardiac .damaged hearts? There are some practical aspects myocytes divide after myocardial infarction. N. Engl. J.of producing a sufficient number of cells for clinical Med. 344, 1750-1757.application. The repair of one damaged human 2. Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A.,heart would likely require millions of cells. The unique Yanuka, O., Amit, M., Soreq, H., and Benvenisty, N. (2000).capacity for embryonic stem cells to replicate in Differentiation of human embryonic stem cells intoculture may give them an advantage over adult embryoid bodies comprising the three embryonic germ layers. Mol. Med. 6, 88-95.stem cells by providing large numbers of replace-ment cells in tissue culture for transplantation purpos- 3. Jackson, K.A., Majka, S.M., Wang, H., Pocius, J., Hartley, C.J., Majesky, M.W., Entman, M.L., Michael, L.H., Hirschi,es. Given the current state of the science, it is unclear K.K., and Goodell, M.A. (2001). Regeneration of ischemichow adult stem cells could be used to generate cardiac muscle and vascular endothelium by adult stemsufficient heart muscle outside the body to meet cells. J. Clin. Invest. 107, 1-8.patients’ demand [7]. 4. Kehat, I., Kenyagin-Karsenti, D., Druckmann, M., Segev, H., Amit, M., Gepstein, A., Livne, E., Binah, O., Itskovitz-Eldor, J.,Although there is much excitement because and Gepstein, L. (2001). Human embryonic stem cells canresearchers now know that adult and embryonic differentiate into myocytes portraying cardiomyocyticstem cells can repair damaged heart tissue, many structural and functional properties. J. Clin. Invest. (in press)questions remain to be answered before clinical 5. Kessler, P and Byrne, B.J. (1999). Myoblast cell grafting .D.applications can be made. For example, how long into heart muscle: cellular biology and potential applica-will the replacement cells continue to function? Do tions. Annu. Rev. Physiol. 61, 219-242. 91
  • Can Stem Cells Repair a Damaged Heart?6. Kocher, A.A., Schuster, M.D., Szabolcs, M.J., Takuma, S., 9. Orlic, D., Kajstura, J., Chimenti, S., Jakoniuk, I., Anderson, Burkhoff, D., Wang, J., Homma, S., Edwards, N.M., and S.M., Li, B., Pickel, J., McKay, R., Nadal-Ginard, B., Bodine, Itescu, S. (2001). Neovascularization of ischemic myocardi- D.M., Leri, A., and Anversa, P (2001). Bone marrow cells . um by human bone-marrow-derived angioblasts prevents regenerate infarcted myocardium. Nature. 410, 701-705. cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat. Med. 7, 430-436. 10. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W.,7. Lanza, R., personal communication. Craig, S., and Marshak, D.R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science. 284,8. Orlic, D., personal communication. 143-147. 92
  • 10. ASSESSING HUMAN STEM CELL SAFETYThe isolation of human stem cells offers the promise WEAVING A STEM CELL SAFETY NETof a remarkable array of novel therapeutics. Biologictherapies derived from such cells—through tissue A diversity of opinion exists among researchers aboutregeneration and repair as well as through the the feasibility of initiating pilot clinical studies usingtargeted delivery of genetic material—are expected human stem cells. Some are of the view that it isto be effective in the treatment of a wide range of reasonable to expect within the next five years thatmedical conditions. Efforts to analyze and assess the human stem cells will be used in transplantationsafety of using human stem cells in the clinical setting settings to replace dead or dying cells within organsare vitally important to this endeavor. such as the failing heart or that genetically modified human stem cells will be created for delivery ofTransplanted human stem cells are dynamic bio- therapeutic genes. Others argue that a good deallogical entities that interact intimately with—and are more information about the basic biology of humaninfluenced by—the physiology of the recipient. Before stem cells needs to be accumulated before theirthey are transplanted, cultured human stem cells are therapeutic potential in humans can be assessed.maintained under conditions that promote either theself-renewing expansion of undifferentiated progeni- Clinical studies involving the transplantation of blood-tors or the acquisition of differentiated properties restoring, or hematopoietic, stem cells have beenindicative of the phenotype the cells will assume. under way for a number of years. Reconstituting theAfter incompletely differentiated human stem cells blood and immune systems through stem cell trans-are transplanted, additional fine-tuning occurs as a plantation is an established practice for treatingconsequence of instructions received from the cells’ hematological malignancies such as leukemia andphysiologic microenvironments within the recipient. lymphoma. Transplanting hematopoietic stem cellsThe capabilities to self-renew and differentiate that resident in the bone marrow or isolated from cordare inherent to human stem cells point simultaneously blood or circulating peripheral blood is used toto their perceived therapeutic potential and to the counter the destruction of certain bone marrow cellschallenge of assessing their safety. caused by high-intensity chemotherapeutic regimens used to battle various solid tumors. Moreover, clinicalAssessing human stem cell safety requires the imple- trials are being conducted to assess the safety andmentation of a comprehensive strategy. Each step in efficacy of using hematopoietic stem cell transplan-the human stem cell development process—begin- tation to treat various autoimmune conditions includ-ning with identifying and evaluating suitable human ing multiple sclerosis, lupus, and rheumatoid arthritis.stem cell sources—must be carefully scrutinized.Included in this global assessment are the derivation, Although precedents exist for the clinical use ofexpansion, manipulation, and characterization of human stem cells, there is considerable reluctancehuman stem cell lines, as well as preclinical efficacy to proceed with clinical trials involving human stemand toxicity testing in appropriate animal models. cells derived from embryonic and fetal sources. ThisBeing able to trace back from the cell population hesitancy extends to adult human stem cells of non-prepared for transplantation to the source of the hematopoietic origin, even though, by contrast, theirfounder human stem cells also allows each safety plasticity is generally considered to be lower than thatcheckpoint to be connected, one to the other. of their embryo- and fetus-derived counterparts. For 93
  • Assessing Human Stem Cell Safetyhuman stem cells to advance to the stage of clinical intended for transplantation are derived from aninvestigation, a virtual safety net composed of a allogeneic donor—that is, someone other that thecore set of safeguards is required (see Table 10.1. recipient—and especially if the cells are obtainedSafeguards for clinical applications of human stem from a master cell bank that has been establishedcells, by source of cell). using human embryonic stem or human embryonic germ cells.Safety Assurance Begins with AdequateDonor Screening The purpose of pedigree evaluation and/or geneticWhether human stem cells are of embryonic, fetal, testing is to establish whether the human stem cellsor adult origin, donor sources must be carefully in question are suitable for use in the context of ascreened. Routine testing should be done to guard particular clinical situation. For example, embryosagainst the inadvertent transmission of infectious derived from a donor with a family history of cardio-diseases. Additionally, pedigree assessment and vascular diseases may not be the best suited for themolecular genetic testing appear to be warranted. derivation of cardiac muscle cells intended to repairThis is arguably the case when human stem cells damaged heart tissue. Similarly, the use of molecular Table 10.1. Safeguards for clinical applications of human stem cells, by source of cell* Adult: Adult: Safeguard Embryo Fetus Autologous Allogeneic (self) (nonself) Screen donors ++ ++ + ++ • Infectious-agent testing • Pedigree assessment • Molecular genetic testing Use controlled, standardized practices and ++ ++ ++ ++ procedures for establishing stem cell lines Develop alternatives to culturing on cell-feeder ++ ++ NA NA layer Perform detailed characterization of ++ ++ ++ ++ tem cell lines • Morphology • Cell-surface antigens • Biochemical markers • Gene expression • Karyotype analysis • Biologic activity Conduct preclinical animal testing ++ ++ ++ ++ • Proof of concept: disease models – Cell integration – Cell migration • Comprehensive toxicity ++ ++ ++ ++ • Proliferative potential ++ ++ + + Monitor patient and do long-term follow-up ++ ++ ++ ++ ++= more important; +=less important; NA=not applicable. 94
  • Assessing Human Stem Cell Safetygenetic analysis could detect a mutation in the gene cells are permitted to achieve before subdividing willfor alpha-synuclein. This gene is known to be respon- all affect the characteristics of human stem cellssible for the rare occurrence of early onset Parkinson’s maintained in culture. Altering the concentrations ofDisease. Detecting such a genetic abnormality in supplemental growth factors and chemical sub-neuronal progenitor cells derived from an established stances, even switching from one supplier to another,embryonic germ cell line could block the use of may lead to changes in cell growth rate, expressionthose cells as a treatment for a number of neuro- of defining cell markers, and differentiation potential.degenerative conditions, including Parkinson’s Disease. Alterations in stem cell properties caused by the use of nonstandardized culture practices are likely toThe number of genes known to be directly respon- affect the behavior and effectiveness of the cellssible for causing disease or anomalous physiologic once transplanted.function is relatively small. Advances in techniques foridentifying, isolating, and analyzing genes, coupled One particular concern is how safe it is to use serumwith the wealth of information destined to become derived from cows as a supplement to cultureavailable as one outcome of the human genome media. Due to the outbreak of bovine spongiformsequencing projects, will raise this number. encephalopathy (BSE) in cattle herds, primarily thoseConsiderably more will also be learned about how raised in the United Kingdom, only serum producedmultiple gene products, each contributing an incre- from cows reared in countries certified to be free ofmental quantity to the overall sum, predispose an BSE should be used. Consumption of beef contami-individual to develop particular diseases. Clearly, it nated with the agent responsible for causing BSE haswill eventually not be possible, or even necessary, to lead to the limited emergence of new variantscreen every source of human stem cells for the Creutzfeldt-Jakob disease (nvCJD) in humans. This dis-entire panoply of disease-associated genes. The ease results in the relentless destruction of brain tissuescreening of targeted genes will be conducted within and is invariably fatal. Placing neural stem cells con-the context of the relevant clinical population. taminated with the BSE infectious agent in a patient’s nervous system to investigate cellular-replacementUsing Controlled, Standardized Practices and therapies for neurological disorders would be bothProcedures for Establishing Cultured Human Stem irresponsible and devastating. Researchers areCell Lines Enhances Safety engaged in a vigorous effort to develop serum-free,To ensure the integrity, uniformity, and reliability of chemically defined media that obviate risks asso-human stem cell preparations intended for clinical ciated with the use of bovine serum.use, it is essential to demonstrate that rigorouslycontrolled, standardized practices and procedures Alternatives to Culturing on a Feeder Layer ofare being followed in establishing and maintaining Animal Cells Improve Safetyhuman stem cell lines in culture. An issue unique to the culturing of human embryonic stem and embryonic germ cells involves the use ofHuman stem cells from virtually every source other mouse embryonic fibroblast feeder cells to keep thethan blood-derived hematopoietic stem cells are embryonic cells in a proliferating, undifferentiatedmaintained in tissue culture for some defined period condition. Human embryonic stem and embryonicof time. This is necessary to obtain a sufficient num- germ cells are seeded directly onto a bed of irradiatedber of cells for use in clinical studies involving trans- mouse feeder cells. Transplanting into humans stemplantation. Culturing human stem cells requires the cell preparations derived from founder cells that haveuse of formulated liquid media supplemented with been in direct, intimate contact with nonhumangrowth factors and other chemical substances that animal cells constitutes xenotransplantation—the usepromote cellular replication and govern the differenti- of organs, tissues, and cells derived from animals toation of the cultured human stem cells. Since human treat human disease. The principal concern of xeno-stem cells are a dynamic, biological entity, failure to transplantation is the unintended transfer of animalstandardize procedures for maintaining and expand- viruses into humans.ing cells in culture could result in unintended alter-ations in the intrinsic properties of the cells. The initial Researchers are devoting considerable attention toseeding density of the cells, the frequency with which developing culture conditions that do not use mousethe culture medium is replenished, and the density feeder cells. In February of this year, scientists from 95
  • Assessing Human Stem Cell SafetyGeron Corporation, a biotech company focusing on in culture for extended periods of time. Continuedthe development of embryonic stem cell technology development and standardization of DNA microarrayfor treating disease, presented findings at a scientific analysis (simultaneous screening for many genes)conference demonstrating that human embryonic and proteomics (protein profiling) technologies willstem cells can be maintained without mouse feeder significantly enhance stem cell characterization.cells. Human embryonic stem cells seeded on a Rigorous and quantitative identification of cell typescommercially available basement membrane matrix within a heterogeneous population of differentiatingin media conditioned by feeder cells retain their human stem cells provides the means to gaugeproliferative potential and capacity to form all three purity of a cellular preparation. In turn, this permitsembryonic germ layers (mesoderm, endoderm, and evaluation of the extent to which purity of a humanectoderm). This suggests that human embryonic stem cell preparation predicts efficacy after trans-stem cells maintained in the absence of direct plantation. It is not necessarily the case that homo-culture on a mouse feeder cell layer are comparable genous populations composed of a single cell typeto human embryonic stem cells co-cultured withmouse feeder cells. will be more effective as a cell-replacement therapy than mixed populations of cells. It is conceivable thatDetailed Characterization of Human Stem Cell the reason differentiation of cultured stem cellsPopulations Reinforces the Safety Net obtained from the brain leads to formation of all theDetailed characterization of cell preparations intend- cell types found within the nervous system (namely,ed for transplantation is critical to the development of neurons, astrocytes, and oligodendrocytes) is thathuman stem cells for clinical use. Identifying the cells their coincidental presence is required to ensurethat make up an human stem cell population intend- maximum survival and functional capability. Theed for clinical study requires identifying cells exhibiting interaction of various phenotypic cell types within athe desired phenotype within the preparation, as well preparation of progenitor cells obtained after theas those that do not. This poses considerable chal- controlled differentiation of cultured human embry-lenges because human embryonic stem and embry- onic stem cells is being actively investigated.onic germ cells have the capacity to give rise to all Once the purity profile has been established for adifferentiated cell types, while adult human stem population of human stem cells generated usingcells, though generally more restricted in their plas- standardized procedures, deviations that occur out-ticity, are capable of generating all cell types that side what is expected due to normal biologic varia-make up the tissue from which they were derived. tion serve as a harbinger that significant, and possiblyOn the basis of the complex biological properties of deleterious, changes may have occurred. Such alter-human stem cells, including their potential to differen- ations could reflect the introduction of genetic muta-tiate along multiple lineages and give rise to a variety tions as a consequence of culture conditions used toof cell types, it is expected that the characterization promote expansion and to induce differentiation ofof stem cell preparations will require a panel of the progenitor cell population.orthogonal assessments. Parameters that will prove Before clinical studies involving human stem celluseful in establishing identity include 1) cell morphology transplantation can be done, it is essential to demon-(visual microscopic inspection of cells to assess their strate that human stem cell preparations possessappearance), 2) expression of unique cell-surface relevant biological activity. The bioassay provides aantigens (as is the case for CD34+ hematopoietic quantitative measure of the potency of a cell prep-stem cells), 3) characterization of biochemical aration and ensures that cells destined for trans-markers such as a tissue-specific enzymatic activity plantation are not inert. Assays may be based on a(e.g., enzymes that produce neurotransmitters for biologic activity such as insulin release from pan-nerve cells), and 4) expression of genes that are creatic islet-like cells, glycogen storage by cellsunique to a particular cell type. Further, analysis of the intended for regeneration of liver tissue, or synchro-nuclear chromosomal karyotype may be used to nous contraction in the case of stem cell-derivedassess genetic stability of established human embry- cardiomyocytes to be used for repairing damagedonic stem and embryonic germ cell lines maintained heart muscle. When cells that have not acquired fully 96
  • Assessing Human Stem Cell Safetydifferentiated functionality are to be transplanted, it In addition to efficacy, evidence for anatomic andmay be appropriate to use surrogate markers that functional integration of transplanted human stempredict the acquisition of the intended biologic cells should be assessed. human stem cells destinedactivity upon further differentiation. (For example, for transplantation may be tagged with a marker,counting tyrosine hydroxylase-expressing neural such as green fluorescent protein, that allows trans-progenitor cells in a mixed population of cells intend- planted cells to be readily identified upon histologicaled to provide dopaminergic neurons for treating examination. A similar approach should be used toParkinson’s Disease could predict the acquisition of evaluate the migration of transplanted human stemrelevant biologic activity after transplantation.) cells from the site of injection into adjacent and more distant tissues. The migration of transplanted humanProof of Concept, Toxicity Testing, and Evaluation stem cells to a nontarget site and subsequent differ-of Proliferative Potential in Animal Models Are entiation into a tissue type that is inappropriate forImportant to the Assessment of Human Stem that anatomic location could be problematic.Cell SafetyA critical element of the safety net is the transplan- Questions about the use of embryonic comparedtation of human stem cells into animals to demon- with adult stem cells with respect to robustness andstrate that the therapy does what it is supposed to do durability should be addressed in animal-transplanta-(“proof of concept”) and to assess toxicity. Admittedly, tion models. Similarly, the issue of whether less-differ-animal models of human disease are imperfect entiated cells will be more effective than more-differ-because most human maladies do not spontan- entiated cells following transplantation should beeously occur in animals. Chemical, surgical, and investigated. Continued advancements in noninva-immunologic methods are used to damage neurons; sive imaging technologies, such as magnetic reso-induce diabetes; simulate heart attacks, stroke, and nance imaging (MRI) and positron emission tomogra-hypertension; or compromise organ function. In situa- phy (PET scanning), will allow these events to betions when focal genetic lesions are known to cause observed in real time with reasonable resolution anddisease, the creation of transgenic mouse colonies in without having to use large numbers of animals.which the culpable gene is either eliminated or over- From the perspective of toxicology, the proliferativeexpressed results in disease models that are capable potential of undifferentiated human embryonic andof faithfully reproducing human-disease-specific embryonic germ cells evokes the greatest level ofpathologies. concern. A characteristic of human embryonic stemHuman stem cells must be transplanted into animal cells is their capacity to generate teratomas whenmodels of human disease. Transplantation of neural transplanted into immunologically incompetentstem cells should demonstrate measurable evidence strains of mice. Undifferentiated embryonic stem cellsof efficacy in models of neurodegenerative disease, are not considered as suitable for transplantation duesuch as Parkinson’s Disease, Huntington’s disease, to the risk of unregulated growth. The question thatand amyotrophic lateral sclerosis (ALS), Alzheimer’s remains is, at what point during differentiation doesdisease, as well as spinal cord injury and stroke. this risk become insignificant, if ever? Identifying theImproved liver function after transplantation of stage at which the risk for tumor formation is mini-hepatocyte precursors should be observed in an mized will depend on whether the process of stemanimal model of hepatic failure. Normalization of cell differentiation occurs only in a forward directionblood insulin concentrations and amelioration of or is reversible. Before clinical trials are begun indiabetic disease symptoms should result from the humans, the issue of unregulated growth potentialtransplantation of pancreatic islet progenitors in a and its relationship to stem cell differentiation mustmouse model of diabetes. It is likely that in all cases, be evaluated. It is essential that careful toxicologyimmunosuppression will be required due to immu- studies are performed that are of the appropriatenologic incompatibility between humans and the duration and that involve transplantation intoanimal model species (usually mouse or rat). immunocompromised animals of undifferentiated or partially differentiated embryonic stem cells, as well as adult stem cells. 97
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  • 11. USE OF GENETICALLY MODIFIED STEM CELLS IN EXPERIMENTAL GENE THERAPIESTo date, only nonembryonic human stem cells have The potential success of gene therapy technologybeen used in cell-based gene therapy studies. The depends not only on the delivery of the therapeuticinherent limitations of these stem cells, as discussed transgene into the appropriate human target cells,below, have prompted scientists to ponder and but also on the ability of the gene to functionexplore whether human embryonic stem cells might properly in the cell. Both requirements poseovercome the current barriers to the clinical success considerable technical challenges.of cell-based gene therapies. Gene therapy researchers have employed two major strategies for delivering therapeutic transgenes intoPRINCIPLES AND PROMISE OF GENE human recipients (see Figure 11.1. Strategies forTHERAPY Delivering Therapeutic Transgenes into Patients). TheGene therapy is a relatively recent, and still highly first is to “directly” infuse the gene into a person.experimental, approach to treating human disease. Viruses that have been altered to prevent them fromWhile traditional drug therapies involve the adminis- causing disease are often used as the vehicle fortration of chemicals that have been manufactured delivering the gene into certain human cell types, inoutside the body, gene therapy takes a very different much the same way as ordinary viruses infect cells.approach: directing a patient’s own cells to produce This delivery method is fairly imprecise and limited toand deliver a therapeutic agent. The instructions for the specific types of human cells that the viral vehiclethis are contained in the therapeutic transgene (the can infect. For example, some viruses commonlynew genetic material introduced into the patient). used as gene-delivery vehicles can only infect cellsGene therapy uses genetic engineering—the intro- that are actively dividing. This limits their usefulness induction or elimination of specific genes by using treating diseases of the heart or brain, because thesemolecular biology techniques to physically mani- organs are largely composed of nondividing cells.pulate genetic material—to alter or supplement the Nonviral vehicles for directly delivering genes intofunction of an abnormal gene by providing a copy cells are also being explored, including the use ofof a normal gene, to directly repair such a gene, or plain DNA and DNA wrapped in a coat of fattyto provide a gene that adds new functions or molecules known as liposomes.regulates the activity of other genes. The second strategy involves the use of living cells toClinical efforts to apply genetic engineering tech- deliver therapeutic transgenes into the body. In thisnology to the treatment of human diseases date to method, the delivery cells—often a type of stem cell,1989. Initially, gene therapy clinical trials focused on a lymphocyte, or a fibroblast—are removed from thecancer, infectious diseases, or disorders in which only body, and the therapeutic transgene is introduceda single gene is abnormal, such as cystic fibrosis. into them via the same vehicles used in the pre-Increasingly however, efforts are being directed viously described direct-gene-transfer method. Whiletoward complex, chronic diseases that involve more still in the laboratory, the genetically modified cellsthan one gene. Prominent examples include heart are tested and then allowed to grow and multiplydisease, inadequate blood flow to the limbs, arthritis, and, finally, are infused back into the patient.and Alzheimer’s disease. 99
  • Use of Genetically Modified Stem Cells in Experimental Gene Therapies DIRECT DELIVERY CELL-BASED DELIVERY Therapeutic Therapeutic transgene The therapeutic The therapeutic transgene transgene is transgene is packaged into a packaged into a delivery vehicle delivery vehicle such as a virus. such as a virus. The therapeutic Target organ transgene is …and injected (e.g., liver) introduced into a into the patient. delivery cell such as a stem cell that is often derived from the patient. The genetically modified cells (e.g., stem cells) are multiplied in the …and readministered laboratory. to the patient.© 2001 Terese Winslow Figure 11.1. Strategies for Delivering Therapeutic Transgenes into Patients. Gene therapy using genetically modified cells offers agent in a regulated fashion. In this case, the thera- several unique advantages over direct gene transfer peutic transgene would be active only in response to into the body and over cell therapy, which involves certain signals, such as drugs administered to the administration of cells that have not been genetically patient to turn the therapeutic transgene on and off. modified. First, the addition of the therapeutic trans- gene to the delivery cells takes place outside the WHY STEM CELLS ARE USED IN patient, which allows researchers an important meas- SOME CELL-BASED GENE THERAPIES ure of control because they can select and work only with those cells that both contain the transgene and To date, about 40 percent of the more than 450 produce the therapeutic agent in sufficient quantity. gene therapy clinical trials conducted in the United Second, investigators can genetically engineer, or States have been cell-based. Of these, approxi- “program,” the cells’ level and rate of production of mately 30 percent have used human stem cells— the therapeutic agent. Cells can be programmed to specifically, blood-forming, or hematopoietic, stem steadily churn out a given amount of the therapeutic cells—as the means for delivering transgenes into product. In some cases, it is desirable to program the patients. cells to make large amounts of the therapeutic Several of the early gene therapy studies using agent so that the chances that sufficient quantities these stem cells were carried out not for therapeutic are secreted and reach the diseased tissue in the purposes per se, but to track the cells’ fate after they patient are high. In other cases, it may be desirable were infused back into the patient. The studies aimed to program the cells to produce the therapeutic 100
  • Use of Genetically Modified Stem Cells in Experimental Gene Therapiesto determine where the stem cells ended up and The only type of human stem cell used in genewhether they were indeed producing the desired therapy trials so far is the hematopoietic stem cell.gene product, and if so, in what quantities and for However, several other types of stem cells are beingwhat length of time. Of the stem cell-based gene studied as gene-delivery-vehicle candidates. Theytherapy trials that have had a therapeutic goal, include muscle-forming stem cells known asapproximately one-third have focused on cancers myoblasts, bone-forming stem cells called(e.g., ovarian, brain, breast, myeloma, leukemia, and osteoblasts, and neural stem cells.lymphoma), one-third on human immunodeficiency Myoblasts appear to be good candidates for use invirus disease (HIV-1), and one-third on so-called gene therapy because of an unusual and advanta-single-gene diseases (e.g., Gaucher’s disease, severe geous biological property: when injected into muscle,combined immune deficiency (SCID), Fanconi they fuse with nearby muscle fibers and become ananemia, Fabry disease, and leukocyte adherence integral part of the muscle tissue. Moreover, sincedeficiency). muscle tissue is generally well supplied with nervesBut why use stem cells for this method of gene thera- and blood, the therapeutic agents produced by thepy, and why hematopoietic stem cells in particular? transgene are also accessible to nerves and theThe major reason for using stem cells in cell-based circulatory system. Thus, myoblasts may not only begene therapies is that they are a self-renewing useful for treating muscle disorders such as muscularpopulation of cells and thus may reduce or dystrophy, but also possibly nonmuscle disorders sucheliminate the need for repeated administrations of as neurodegenerative diseases, inherited hormonethe gene therapy. deficiencies, hemophilia, and cancers.Since the advent of gene therapy research, Several promising animal studies of myoblast-hematopoietic stem cells have been a delivery cell mediated gene therapy have been reported [17]. Forof choice for several reasons. First, although small in instance, this approach was successful in correctingnumber, they are readily removed from the body via liver and spleen abnormalities associated with athe circulating blood or bone marrow of adults or the lysosomal storage disease in mice. Investigators haveumbilical cord blood of newborn infants. In addition, also achieved stable production of the human clot-they are easily identified and manipulated in the ting factor IX deficient in hemophilia at therapeuticlaboratory and can be returned to patients relatively concentrations in mice for at least eight months.easily by injection. Myoblasts engineered to secrete erythropoietin (a hormone that stimulates red blood cell production)The ability of hematopoietic stem cells to give rise to were successful in reversing a type of anemiamany different types of blood cells means that once associated with end-stage renal disease in a mousethe engineered stem cells differentiate, the thera- model of renal failure.peutic transgene will reside in cells such as T and Blymphocytes, natural killer cells, monocytes, Another animal study of myoblast-mediated genemacrophages, granulocytes, eosinophils, basophils, transfer involved a mouse model of familial amy-and megakaryocytes. The clinical applications of otrophic lateral sclerosis (ALS, also known as Louhematopoietic stem cell-based gene therapies are Gehrig’s disease), a fatal disorder characterized bythus also diverse, extending to organ transplantation, progressive degeneration of the brain and spinalblood and bone marrow disorders, and immune cord nerves that control muscle activity. Investigatorssystem disorders. injected myoblasts containing the transgene for a human nerve growth factor into the muscles of theIn addition, hematopoietic stem cells “home,” or ALS mice before the onset of disease symptoms andmigrate, to a number of different spots in the body— motor neuron degeneration. The transgene remainedprimarily the bone marrow, but also the liver, spleen, active in the muscle for up to 12 weeks, and, mostand lymph nodes. These may be strategic locations importantly, the gene therapy successfully delayedfor localized delivery of therapeutic agents for disor- the onset of disease symptoms, slowed muscle atro-ders unrelated to the blood system, such as liver phy, and delayed the deterioration of motor skills [16].diseases and metabolic disorders such asGaucher’s disease. 101
  • Use of Genetically Modified Stem Cells in Experimental Gene TherapiesIn a series of experiments in rodents, a team of inves- might overcome some of these limitations, but furthertigators has been testing neural stem cells as vehicles research will be needed to determine whetherfor cell-based gene therapy for brain tumors known embryonic stem cells are better suited to meet theas gliomas. Gliomas are virtually impossible to treat needs of gene therapy applications than are adultbecause the tumor cells readily invade the surround- stem cells.ing tissue and migrate extensively into the normal One important feature of the optimal cell for deliver-brain. The researchers genetically modified human ing a therapeutic transgene would be its ability toneural stem cells to produce a protein—cytosine retain the therapeutic transgene even as it prolifer-deaminase—that converts a nontoxic precursor drug ates or differentiates into specialized cells. Most of theinto an active form that kills cancer cells. The engi- cell-based gene therapies attempted so far haveneered neural stem cells were then injected into the used viral vehicles to introduce the transgene into thebrains of mice with human-derived gliomas. Within hematopoietic stem cell. One way to accomplish thistwo weeks of the gene therapy and systemic treat- is to insert the therapeutic transgene into the one ofment with the precursor drug, the tumors had shrunkby 80 percent. The animal studies also revealed that the chromosomes of the stem cell. Retroviruses areneural stem cells were able to quickly and accurately able to do this, and for this reason, they are often used as the vehicle for infecting the stem cell and“find” glioma cells, regardless of whether the stem introducing the therapeutic transgene into thecells were implanted directly into the tumors, implant-ed far from the tumors (but still within the brain), or chromosomal DNA. However, mouse retroviruses areinjected into circulating blood outside the brain [1]. only efficient at infecting cells that are actively divid- ing. Unfortunately, hematopoietic stem cells are qui-Another cell-based gene therapy system under escent and seldom divide. The percentage of steminvestigation involves the use of osteoblasts, or bone- cells that actually receive the therapeutic transgeneforming stem cells. In a recent preliminary study has usually been too low to attain a therapeuticexamining a gene therapy approach to bone repair effect. Because of this problem, investigators haveand regeneration, researchers genetically engineered been exploring the use of viral vehicles that canosteoblasts to produce a bone growth factor. The infect nondividing cells, such as lentiviruses (e.g., HIV)osteoblasts were added to a biodegradable matrix or adeno-associated viruses. This approach has notthat could act as a “scaffold” for new bone forma- been entirely successful, however, because oftion. Within a month after the cell-impregnated problems relating to the fact that the cells themselvesscaffold was implanted into mice, new bone forma- are not in an active state [13, 19].tion was detectable. Although this work is in the veryearly stages, it offers hope of an effective alternative One approach to improving the introduction of trans-to conventional bone-grafting techniques [14]. genes into hematopoietic stem cells has been to stimulate the cells to divide so that the viral vehicles can infect them and insert the therapeutic transgene.HOW EMBRYONIC STEM CELLS Inder Verma of the Salk Institute has noted, however,MIGHT PLAY A ROLE IN GENE that this manipulation can change other importantTHERAPY RESEARCH properties of the hematopoietic stem cells, such as plasticity, self-renewal, and the ability to survive andWith one notable exception, no therapeutic effects grow when introduced into the patient [23]. This possi-have been achieved in gene therapy trials to date. bility might be overcome with the use of embryonicThe first successful gene therapy occurred in a recent stem cells if they require less manipulation. And inFrench study in which a therapeutic transgene for fact, some preliminary data suggest that retroviralcorrecting X-linked severe combined immune vectors may work more efficiently with embryonicdeficiency was introduced into the bone marrow stem cells than with the more mature adult stemcells of children, resulting in improved function of their cells. For example, researchers have noted that retro-immune systems and correction of the disease [5]. viral vectors introduce transgenes into human fetalThis encouraging success aside, the generally disap- cord blood stem cells more efficiently than into cordpointing results are due, in part, to the inherent limita- blood stem cells from newborns, and that the fetaltions of adult and cord blood stem cells. In principle cord blood stem cells also had a higher proliferativeat least, the use of human embryonic stem cells 102
  • Use of Genetically Modified Stem Cells in Experimental Gene Therapiescapacity (i.e., they underwent more subsequent cell an enzyme known as telomerase. Low levels ofdivisions). This suggests that fetal cord blood stem cells telomerase activity result in short telomeres and, thus,might be useful in cell-based in utero gene therapy fewer rounds of cell division—in other words, shorterto correct hematopoietic disorders before birth [15, 21]. longevity. Higher levels of telomerase activity result in longer telomeres, more possible cell divisions, andIn some cases—such as a treatment of a chronic overall longer persistence. Mouse embryonic stemdisease—achieving continued production of the ther- cells have been found to have longer telomeres andapeutic transgene over the life of the patient will be higher levels of telomerase activity compared withvery important. Generally, however, gene therapies adult stem cells and other more specialized cells inusing hematopoietic stem cells have encountered a the body. As mouse embryonic stem cells give rise tophenomenon known as “gene silencing,” where, over hematopoietic stem cells, telomerase activity levelstime, the therapeutic transgene gets “turned off” due drop, suggesting a decrease in the self-renewingto cellular mechanisms that alter the structure of the potential of the hematopoietic stem cells [3, 4]. (Forarea of the chromosome where the therapeutic more detailed information regarding telomeres andgene has been inserted [6, 7, 11, 22, 24]. Whether telomerase, see Figure C.2. Telomeres andthe use of embryonic stem cells in gene therapy Telomerase.)could overcome this problem is unknown, althoughpreliminary evidence suggests that this phenomenon Human embryonic stem cells have also been shownmay occur in these cells as well [8, 18]. to maintain pluripotency (the ability to give rise to other, more specialized cell types) and the ability toPersistence of the cell containing the therapeutic proliferate for long periods in cell culture in the labo-transgene is equally important for ensuring continued ratory [2]. Adult stem cells appear capable of only aavailability of the therapeutic agent. Verma noted limited number of cell divisions, which would preventthat the optimal cells for cell-mediated gene transfer long-term expression of the therapeutic gene need-would be cells that will persist for “the rest of the ed to correct chronic diseases. “Embryonic stem cellspatient’s life; they can proliferate and they would can be maintained in culture, whereas that is nearlymake the missing protein constantly and forever” [23]. impossible with cord blood stem cells,” says RobertPersistence, or longevity, of the cells can come about Hawley of the American Red Cross Jerome H. Hollandin two ways: a long life span for an individual cell, or Laboratory for Biomedical Sciences, who is develop-a self-renewal process whereby a short-lived cell ing gene therapy vectors for insertion into humanundergoes successive cell divisions while maintaining hematopoietic cells [12]. “So with embryonic stemthe therapeutic transgene. Ideally, then, the geneti- cells, you have the possibility of long-term mainte-cally modified cell for use in cell-based gene therapy nance and expansion of cell lines, which has notshould be able to self-renew (in a controlled manner been possible with hematopoietic stem cells.”so tumors are not formed) so that the therapeuticagent is available on a long-term basis. This is one of The patient’s immune system response can bethe reasons why stem cells are used, but adult stem another significant challenge in gene therapy. Mostcells seem to be much more limited in the number cells have specific proteins on their surface that allowof times they can divide compared with embryonic the immune system to recognize them as either “self”stem cells. The difference between the ability of adult or “nonself.” These proteins are known as major histo-and embryonic stem cells to self-renew has been compatibility proteins, or MHC proteins. If adult stemdocumented in the mouse, where embryonic stems cells for use in gene therapy cannot be isolated fromcells were shown to have a much higher proliferative the patient, donor cells can be used. But because ofcapacity than do adult hematopoietic stem cells [25]. the differences in MHC proteins among individuals, the donor stem cells may be recognized as nonselfResearchers are beginning to understand the biolo- by the patient’s immune system and be rejected.gical basis of the difference in proliferative capacitybetween adult and embryonic stem cells. Persistence John Gearhart of Johns Hopkins University and Peterof cells and the ability to undergo successive cell Rathjen at the University of Adelaide speculate thatdivisions are in part, at least, a function of the length embryonic stem cells may be useful for avoidingof structures at the tips of chromosomes called such immune reactions [10, 20]. For instance, it maytelomeres. Telomere length is, in turn, maintained by be possible to establish an extensive “bank” of 103
  • Use of Genetically Modified Stem Cells in Experimental Gene Therapiesembryonic stem cell lines, each with a different set Achieving clinical success with cell-based geneof MHC genes. Then, an embryonic stem cell that is therapy will require new knowledge and advances inimmunologically compatible for a patient could be several key areas, including the design of viral andselected, genetically modified, and triggered to nonviral vehicles for introducing transgenes into cells,develop into the appropriate type of adult stem cell the ability to direct where in a cell the transgene isthat could be administered to the patient. By gene- introduced, the ability to direct the genetically modi-tically modifying the MHC genes of an embryonic fied stem cells or the secreted therapeutic agent tostem cell, it may also be possible to create a “univer- diseased tissues, optimization and regulation of thesal” cell that would be compatible with all patients. production of the therapeutic agent within the stemAnother approach might be to “customize” embry- cell, and management of immune reactions to theonic stem cells such that cells derived from them gene therapy process. The ability of embryonic stemhave a patient’s specific MHC proteins on their cells to generate a wide variety of specialized cellsurface and then to genetically modify them for use types and being able to maintain them in the labor-in gene therapy. Such approaches are hypothetical atory would make embryonic stem cells a promisingat this point, however, and research is needed to model for exploring critical questions in many ofassess their feasibility. these areas.Ironically, the very qualities that make embryonic “There are possibilities of long-term maintenance andstem cells potential candidates for gene therapy (i.e., expansion of embryonic stem cells and of differen-pluripotency and unlimited proliferative capacity) also tiation along specific lineages that have not beenraise safety concerns. In particular, undifferentiated possible with hematopoietic stem cells,” Zanjani says.embryonic stem cells can give rise to teratomas, “And if they [embryonic stem cells] could be used [intumors composed of a number of different tissue the laboratory] as a model for differentiation, youtypes (see Chapter 10. Assessing Human Stem Cell could evaluate … vectors for gene delivery and getSafety). It may thus be preferable to use a differen- an idea of how genes are translated in patients.”tiated derivative of genetically modified embryonic Cynthia Dunbar, a gene therapy researcher at thestem cells that can still give rise to a limited number National Institutes of Health, similarly notes thatof cell types (akin to an adult stem cell). Cautions embryonic stem cells could be useful not only inEsmail Zanjani of the University of Nevada, “We could screening new viral and nonviral vectors designed todifferentiate embryonic stem cells into, say, liver cells, introduce therapeutic transgenes into cells, but espe-and then use them, but I don’t see how we can take cially for testing levels of production of the therapeu-embryonic stem cells per se and put genes into tic agent after the embryonic stem cells differentiatethem to use therapeutically” [26]. in culture [9]. Explains Dunbar, “These behaviors are hard to predict for human cells based on animalFurther research is needed to determine whether the studies … so this would be a very useful laboratorydifferentiated stem cells retain the advantages, such tool.” Indeed, the major contribution of embryonicas longer life span, of the embryonic stem cells from stem cells to gene therapy may be to advance thewhich they were derived. Because of the difficulty in general scientific knowledge needed to overcomeisolating and purifying many of the types of adult many of the current technical hurdles to successfulstem cells, embryonic stem cells may still be better therapeutic gene transfer.targets for gene transfer. The versatile embryonicstem cell could be genetically modified, and then, intheory, it could be induced to give rise to all varieties REFERENCESof adult stem cells. Also, since the genetically modi- 1. Aboody, K.S., Brown, A., Rainov, N.G., Bower, K.A., Liu, S.,fied stem cells can be easily expanded, large, pure Yang, W., Small, J.E., Herrlinger, U., Ourednik, V., Black, P.M., Breakefield, X.O., and Snyder, E.Y. (2000). Neural stem cellspopulations of the differentiated cells could be pro- display extensive tropism for pathology in adult brain:duced and saved. Even if the differentiated cells evidence from intracranial gliomas. Proc. Natl. Acad. Sci.were not as long-lived as the embryonic stem cells, U. S. A. 97, 12846-12851.there would still be sufficient genetically modifiedcells to give to the patient whenever the needarises again. 104
  • Use of Genetically Modified Stem Cells in Experimental Gene Therapies 2. Amit, M., Carpenter, M.K., Inokuma, M.S., Chiu, C.P Harris, ., 15. Luther-Wyrsch, A., Costello, E., Thali, M., Buetti, E., Nissen, C., C.P Waknitz, M.A., Itskovitz-Eldor, J., and Thomson, J.A. ., Surbek, D., Holzgreve, W., Gratwohl, A., Tichelli, A., and (2000). Clonally derived human embryonic stem cell lines Wodnar-Filipowicz, A. (2001). Stable transduction with lenti- maintain pluripotency and proliferative potential for viral vectors and amplification of immature hematopoietic prolonged periods of culture. Dev. Biol. 227, 271-278. progenitors from cord blood of preterm human fetuses. Hum. Gene. Ther. 12, 377-389. 3. Armstrong, L., Lako, M., Lincoln, J., Cairns, P.M., and Hole, N. (2000). mTert expression correlates with telomerase activity 16. Mohajeri, M.H., Figlewicz, D.A., and Bohn, M.C. (1999). during the differentiation of murine embryonic stem cells. Intramuscular grafts of myoblasts genetically modified to Mech. Dev. 97, 109-116. secrete glial cell line-derived neurotrophic factor prevent motoneuron loss and disease progression in a mouse 4. Betts, D.H., Bordignon, V., Hill, J.R., Winger, Q., Westhusin, model of familial amyotrophic lateral sclerosis. Hum. Gene M.E., Smith, L.C., and King, W.A. (2001). Reprogramming of Ther. 10, 1853-1866. telomerase activity and rebuilding of telomere length in cloned cattle. Proc. Natl. Acad. Sci. U. S. A. 98, 1077-1082. 17. Ozawa, C.R., Springer, M.L., and Blau, H.M. (2000). A novel means of drug delivery: myoblast-mediated gene therapy 5. Cavazzana-Calvo, M., Hacein-Bey, S., de Saint, B.G., Gross, and regulatable retroviral vectors. Annu. Rev. Pharmacol. F., Yvon, E., Nusbaum, P Selz, F., Hue, C., Certain, S., ., Toxicol. 40, 295-317. Casanova, J.L., Bousso, P Deist, F.L., and Fischer, A. (2000). ., Gene therapy of human severe combined immuno- 18. Pannell, D., Osborne, C.S., Yao, S., Sukonnik, T., Pasceri, P ., deficiency (SCID)-X1 disease. Science. 288, 669-672. Karaiskakis, A., Okano, M., Li, E., Lipshitz, H.D., and Ellis, J. (2000). Retrovirus vector silencing is de novo methylase 6. Challita, P and Kohn, D.B. (1994). Lack of expression from .M. independent and marked by a repressive histone code. a retroviral vector after transduction of murine hemato- EMBO J. 19, 5884-5894. poietic stem cells is associated with methylation in vivo. Proc. Natl. Acad. Sci. U. S. A. 91, 2567-2571. 19. Park, F., Ohashi, K., Chiu, W., Naldini, L., and Kay, M.A. (2000). Efficient lentiviral transduction of liver requires cell 7. Chen, W.Y. and Townes, T.M. (2000). Molecular mechanism cycling in vivo. Nat. Genet. 24, 49-52. for silencing virally transduced genes involves histone deacetylation and chromatin condensation. Proc. Natl. 20. Rathjen, P.D., Lake, J., Whyatt, L.M., Bettess, M.D., and Acad. Sci. U. S. A. 97, 377-382. Rathjen, J. (1998). Properties and uses of embryonic stem cells: prospects for application to human biology and 8. Cherry, S.R., Biniszkiewicz, D., van Parijs, L., Baltimore, D., and gene therapy. Reprod. Fertil. Dev. 10, 31-47. Jaenisch, R. (2000). Retroviral expression in embryonic stem cells and hematopoietic stem cells. Mol. Cell. Biol. 20, 21. Shields, L.E., Kiem, H.P and Andrews, R.G. (2000). Highly ., 7419-7426. efficient gene transfer into preterm CD34+ hematopoietic progenitor cells. Am. J. Obstet. Gynecol. 183, 732-737. 9. Dunbar, C., personal communication. 22. Struhl, K. (1998). Histone acetylation and transcriptional10. Gearhart, J. (1998). New potential for human embryonic regulatory mechanisms. Genes. Dev. 12, 599-606. stem cells. Science. 282, 1061-1062. 23. Verma, I., personal communication.11. Halene, S. and Kohn, D.B. (2000). Gene therapy using hematopoietic stem cells: Sisyphus approaches the crest. 24. Wade, P.A., Pruss, D., and Wolffe, A.P (1997). Histone . Hum. Gene Ther. 11, 1259-1267. acetylation: chromatin in action. Trends Biochem. Sci. 22, 128-132.12. Hawley, R., personal communication. 25. Yoder, M.C. and Hiatt, K. (1999). Murine yolk sac and bone13. Korin, Y.D. and Zack, J.A. (1998). Progression to the G(1)b marrow hematopoietic cells with high proliferative potential phase of the cell cycle is required for completion of display different capacities for producing colony-forming human immunodeficiency virus type 1 reverse transcription cells ex vivo. J. Hemato. Stem Cell Res. 8, 421-430. in T cells. J. Virol. 72, 3161-3168. 26. Zanjani, E., personal communication.14. Laurencin, C.T., Attawia, M.A., Lu, L.Q., Borden, M.D., Lu, H.H., Gorum, W.J., and Lieberman, J.R. (2001). Poly(lactide-co- glycolide)/hydroxyapatite delivery of BMP-2-producing cells: a regional gene therapy approach to bone regeneration. Biomaterials. 22, 1271-1277. 105
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  • APPENDIX A: EARLY DEVELOPMENTHow does a single cell—the fertilized egg—give rise hydra, the microscopic roundworm Caenorhabditisto a complex, multicellular organism? The question elegans, and the fruit fly Drosophila melanogaster;reflects one of the greatest mysteries of life, and rep- and vertebrates such as amphibians, chick embryosresents a fundamental challenge in developmental and more recently, zebrafish Danio rerio, which arebiology. As yet, knowledge about the processes by transparent as embryos and allow the detailedwhich a fertilized egg divides (cleavage), forms a ball monitoring of cell differentiation and migration duringof cells (morula), develops a cavity (blastocyst stage), development. The vast majority of studies on embryo-forms the three primary germ layers of cells that will genesis in mammals has been conducted in mice.ultimately give rise to all the cell types of the body For obvious ethical reasons, detailed research on(gastrula stage), and ultimately generates all the human embryos has been limited. But the study ofspecialized tissues and organs of a mature organism embryogenesis in all of these systems yields as manyis far from complete. Little is known about the specific questions as answers.genes that regulate these early events or how inter-actions among cells or how cellular interactions with For example, what signals the earliest cell differen-other factors in the three-dimensional environment of tiation events in the embryo? What regulates thethe early embryo affect development. The processes activity of genes that are important for embryonicby which a fertilized egg becomes an embryo, development? How and when are the axes of thecalled embryogenesis, include coordinated cell embryo’s body—anterior-posterior (head-tail), dorsal-division, cell specialization, cell migration, and ventral (back-belly), left-right—determined? What rolegenetically programmed cell death [24, 35]. does genetically controlled cell death, also known as apoptosis, play in embryogenesis? What influencesA description of the stages of early embryogenesis in the cell cycle, the controlled series of molecularhumans and mice follows. It includes an explanation events that leads to cell division or the cessation ofof some of the more technical terms and concepts cell division?that are used throughout the document. It alsoincludes a selective discussion of some of the genes, Much of the information about human embryonicmolecules, signaling pathways, and other influences development comes from studies of embryogenesison early embryonic development in the living organ- in the mouse. Like mammalian embryonic develop-ism (in vivo) that are used in experiments with stem ment in general, many aspects of embryogenesis incells maintained in the laboratory (in vitro). mice resembles that of humans, but development in mice also differs in several important respects from human development. For example, embryonic andEXPERIMENTAL SYSTEMS USED TO fetal development in mice takes 18 to 20 days; inUNDERSTAND EMBRYOGENESIS humans, the process takes nine months. The placentaMany kinds of experimental systems have been used forms and functions differently in the two species. Into understand how a fertilized egg produces a blas- humans, an embryonic disk develops after thetocyst, the first structure in which any cell specializa- embryo implants in the uterine wall, whereas in micetion occurs, and a gastrula, in which the three embry- an egg cylinder forms. The yolk sac of a mouseonic germ layers—endoderm, mesoderm, and embryo persists and functions throughout gestation;ectoderm—first appear. They include experiments in humans, the yolk sac functions only in earlywith yeast cells; invertebrates such as tiny, jellyfish-like embryogenesis [28]. The primary roles of the human A-1
  • Appendix A: Early Development embryonic yolk sac are to initiate hematopoiesis and A FERTILIZED EGG FORMS A help in the formation of the primary germ cells, which BLASTOCYST will ultimately differentiate into eggs and sperm in the adult. And even for mice, knowledge about the genes, Prior to fertilization in humans and mice, the egg factors, and signal-transduction pathways that control (oocyte) enlarges, divides by meiosis, and matures in embryonic development is limited. Signal transduction its ovarian follicle until it reaches a stage of meiotic is a series of molecular events triggered by a signal at division called metaphase II (see Figure A.1. Cell the surface of the cell and leading to a response by Cycle). At this point, the follicle releases the oocyte the cell—the secretion of a hormone, or a change in into the oviduct, one of two tube-like structures that the activity of a particular gene, for instance [20]. lead from the ovaries to the uterus. The mature oocyte, a haploid cell that contains half the normal Other sources of information about human develop- number of chromosomes, is surrounded by a protec- ment include studies of human embryonal carcino- tive coat of noncellular material (made of extra- ma (EC) cells maintained in vitro and of histological cellular matrix and glycoproteins), called the zona sections of human embryos. (EC cells are derived pellucida. For fertilization to occur, a haploid sperm from unusual tumors called teratocarcinomas, which cell must bind to and penetrate the zona pellucida, may form spontaneously in the human testis or fuse with the cell membrane of the oocyte, enter the ovary.) Also, within the past 20 years, clinics and oocyte cytoplasm, and fuse its pronucleus with the research institutes in many countries have developed oocyte pronucleus. Fusion of the sperm and egg in vitro conditions that allow fertilization and blastocyst pronuclei restores the number of chromosomes that formation. Thus, the study of methods to improve is typical of a given species. In humans, the normal pregnancy rates following in vitro fertilization (IVF) has diploid number of chromosomes for all the cells of yielded important information about early human the body (somatic cells) is 46 (23 pairs of chromo- embryogenesis. somes). Mature sperm and egg cells (germ cells) Cdk1/CycA, CycB Cdk 2,3,4/CycD (Mitosis) M (Gap 2) G2 G1 (Gap 1) S© 2001 Terese Winslow, Lydia Kibiuk Cdk2/CycE Cdk2/CycA Figure A.1. Cell Cycle. A-2
  • Appendix A: Early Developmentin contrast, carry only 23 chromosomes, the haploid By the 16-cell stage, the compacted embryo isnumber [1]. termed a morula. In mice, the first evidence that cells have become specialized occurs when the outerUnder normal conditions, fertilization of the human cells of the 16-cell morula divide to produce an outeroocyte occurs in the oviduct, near the ovary. A rim of cells—the trophectoderm—and an inner corehuman egg is many times larger than a sperm cell, of cells, the inner cell mass [19]. Although the signalswhich means the oocyte contributes most of the within the 16-cell morula that regulate the differentia-cytoplasm of the zygote, another name for the fertil- tion of the trophectoderm are largely unknown, it isized egg. As a result, any maternal gene products in clear that the outer cells of the morula are polarized.the zygote cytoplasm influence its first few divisions, That is, one side of the cell differs from the other side.called cleavages. Within several days, and after Thus, in the first differentiation event of embryogene-several cleavages, the genome (all the DNA or sis, the outer, polar cells give rise to trophectodermhereditary information in the cell’s chromosomes) of and the inner, apolar cells become the inner cellthe zygote becomes activated and controls subse- mass. This suggests that individual cells of the earlyquent embryonic development [19, 28]. Also, during embryo exhibit more intrinsic polarity than hadthese initial cleavages, the resulting daughter cells do been thought [27].not increase in size. Rather, as early cell division pro-ceeds, the amount of cytoplasm of each daughter Ultimately, the cells of the inner cell mass will give risecell is reduced by half, and the total volume of the to all the tissues of the embryo’s body, as well as toearly embryo remains unchanged from that of the the nontrophoblast tissues that support the develop-fertilized egg [30, 35]. ing embryo. The latter are referred to as extra- embryonic tissues and include the yolk sac, allantois,After fertilization, the zygote makes its way to the and amnion. The trophectoderm, in turn, will gener-uterus, a journey that takes three to four days in mice ate the trophoblast cells of the chorion, the embryo’sand five to seven days in humans. As it travels, the contribution to the extraembryonic tissue known aszygote divides. The first cleavage produces two the placenta [19, 28].identical cells and then divides again to produce fourcells. If these cells separate, genetically identical The cells of the inner cell mass and trophectodermembryos result, the basis of identical twinning. Usually, continue to divide. Information gained from the studyhowever, the cells remain together, dividing asynchro- of mouse embryos suggests that the two tissues neednously to produce 8 cells, 16 cells, and so on [19]. to interact; the inner cell mass helps maintain theEach early round of cell division takes approximately ability of trophectoderm cells to divide, and the36 hours, according to information gleaned from the trophectoderm appears to support the continuedstudy of human embryos in vitro [34]. In humans and development of the inner cell mass [32]. Secretedmice, at about the eight-cell stage, the embryo paracrine factors (molecular signals that affect othercompacts, meaning that the formerly “loose” ball of cell types), including fibroblast growth factor-4cells comes together in a tight array that is inter- (FGF-4), which is released from inner cell mass cellsconnected by gap junctions. These specialized [46], help direct embryogenesis at this stage. FGF-4membrane structures consist of an array of six protein signaling also helps regulate the division and differ-molecules called connexins, which form a pore that entiation of trophectoderm cells [29].allows the exchange of ions and small molecules By embryonic day 3 (E3.0) in the mouse and days 5between cells [27]. to 6 in human development [14], the embryo devel-Recent information from studies of mouse embryos ops a cavity called the blastocoel. It fills with a wateryindicates that even at this early stage of embryo- fluid secreted by trophectodermal cells and trans-genesis, the anterior-posterior axis of the embryo has ported in from the exterior. As a result of cavitationbeen established, a point of some concern for in and the physical separation and differentiation of thevitro fertilization techniques, which disrupt early trophectoderm from the inner cell mass, the morulapatterning events. The establishment of the anterior- becomes a blastocyst. Its chief structural features areposterior axis is critical to normal fetal development, the outer sphere of flattened trophectoderm cellsbecause it helps determine the overall body plan of (which become the trophoblast), the small, roundthe embryo [3, 15, 18]. A-3
  • Appendix A: Early Development DAY 2 Uterus DAY 1 DAY 3–4 First cleavage 2-cell stage 4-cell stage 8-cell uncompacted DAY 4 morula 8-cell compacted Fertilized egg morula (zygote) DAY 5 Trophectoderm Ovary Early Blastocoel DAY 0 blastocyst Inner Fertilization cell mass DAY 6–7 Zona pellucida Late-stage blastocyst (hatching) DAY 8–9 Ovulation Oocyte Epiblast© 2001 Terese Winslow Implantation of the blastocyst Hypoblast Figure A.2. Development of the Preimplantation Blastocyst in Humans. cells of the inner cell mass, and the fluid-filled istry of the cells change as they increase in number blastocoel [3, 19]. and begin to differentiate. For example, the primary sources of energy for the cleavage-stage embryo By E4.0 in mice, and between 5 to 7 days post- are pyruvate, lactate, and amino acids—simple fertilization in humans, the blastocyst reaches the molecules that play important roles in various meta- uterus. It has not yet implanted into the uterine wall bolic pathways. But after compaction of the morula, and is therefore still a pre-implantation embryo. When glucose is taken up by the embryo and used as a it arrives in the uterus, the blastocyst “hatches” out of primary source of energy [15]. Indeed, mammalian the zona pellucida, the structure that originally blastocysts may have a unique transporter molecule, surrounded the oocyte and that also prevented the GLUT8, that ferries glucose into the blastocyst. GLUT8 implantation of the blastocyst into the wall of the appears in the blastocyst at the same time as the oviduct [19]. (An embryo that does implant in the receptor for insulin-like growth factor–1 (IGF-1). Thus, oviduct results in a tubal pregnancy, which can result the blastocyst, which requires a great deal of energy in severe hemorrhaging.) at this stage of development, is equipped to respond The nutritional requirements of the embryo change to insulin by taking up glucose [7]. markedly during the time from zygote formation to These and other observations about the preimplan- the compaction of the morula, to the development tation blastocyst have led to recommendations of the blastocyst. Also, the physiology and biochem- A-4
  • Appendix A: Early Development E2.5 Zona E2.0 pellucida E3.0 Inner 4-cells cell mass E4.0 8-cells E1.5 uncompacted Trophoblast 2-cells 8-cells Epiblast compacted Hypoblast Ovary Early E4.5 blastocyst (hatching) E1.0 Late Oocyte blastocyst Uterus Fertilized Implantation egg (zygote) E0 Ovulation Fertilization E0.5© 2001 Terese Winslow Figure A.3. Development of the Preimplantation Blastocyst in Mice from Embryonic Day 0 (E0) Through Day 5 (E5.0). about the importance of adapting the culture of ES cells occurs only in a laboratory culture dish conditions to accommodate the changing nutritional [41]. ES cells that are grown in the laboratory most requirements of the embryo when animal embryos closely resemble cells of the epiblast [5], but ES cells are grown in the laboratory [16]. are not identical to epiblast cells [42]. The term epiblast refers to all the pluripotent cell populations It is at this stage of embryogenesis—near the end of that follow the formation of the primitive endoderm the first week of development in humans and about and precede the formation of the gastrula [23]. Like E4.0 in mice—that embryonic stem (ES) cells can be the epiblast cells of the embryo, ES cells in culture derived from the inner cell mass of the blastocyst. have the potential to give rise to all the cell types Human ES cells are derived from embryos generated of the body. However, unlike the epiblast cells of through in vitro fertilization procedures and donated the embryo, ES cells in vitro cannot give rise to a for research. An embryo at this stage of development complete organism. They do not have the three- in vivo would not yet be physically connected to the dimensional environment that is essential for uterine wall; it would still be a preimplantation embryonic development in vivo, and they lack the embryo. trophectoderm and other tissues that support fetal ES cells, per se, may be an in vitro phenomenon. development in vivo (see Chapter 2. The Embryonic Some scientists argue that the apparent immortality Stem Cell). A-5
  • Appendix A: Early DevelopmentTHE BLASTOCYST IMPLANTS IN THE comprise the trophectoderm and the primitiveUTERINE WALL endoderm. The preimplantation mouse embryo consists of approximately 200 cells, approximately 20Many of the molecular and cellular events that occur to 25 of which are inner cell mass or epiblast cellsduring the second week of human embryonic devel- [20, 23]. The day 5, preimplantation human embryoopment, and at the end of the first week of embryo- contains 200 to 250 cells, only 30 to 34 of which aregenesis in the mouse, help establish the placenta. inner cell mass cells [4].The placenta connects the fetal and maternal blood-streams and provides nutrients to the embryo The extraembryonic cells of both species differentiatethroughout the remainder of gestation. into the tissues that will convey nutrients to the embryo and remove its waste products. For example,On or about postfertilization days 8 to 9 in humans some of the trophoblast cells invade the epithelial(and E4.5 in the mouse), the ball-shaped embryo lining of the uterus (also known as the decidua), andimplants into in the uterine wall (see Figure A.2. form a multinucleated tissue called a syncitium. ThisDevelopment of the Preimplantation Blastocyst in syncytiotrophoblast, as it is called, then developsHumans). The inner cell mass of the human embryo lacunae (cavities). By postfertilization day 10 to11 inat this stage has split into layers. One is the hypoblast, humans, the syncytiotrophoblast becomes suppliedwhich lies next to the blastocoel and gives rise to the with maternal blood vessels. The fusion of the embry-primitive endoderm. (Later, the primitive endoderm onic chorion and the maternal decidua and vascularwill give rise to the outer layer of the yolk sac, a tissue generates the placenta [19, 29].curious reminder of reptilian ancestry in mammalianembryos.) The other cell layer that develops from the The formation of the placenta is a critical process ininner cell mass is the epiblast. It will give rise to all the human embryogenesis. Without a healthy placenta,cells of the embryo’s body [19, 23]. the embryo does not survive; its malformation can trigger a spontaneous abortion [49]. The placentaThe epiblast can be thought of as the group of cells anchors the developing embryo to the uterine wallthat succeeds the inner cell mass. Pluripotent cells and connects it to the maternal bloodstream, thusare defined differently in scientific articles and text supplying the embryo with ions and metabolites andbooks. In general, however, pluripotent cells are providing a waste-removal mechanism for thecapable of giving rise to all the kinds of cells that embryo [10, 26]. Later, the umbilical cord connectsoccur in the mature organism. So at this stage of the embryo to the chorion portion of the placenta.embryogenesis, the only pluripotent cells are the The cord contains the fetal arteries and veins. Usually,undifferentiated cells of the epiblast. the maternal and fetal blood do not mix directly. Instead, soluble substances pass through fingerlikeBy E6.0 in the mouse, three differentiated cell types projections called villi that have embedded in theexist: the trophoblast, the epiblast (also called the uterine wall, and that have also developed from theembryonic ectoderm or primitive ectoderm at this trophoblast of the embryo [19].stage), and the primitive endoderm (see Figure A.3.Development of the Preimplantation Blastocyst in In mice, some of the genes that regulate the devel-Mice). During the next major phase of development, opment of the placenta have been identified. Onetermed gastrulation, the embryonic ectoderm will is the Mash2 gene, which is expressed in the tropho-differentiate into the three primary germ layers— blast cells of the embryo after it implants into theendoderm, mesoderm, and ectoderm. Thus, the uterine wall. If Mash2 is inactivated, the placentaembryonic ectoderm has succeeded the epiblast as does not form and the embryo dies (at E10.5 in thethe tissue that will generate the body of the embryo. mouse) [21, 45]. However, it is not known whether theThe primitive endoderm differentiates into parietal same genes that regulate placenta formation in theand visceral endoderm, the anterior region of which mouse act in humans.will help regulate the development of the body planduring gastrulation [23]. Meanwhile, during postfertilization days 7 to 14 of human development, the epiblast splits to form thePrior to gastrulation, the majority of cells (approxi- amnionic cavity. The cavity fills with fluid and cushionsmately 75 percent) in the preimplantation blastocyst the embryo throughout gestation. A-6
  • Appendix A: Early DevelopmentTHE BLASTOCYST BECOMES Meanwhile, the anterior region of the mesoderm thatA GASTRULA develops from the primitive streak is preparing to give rise to the heart. The anterior epiblast is generatingAt the start of the third week of human development, the neuroectoderm and the ectoderm that coversand about E6.0 in the mouse (the egg-cylinder the surface of the embryo. The ectodermal tissuestage), the cells of the epiblast begin to differentiate. that lies dorsal to the notochord will generate theBy the end of the third week, they will have generated neural plate, which will round up to form the neuralthe three primary germ layers of the embryo—endo- tube, the precursor to the central nervous systemderm, mesoderm, and ectoderm. A detailed descrip- (brain and spinal cord) [23].tion of all the events of this critical stage of differen-tiation—known as gastrulation—is beyond the scope Thus, by the end of the third week of embryonicof this report. However, the onset of gastrulation is development in humans, and by E8.0 in the mouse,triggered at the posterior end of the embryo with the the primitive ectoderm of the postimplantationformation of a structure called the node (from blastocyst has generated the ectoderm, mesoderm,Hensen’s node in chick embryogenesis). The node, and endoderm of the gastrula (see Figure A.4.together with another important signaling center, the Development of Human Embryonic Tissues). Theseanterior visceral endoderm (AVE), helps regulate the and other complex processes result in the formationformation of the pattern of the embryo’s body at this of the tissues and organs that occur in an adultstage of development [19]. mammal (see Figure 1.1. Differentiation of Human Tissues). They require the activation and inactivationThe process of gastrulation begins between days 14 of specific genes at specific times, highly integratedand 16 of human development and at about E6.5 in cell-cell interactions, and interactions between cellsthe mouse. At that time, a primitive streak forms in a and their noncellular environment, the extracellularspecific region of the epiblast along the posterior axis matrix [3, 19].of the embryo. Little is known about the signals thatregulate the generation of the primitive streak, In general, the embryonic “outer” layer, or ectoderm,although the genes goosecoid, T, Evx-1, and follistatin gives rise to the following tissues: central nervousare expressed [23]. Nevertheless, the forward migra- system (brain and spinal cord) and peripheral nervoustion of the posterior epiblast cells occurs as their system; outer surface or skin of the organism; corneacell-cell contacts break down, and they release and lens of the eye; epithelium that lines the mouthenzymes that digest the basement membrane that and nasal cavities and the anal canal; epitheliumlies underneath. This allows the epiblast cells to of the pineal gland, pituitary gland, and adrenalmigrate into the space between the epiblast and medulla; and cells of the neural crest (which givesthe visceral endoderm [6]. rise to various facial structures, pigmented skin cells called melanocytes, and dorsal root ganglia, clustersThe forward-moving epiblast cells also spread later- of nerve cells along the spinal cord). The embryonically, a migration that induces the formation of the “middle” layer, or mesoderm, gives rise to skeletal,mesoderm and the notochord. The notochord is a smooth, and cardiac muscle; structures of thetemporary, rod-like structure that develops along the urogenital system (kidneys, ureters, gonads, anddorsal surface of the embryo and will ultimately reproductive ducts); bone marrow and blood; fat;connect the anterior visceral endoderm (AVE) and bone, and cartilage; other connective tissues; andthe node. Cells at the anterior end of the notochord the lining of the body cavity. The embryonic “inner”will eventually underlie the forebrain [19]. layer, or endoderm, gives rise to the epithelium of the entire digestive tract (excluding the mouth and analAt the anterior end of the primitive streak is the node, canal); epithelium of the respiratory tract; structuresa two-layered structure and important signaling associated with the digestive tract (liver and pan-center in the embryo. The ventral layer of cells in the creas); thyroid, parathyroid, and thymus glands;node comes from the epiblast and generates the epithelium of the reproductive ducts and glands;notochordal plate, which then forms the notochord. epithelium of the urethra and bladder [19].Endoderm, which will give rise to the gut, also devel-ops near the node, along the sides of the notochord. A-7
  • Appendix A: Early Development Fertilized egg DAY 0 Extraembryonic Embryonic Fertilized egg Tissues Tissues Blastocyst DAY 5 Blastocyst DAY Inner cell mass 6-7 Trophoblast Inner cell mass Trophoblast DAY Epiblast 8-9 Hypoblast Cytotrophoblast Hypoblast Epiblast Syncytiotrophoblast DAY Cytotrophoblast 12 Syncytiotro- Extraembryonic Amniotic Primitive phoblast endoderm ectoderm ectoderm DAY 14 Gastrula Yolk sac Primitive streak DAY Primitive 15 streak Extraembryonic Embryonic Embryonic Embryonic mesoderm mesoderm endoderm ectoderm Embryonic ectoderm DAY 16© 2001 Terese Winslow Embryonic mesoderm Embryonic endoderm Figure A.4. Development of Human Embryonic Tissues. A-8
  • Appendix A: Early Development PRIMORDIAL GERM CELLS ARE THE parent—a process termed genomic imprinting PRECURSORS TO EGGS AND SPERM (discussed below). Not to be forgotten in this developmental scheme Another feature that distinguishes primordial germ are the primordial germ (PG) cells, which will give rise cells from somatic cells is their continuous expression to eggs and sperm in the adult organism (see Figure of Oct-4, a transcription factor produced by prolifer- A.5. Development of Mouse Embryonic Primordial ating, unspecialized cells. Thus, the regulation of PG Germ Cells). Prior to gastrulation, at about the time of cell fate in the mammalian embryo is a result of the primitive streak formation, these precursor cells split local environment of the cells, a recurring theme in off from the proximal region of the epiblast and mammalian embryogenesis, and the expression of migrate into the extraembryonic mesoderm (which genes in the PG cells [37]. Later in development, the generates the yolk sac and allantois). It is not until the PG cells embark in another migration and ultimately proximal epiblast cells reach the extraembryonic come to rest in the genital ridge, the tissue that will mesoderm that they are committed to becoming give rise to the gonads: testes in males and ovaries PG cells. Their location in this tissue—which is remote in females [35]. In the testis, the PG cells give rise to from the rest of the embryo’s body, or somatic, spermatagonial stem cells that reside in the testis cells—may allow PG cells to avoid some of the throughout the life of the male. They continuously events that drive somatic cells through the process of renew themselves and differentiate through the differentiation. One such event is DNA methylation, a process of spermatogenesis into mature, functional means of silencing genes inherited from one sperm cells. There is no evidence, however, that they have pluripotential properties [39]. Fertilization Sperm Blastocyst Oocyte Maturity Primordial germ cell E5.5 precursors Fetal growth Migration of E7.5 Primordial germ cells© 2001 Terese Winslow, Caitlin Duckwall Primordial germ cells Genital ridge E10.5 Primordial E8.5 germ cells Figure A.5. Development of Mouse Embryonic Primordial Germ Cells. A-9
  • Appendix A: Early DevelopmentGENES, MOLECULES, AND OTHER initiate the process of making a molecule of messen-SIGNALS ARE IMPORTANT IN EARLY ger mRNA. As indicated above, the sequence of bases in DNA—the order of A, T, C, and G—dictatesEMBRYOGENESIS the sequence of the mRNA which will be formed.This overview of the processes of blastocyst formation, Thus, an A in DNA can bind only to a U in mRNA. Theimplantation, and gastrulation has ignored most of DNA base G will bind only to the RNA base C, and sothe crucial signals that direct embryonic develop- on. RNA polymerase connects these bases togetherment. These signals include genes expressed by cells in a process called elongation.at different stages of development, molecular factors The second major stage of the process of makingsecreted by cells, complex molecular signaling proteins based on the code of DNA is called trans-systems that allow cells to respond to secreted lation. During translation, the mRNA—which wasfactors, specialized membrane junctions that generated in the nucleus of a cell and now carries itsconnect cells and allow them to communicate, transcript of the DNA code—moves to the cytoplasm,components of the noncellular environment (known where it attaches temporarily to tiny structures calledas the extracellular matrix), and genomic imprinting. ribosomes. There, molecules of mRNA direct theThese signals—as well as their origins and effects— assembly of small molecules called amino acids (ofare the least understood elements of embryonic which 20 kinds exist) into proteins. Each amino acid isdevelopment in any organism. specified by a code of three bases. The helpers in this effort are molecules of transfer RNA (tRNA). EachGENE TRANSCRIPTION, TRANS- tRNA molecule contains its own triplet code (toLATION, AND PROTEIN SYNTHESIS match the mRNA code), and each tRNA ferries a particular kind of amino acid to the mRNA-ladenA gene is a linear segment of a DNA molecule that ribosomes.encodes one or more proteins. The process occurs inthree major steps (see Figure A.6. Gene Transcription, Then, in the third step of protein synthesis, the aminoTranslation, and Protein Synthesis). The DNA, which is acids are linked through chemical bonds to createdouble-stranded, unwinds and copies its triplet code a protein molecule. Proteins typically consist of(varying sequences of the four nitrogen bases ade- hundreds of amino acids. Thus, the sequence ofnine (A), thymine (T), cytosine (C), and guanine (G)) bases in DNA determines the sequence of mRNA,into a messenger RNA (mRNA) molecule. In RNA, which then determines the linear sequence of aminouracil (U) is substituted for T. The process is called acids in a protein. Depending on its sequence oftranscription because the triplet code of a DNA amino acids, a protein may fold, twist, bend, pleat,molecule is transcribed into the triplet code of an coil, or otherwise contort itself until it assumes themRNA molecule. A gene that makes an mRNA tran- three-dimensional shape that makes it functional.script is active; the gene is said to be expressed. In the body, proteins make up most of the structuralThe process of initiating transcription is complex. elements of cells and tissues. They also function asIt requires the binding of certain proteins, called enzymes, which regulate all of the body’s chemicaltranscription factors, to regions of the DNA near the reactions.site where transcription begins. Transcription factorsbind at sequences of DNA called the promoter Gene Expression and Factors in theenhancer region. The factors can activate or repress Preimplantation Blastocysttranscription. Although some transcription factors bind It is difficult to identify the genes and factors in vivodirectly to the DNA molecule, many bind to other that affect the earliest events in mammalian devel-transcription factors. Thus, protein-DNA interactions opment; maintain the undifferentiated, proliferatingand protein-protein interactions regulate gene state of inner cell mass or epiblast cells; regulateactivity. Their interactions then activate or block the implantation; and direct the differentiation of cellsprocess of transcription. along specific developmental pathways, or cell line- ages. The embryo itself is very small and, in vivo, isTranscription actually begins when the enzyme RNA almost wholly inaccessible to study. Therefore, manypolymerase II binds to the promoter region of DNA to A-10
  • Appendix A: Early Development Cell Nucleus Transcription Translation Cytoplasm Nuclear Membrane Beginning Nearly completed of protein protein DNA Newly formed messenger RNA (mRNA) mRNA Bases Ribosome Double helix unwinds Codon Nucleotide THE ROLE OF THE RIBOSOME IN TRANSLATION Amino acid binds to growing protein chain Alanine Proline Proline Amino Newly formed Argin acid protein chain ine Isole Leuc ucin in e e tRNA mRNA tRNA binds Nucleus to codon Ribosome Codon© 2001 Terese Winslow Cytoplasm Completed protein Alanine Proline Proline Arginine Leucine Isoleucine Figure A.6. Gene Transcription, Translation, and Protein Synthesis. of the genetic and molecular influences that are now gastrulation in the mouse. The gene for Oct-4, known to regulate early embryogenesis in vivo were Pou5f1, is expressed in primordial germ cells, however identified by studying mouse embryonic stem cells [12, 32]. in vitro. Oct-4 is a member of the class 5 POU (for Pit, Oct, For instance, Oct-4 is a transcription factor that has and Unc) family of transcription factors, which bind come to be recognized as a prototypical marker of promoter or enhancer sites in DNA. These proteins undifferentiated, dividing cells. It is necessary for regulate gene transcription. The transcription factor maintaining the undifferentiated state and prolifera- Oct-4 can activate or repress gene expression; it tion of cells of the inner cell mass and the epiblast. binds to DNA at a distance from the start of transcrip- Most of the studies of Oct-4 have been conducted tion. Hence, depending on the target gene, Oct-4 in mouse embryos and ES cells. Oct-4 is expressed in may require the presence of co-activator proteins the mouse oocyte, it disappears during the first cleav- such as the E1A-like transcription factors and the age of the zygote, and it reappears in the four-cell Sox2 protein [37]. mouse embryo as the genome of the zygote begins Another target of Oct-4 in mouse embryogenesis is to control embryonic development. Oct-4 persists in the Fgf4 gene. It encodes fibroblast growth factor-4 the inner cell mass of the blastocyst, but does not (FGF-4), a growth factor protein that is expressed occur in differentiated trophectoderm cells, nor does together with Oct-4 in the inner cell mass and it occur other differentiated cell types that arise after A-11
  • Appendix A: Early Developmentepiblast [32]. FGF-4 is a paracrine signal, meaning the dorsal surface, with the largest concentration ofthat it is released from one cell type and it acts on neuronal tissue—the brain—at the anterior end ofanother. In this case, FGF-4 is released from proliferat- the embryo. The limbs develop symmetrically anding inner cell mass cells and it affects the surrounding bilaterally, whereas the heart—although it begins astrophectoderm. FGF-4 may also act as an autocrine a symmetrical structure—ultimately comes to pointsignal, meaning that it may affect the same inner cell toward the left side of the trunk. Some internal struc-mass cells that released it [13]. tures are paired (the kidneys, lungs, adrenal glands, testes, and ovaries), whereas many are not (the heart,A series of recent experiments indicates that the level gut, pancreas, spleen, liver, and uterus) [19].of Oct-4 expression—not simply its presence orabsence in a cell—determines how mouse embry- Information about the establishment of these bodyonic stem cells differentiate and whether they axes and their role in development is far from com-continue to proliferate [33] (see Appendix B. Mouse plete. For example, the anterior-posterior axis of theEmbryonic Stem Cells). mouse blastocyst may be determined before it implants and is certainly established before gastru-Two proteins, leptin and STAT3, which are produced lation [15, 17]. An unanswered question, however, isby maternal granulosa cells that surround the oocyte, whether this early embryonic axis helps determine theare apparently secreted into the oocyte as it matures later development of the embryo. The early axis mayin its ovarian follicle. By the time the mouse or human play a role in primitive streak formation, and requireszygote reaches the four-cell stage, leptin and STAT3 the expression of Wnt, which helps regulate theare concentrated in what may be the founder cell of formation of one of embryo’s chief signaling centers:the embryonic trophectoderm. Later, when the tro- the node [38].phectoderm differentiates and separates from theinner cells mass of the blastocyst, leptin and STAT3 are As indicated above, an important group of cells thatexpressed only in the trophectoderm, where they play produces molecular signals that help determine thea critical role during implantation [12]. anterior-posterior axis of the mouse embryo is the anterior visceral endoderm (AVE). The AVE expressesLeukemia inhibitory factor (LIF), a cytokine, also plays different genes along its length. At E5.0 in the mouse,an important role during implantation. The LIF gene is for example, the Hex gene—a member of the familyexpressed in cultured mouse [31] and bovine blasto- of homeobox genes that help regulate body pattern-cysts, as is the gene for its receptor. The mRNA for the ing of the mouse embryo—is expressed in the distalreceptor for LIF is expressed in human blastocysts [12, visceral endoderm. These cells migrate to become47]. LIF, therefore, seems to be important for early the AVE, which forms on the opposite side of themammalian blastocyst development, as well as embryo from the primitive streak, thus establishing theimplantation. It is also essential for the survival of the anterior-posterior axis of the fetus [17].primordial germ cells, which will become eggs andsperm in the mature organism [22]. And if mouse Then, between E6.0 and E7.0 in the mouse, theembryonic stem (ES) cells are cultured from the inner anterior region of the AVE, where the heart will form,cell mass of a blastocyst without the presence of expresses Mrg1. The medial region expresses the“feeder” layers of cells, they require the addition of LIF transcription factor genes Otx2 and Lim1, as well asto the culture medium in order to survive and pro- other genes. The region of the AVE that lies next to theliferate [40]. Curiously, cultures of human ES cells do part of the epiblast that will give rise to oral ectodermnot respond to LIF [36, 48]. and the forebrain expresses Hesx1, another home- obox gene. Collectively, the AVE and the genes itRegulation of Body Patterning in the Embryo expresses help regulate the development of theAs the embryo forms, its overall body pattern is deter- anterior end of the embryo [3].mined by the establishment of three clear axes—theanterior-posterior axis (head-tail), the dorsal-ventral Other genes, notably Bmp4, also help shape the(back-belly) axis, and left-right asymmetry. The estab- mouse embryo prior to gastrulation. BMP stands forlishment of these body axes at the correct time is bone morphogenetic protein, a family of proteinsfundamental to normal embryonic development. For that help regulate the differentiation of mesenchymalinstance, the central nervous system develops along cells, which are derived from mesoderm, including A-12
  • Appendix A: Early Developmentbone-forming osteoblasts, and adipocytes, which are the early differentiation of the visceral endoderm [18].fat cells. They also play a role in CNS development. Other genes are expressed in the pregastrulationBmp4, which is expressed in the extraembryonic epiblast; examples are Brachyury and Cripto, whichectoderm next to the epiblast and also in the inner encode secreted growth factors. Still others, includingcell mass of the E3.5 and E4.5 mouse blastocyst, Nodal and Otx2, are expressed in both the epiblastmay activate genes in epiblast cells that then and the visceral endoderm [18].migrate to form the primitive streak. Wnt3 apparentlyhelps induce the formation of both the primitive A host of genes is expressed along the primitivestreak and the node in mammals, although there is streak. These include HNF-3ß in the notochord, node,no evidence indicating that Wnt3 expression is and floor plate (which will underlie the forebrain);required for mesoderm induction. However, formation nodal, goosecoid, T, and Lim-1, in the node;of the embryo’s head region, obviously a key anterior Follistatin and T for the remainder of the streak; andstructure, seems to require inhibition of the activities of FGF-4 just caudal to the node.Wnt and Bmp4—a potential role of the AVE [3, 18]. It is far easier to monitor the expression of particularTherefore, coordinating the embryo’s “decisions” genes than it is to identify their function(s) duringabout its body pattern is a hierarchy of genes. development. One of the most useful kinds of experi-Overall, the Hox genes specify anterior-posterior polar- ments for determining the function of a gene involvesity. Their normal function can be subverted by retinoic its permanent inactivation—to create a knockoutacid, which can activate Hox genes in inappropriate mouse, for example—followed by studies of impairedplaces. Less is known about the establishment of the functions in the gene-deficient animal. Similardorsal-ventral axis. It may be determined in the blas- research strategies obviously cannot be used totocyst, or even in the oocyte [16]; it is clearly estab- determine the functions of specific genes in humanlished when the notochord develops. Genes such as embryogenesis. However, it is possible to identifyNodal and Lefty help determine left-right asymmetry. human genes that are important for development byGenes that regulate body patterning in embryonic studying heritable abnormalities or congenital defectsdevelopment are well conserved throughout evolu- that have a genetic basis. Then, the function of thetion among both vertebrates and invertebrates [19]. human genes—which almost certainly will have simi- lar effects in mice—can be assessed in more detailRegulation of Cell Differentiation in Early by generating knockout mice that lack the gene.EmbryogenesisMyriad other genetic and molecular signals conspireto regulate cell differentiation in the embryo. Factors THE CELL CYCLEin a cell’s environment bind to receptor molecules in Many cells of the early embryo are in a constantits membrane and activate a series of intracellular state of dividing or of preparing to divide. The seriesresponses that may result in gene activation or inacti- of molecular events that regulate these processes isvation. The process by which a cell responds to an called the cell cycle (see Figure A.1. Cell Cycle).external signal is called signal transduction, and isitself the subject of many articles and books. The cell cycle includes four main phases: DNA syn- thesis (S phase), G2 (a gap phase during which theOne of the earliest genes to be involved in cell differ- cell increases in size and prepares to divide), cellentiation in the preimplantation blastocyst are those division (also called mitosis, M phase), and G1 (a gapthat encode the GATA class of transcription factors. phase of cell growth and replication of the centrioles).GATA-6 is expressed in some inner cell mass cells of When a cell exits the cell cycle, to differentiate, forthe E3.5 mouse blastocyst; GATA-4 is expressed in the example, it is said to be in G0. Progression throughE5.5 parietal and visceral endoderm. GATA-6 expres- the cell cycle is regulated by the activation of cyclin-sion is required for the formation of the visceral dependent kinases (Cdks), enzymes that attachendoderm; the role of GATA-4 is less clear. Other genes phosphate groups onto other proteins. Particular Cdkssuch as HNF-4, which encodes a transcription factor, and their associated cyclins regulate the transitionand STAT3, which encodes a protein important in a from one phase of the cell cycle to the next. Forcytokine signaling pathway, are expressed later, during example, in mammalian cells, Cdk2 and cyclin E A-13
  • Appendix A: Early Developmentregulate the transition from G1 to S, whereas Cdk1 causes the death of skin cells between fingers andand cyclins A and B regulate the transition from S to toes—the typical “webbing” of fetal digits [19].G2. And recently, it has become clear that the cell Many of the genes that regulate apoptosis werecycle has several checkpoint mechanisms, during discovered in studies of the microscopic roundworm,which the cell stops its progression through the cycle C. elegans. The mammalian counterparts of thesewhile it repairs damaged DNA [11]. genes are very similar in terms of their DNAThe activity of the cell cycle varies, depending on the sequences, and are called homologues. For exam-status of the cell and the cues—such as cytokine ple, in C. elegans, the ced-4 and ced-3 genes arestimulation—the cell receives from its environment. activated (in that order) prior to apoptosis. They, inSome cells “cycle” quickly, dividing in a matter of turn, activate enzymes called caspases, whichhours. Others cycle slowly, and some do not cycle at actually trigger apoptosis. But the regulatory pathwayall. The epiblast cells of the postimplantation E5.5 to that leads to cell death is complex. AnotherE6.0 mouse blastocyst, for example, have a mean apoptosis-control gene called ced-9 can blockcycle time of 11.5 hours. But a day later, at E6.5 to activation of ced-4 and ced-3, and thereby “rescue”E7.0, epiblast cells have a mean cell cycle time of a cell from apoptosis. The mammalian homologuesonly 4.4 hours [23]. In contrast, the cycle time for cells of ced-9 are members of the BCL-2 gene family,in the cleavage-stage human embryo—a much which prevent apoptosis in mammalian cells—earlier developmental stage—is approximately 36 and in C. elegans, if they are introduced into cellshours [34]. And cells that are terminally differentiat- from the worm [19].ed—mature nerve cells in the brain, for example— Another mammalian apoptosis gene, Apaf-1 workshave stopped dividing altogether. What factors regu- with caspase-9 to bring about cell death. It is interest-late the cell cycle during development, or how the ing to note that the silencing of the Apaf-1 gene—cell cycle alters gene expression or any other event rather than a mutation in its DNA sequence—wasin embryogenesis, remains largely unknown. recently linked to cancer metastasis [43]. In fact, several of the genes that normally regulate apoptosisCELL DEATH IS A NORMAL PROCESS inhibit the formation of tumors because they triggerDURING EMBRYOGENESIS the death of cells with damaged DNA that mightIt is a general characteristic of undifferentiated cells— otherwise replicate to produce a tumor. Because ofincluding embryonic cells in vivo or in vitro— that their normal, protective function against the develop-when they stop dividing, they differentiate, become ment of cancer, such genes are termed tumor-quiescent or senescent (stop their progress through suppressor genes. Many tumor-suppressor genes,the cell cycle and enter a period of temporary or including Apaf-1, are associated with the so-calledpermanent “rest”), or die. In vivo or in vitro, the p53 tumor-suppressor pathway. If even one of theprocess of cell death can occur by necrosis or apop- apoptosis-regulating genes becomes mutated, thetosis. The latter is a form of genetically controlled cell tumor-suppressor pathway can fail, a step toward thedeath that, in itself, is an important aspect of normal development of cancer.embryonic development in vivo. When the geneticprogram for apoptosis becomes activated, the cell SOME COMPARISONS BETWEENcommits a form of molecular suicide. Its DNA dis- EMBRYOGENESIS AND ONCOGENESISintegrates in a characteristic manner, blebs (small There are many molecular links between the regula-pouches) form in the cell membrane, and the cell tion of normal embryogenesis and the induction ofdies. The genetic controls for apoptosis differ, cancer, which is called oncogenesis. A compre-depending on the cell type, but all involve activating hensive review of the similarities between the twoproteases called caspases, enzymes that destroy the exceeds the scope of this report. However, it is usefulprotein components of cells. to point out that at least some of the genes, factors,As the body of an embryo develops, apoptosis helps and cell-cell interactions critical for normal embry-shape it. For example, apoptosis helps control the onic development also play a role in—or are alteredspacing of nerve cells in the brain and spinal cord; it in—tumor development. The example cited abovehelps generate the space in the middle ear, and it A-14
  • Appendix A: Early Developmentindicates that some of the genes that function during only if the mutations occur in the germ line. Somaticapoptosis in the embryo also protect the mature mutations of BRCA1 and BRCA2 are not linked toorganism from developing tumors. breast cancer [8].A different, and obvious, parallel between embryo-genesis and oncogenesis can be observed in the DNA METHYLATION AND GENOMICspontaneous formation of tumors in the gonads of IMPRINTING AFFECT EMBRYONICmammals, including humans. These unusual tumors, DEVELOPMENTwhich include teratomas, embryonal carcinomas, DNA methylation is the process of adding methyland teratocarcinomas, develop from the germ cells groups to specific cytosine residues in the promoterin the testes or ovaries. The tumors have provoked a regions of DNA. DNA methylation is a genome-widegreat deal of interest because they often contain phenomenon; it occurs in many genes dependinghighly differentiated cells and tissues such as teeth, on the stage of development and the differentiationhair, neural cells, and epithelial cells. The structures status of a cell. When the methyl groups are boundare disorganized, but often recognizable [2]. at their designated sites in DNA, transcription factorsAlthough teratomas are benign, embryonal carcino- cannot bind to the DNA and gene transcription ismas and teratocarcinomas are highly malignant. The turned off. Also, DNA methylation causes a rearrange-latter contain a kind of stem cell, called an embry- ment of the structure of chromatin, the combinationonal carcinoma (EC) cell, which in mice and humans of DNA and protein that forms the chromosomes.resembles embryonic stem (ES) cells. Human EC cells, DNA methylation patterns change during develop-unlike ES cells, typically have abnormal chromo- ment, and their rearrangement in different tissues atsomes. The chromosomes in mouse EC cells may different times is an important method for controllingappear to be normal, although they carry genetic gene expression [19].defects. Nevertheless, mouse EC cells can contribute Also important to embryonic development is theto normal embryonic development if they are intro- process of genomic imprinting, which causes certainduced into a mouse blastocyst, which is then to be genes turned on or off, depending on whetherimplanted in the uterus of a pseudopregnant female they are inherited from the mother or the father.[2] (see Table A.1. Comparison of Mouse, Monkey, Several mechanisms of genomic imprinting exist inand Human ES, EG, and EC Cells). mammals. A common method of imprinting is DNAOther genes recently identified as important in the methylation. Once methylated, or “marked,” a genedevelopment of human cancers are also active may be activated or inactivated. Thus, the process ofduring embryonic development. For instance, the marking a gene as being inherited from either thehuman breast cancer genes, BRCA1 and BRCA2, father or the mother is genomic imprinting [19].and their counterparts in mice are expressed in the For most of the genes known to undergo imprinting,three primary germ layers during embryogenesis, specific regulatory regions have been identifiedparticularly in cell types undergoing the most rapid where methylation takes place. The methylationproliferation. The expression of these genes is marks are acquired during gametogenesis, thedependent on the stage of the cell cycle, with peak process of sperm and egg formation, and theyexpression during the G1/S transition and lowest persist during the development of the pre- and post-expression in cells in the G1 or G0 phase. In mouse implantation embryo [9, 44]. In contrast, the genesand nonhuman primate (cynomolgus monkey) of nonimprinted embryos acquire their methylationembryos, the temporal and spatial patterns of Brca1 patterns after blastocyst implantation, as do ESand Brca2 expression are virtually identical, despite cells in vitro [50].the fact that the coding sequences for the genesand their promoters differ between the species. In The genomes of germ cells and the zygote are large-humans, BRCA1 and BRCA2 probably function during ly demethylated, although the sites associated withthe development of mammary epithelium, although parental-specific imprints remain methylated. In thelittle is known about their role in this process. Mutant preimplantation blastocyst, the nonimprinted genesforms of the genes appear to cause breast cancer of undifferentiated cells remain demethylated, which A-15
  • Appendix A: Early Development Table A.1. Comparison of Mouse, Monkey, and Human Pluripotent Stem Cells Marker Mouse EC/ Monkey Human Human Human Name ES/EG cells ES cells ES cells EG cells EC cells SSEA-1 + – – + – SSEA-3 – + + + + SEA-4 – + + + + TRA-1-60 – + + + + TRA-1-81 – + + + + Alkaline + + + + + phosphatase Oct-4 + + + Unknown + Telomerase activity + ES, EC Unknown + Unknown + Feeder-cell ES, EG, Yes Yes Yes Some; relatively low dependent some EC clonal efficiency Factors which aid LIF and other Co-culture with Feeder cells + LIF, bFGF, Unknown; in stem cell factors that act feeder cells; other serum; feeder forskolin low proliferative self-renewal through gp130 promoting factors layer + capacity receptor and can have not been serum-free substitute for identified medium + bFGF feeder layer Growth Form tight, Form flat, loose Form flat, loose Form rounded, Form flat, loose characteristics rounded, aggregates; aggregates; multi-layer clumps; aggregates; in vitro multi-layer clumps; can form EBs can form EBs can form EBs can form EBs can form EBs Teratoma + + + – + formation in vivo Chimera + Unknown + – + formation KEY ES cell = Embryonic stem cell TRA = Tumor rejection antigen-1 EG cell = Embryonic germ cell LIF = Leukemia inhibitory factor EC cell = Embryonal carcinoma cell bFGF = Basic fibroblast growth factor SSEA = Stage-specific embryonic antigen EB = Embryoid bodiesmeans that most of their genes are capable of being parentally specified DNA methylation patterns. Thisexpressed. But before gastrulation, as the three germ phenomenon of erasing the marks for genomiclayers prepare to differentiate, the DNA of the imprinting occurs as the PGCs migrate to theembryo’s somatic cells becomes remethylated and gonadal ridges, which in the mouse occurs on E13.5genes are selectively turned on or off. The only cells [23]. Then, as the germ cells mature, their genomesthat escape this phenomenon are the primordial acquire new imprints due to the activity of a specificgerm cells (PGCs). They gradually remove their DNA methyltransferase, which adds methyl groupsgenomic imprinting marks, which exist in the form of to DNA [50]. A-16
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Blastocyst transfer after enzymatic Hogan, B., Gardner, R.L., Smith, A., Klar, A.J.S., Henrique, D., treatment of the zona pellucida: improving in-vitro fertiliza- D’Urso, G., Datta, S., Holliday, R., Astle, C.M., Chen, J., Harrison, tion and understanding implantation. Hum. Reprod. 13, D.E., Xie, T., Spradling, A., Andrews, P .W., Przyborski, S.A., 2926-2932. Thomson, J.A., Kunath, T., Strumpf, D., Rossant, J., Tanaka, S., Orkin, S.H., Melchers, F., Rolink, A., Keller, G., Pittenger, M.F.,15. Gardner, R.L. (1997). The early blastocyst is bilaterally Marshak, D.R., Flake, A.W., Panicker, M.M., Rao, M., Watt, F.M., symmetrical and its axis of symmetry is aligned with the Grompe, M., Finegold, M.J., Kritzik, M.R., Sarvetnick, N., and animal-vegetal axis of the zygote in the mouse. Winton, D.J. (2001e). Stem cell biology, Marshak, D.R., Development. 124, 289-301. Gardner, R.L., and Gottlieb, D. eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press). A-17
  • Appendix A: Early Development31. Nichols, J., Davidson, D., Taga, T., Yoshida, K., Chambers, 42. Smith, A.G. (2001). Origins and properties of mouse I., and Smith, A. (1996). Complementary tissue-specific embryonic stem cells. Annu. Rev. Cell. Dev. Biol. 1-22. expression of LIF and LIF-receptor mRNAs in early mouse embryogenesis. Mech. Dev. 57, 123-131. 43. Soengas, M.S., Capodieci, P Polsky, D., Mora, J., Esteller, ., M., Opitz-Araya, X., McCombie, R., Herman, J.G., Gerald,32. Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe- W.L., Lazebnik, Y.A., Cordon-Cardo, C., and Lowe, S.W. Nebenius, D., Chambers, I., Scholer, H., and Smith, A. (2001). Inactivation of the apoptosis effector Apaf-1 in (1998). Formation of pluripotent stem cells in the malignant melanoma. Nature. 409, 207-211. mammalian embryo depends on the POU transcription factor Oct4. Cell. 95, 379-391. 44. Surani, M.A. (1998). Imprinting and the initiation of gene silencing in the germ line. Cell. 93, 309-312.33. Niwa, H., Miyazaki, J., and Smith, A.G. (2000). Quantitative expression of Oct-3/4 defines differentiation, dedifferen- 45. Tanaka, M., Gertsenstein, M., Rossant, J., and Nagy, A. tiation or self-renewal of ES cells. Nat. Genet. 24, 372-376. (1997). Mash2 acts cell autonomously in mouse spon- giotrophoblast development. Dev. Biol. 190, 55-65.34. Odorico, J.S., Kaufman, D.S., and Thomson, J.A. (2001). Multilineage Differentiation from Human Embryonic Stem 46. Tanaka, S., Kunath, T., Hadjantonakis, A.K., Nagy, A., and Cell Lines. Stem Cells. 19, 193-204. Rossant, J. (1998). Promotion of trophoblast stem cell proliferation by FGF4. Science. 282, 2072-2075.35. Pelton, T.A., Bettess, M.D., Lake, J., Rathjen, J., and Rathjen, P (1998). Developmental complexity of early mammalian .D. 47. Teruel, M., Smith, R., and Catalano, R. (2000). Growth pluripotent cell populations in vivo and in vitro. Reprod. factors and embryo development. Biocell. 24, 107-122. Fertil. Dev. 10, 535-549. 48. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A.,36. Pera, M.F., Reubinoff, B., and Trounson, A. (2000). Human Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998). embryonic stem cells. J. Cell Sci. 113 ( Pt 1), 5-10. Embryonic stem cell lines derived from human blastocysts. Science. 282, 1145-1147.37. Pesce, M., Anastassiadis, K., and Scholer, H.R. (1999). Oct-4: lessons of totipotency from embryonic stem cells. Cells 49. Trounson, A.O., Gardner, D.K., Baker, G., Barnes, F.L., Tissues Organs. 165, 144-152. Bongso, A., Bourne, H., Calderon, I., Cohen, J., Dawson, K., Eldar-Geve, T., Gardner, D.K., Graves, G., Healy, D., Lane,38. Rossant, J., personal communication. M., Leese, H.J., Leeton, J., Levron, J., Liu, D.Y., MacLachlan, V., Munné, S., Oranratnachai, A., Rogers, P Rombauts, L., .,39. Shinohara, T., Orwig, K.E., Avarbock, M.R., and Brinster, R.L. Sakkas, D., Sathananthan, A.H., Schimmel, T., Shaw, J., (2000). Spermatogonial stem cell enrichment by multi- Trounson, A.O., Van Steirteghem, A., Willadsen, S., and parameter selection of mouse testis cells. Proc. Natl. Acad. Wood, C. (2000). Handbook of in vitro fertilization, (Boca Sci. U. S. A. 97, 8346-8351. Raton, London, New York, Washington, D.C.: CRC Press).40. Smith, A.G., Heath, J.K., Donaldson, D.D., Wong, G.G., 50. Tucker, K.L., Beard, C., Dausmann, J., Jackson-Grusby, L., Moreau, J., Stahl, M., and Rogers, D. (1988). Inhibition of Laird, P.W., Lei, H., Li, E., and Jaenisch, R. (1996). Germ-line pluripotential embryonic stem cell differentiation by purified passage is required for establishment of methylation and polypeptides. Nature. 336, 688-690. expression patterns of imprinted but not of nonimprinted genes. Genes Dev. 10, 1008-1020.41. Smith, A. (2001). Embryonic stem cells. Marshak, D.R., Gardner, D.K., and Gottlieb, D. eds. Cold Spring Harbor Laboratory Press, 205-230. A-18
  • APPENDIX B: MOUSE EMBRYONIC STEM CELLSMOUSE EMBRYONIC STEM determine, to a great extent, whether mouse ES cellsCELL CULTURES can be derived from a given blastocyst.The techniques for culturing mouse embryonic stem Generating cultures of mouse or human ES cells that(ES) cells from the inner cell mass of the preimplan- remain in a proliferating, undifferentiated state is atation blastocyst were first reported 20 years ago [6, multistep process that typically includes the following.11], and versions of these standard procedures are First, the inner cell mass of a preimplantation blasto-used today in laboratories throughout the world. cyst is removed from the trophectoderm that sur-Additionally, studies of embryonal carcinoma (EC) rounds it. (For cultures of human ES cells, blastocystscells from mice and humans [2, 30] have helped are generated by in vitro fertilization and donated forestablish parameters for growing and assessing ES research.) The small plastic culture dishes used tocells. It is striking that, to date, only three species of grow the cells contain growth medium supplementedmammals have yielded long-term cultures of self- with fetal calf serum, and are sometimes coated withrenewing ES cells: mice, monkeys, and humans [21, a “feeder” layer of nondividing cells. The feeder cells34, 35, 36] (see Figure B.1. Origins of Mouse are often mouse embryonic fibroblast (MEF) cells thatPluripotent Stem Cells). have been chemically inactivated so they will not divide. Mouse ES cells can be grown in vitro withoutIn mice, the efficiency of generating ES cells is influ- feeder layers if the cytokine leukemia inhibitory factorenced by the genetic strain of laboratory mice and (LIF) is added to the culture medium (see below).individual factors that affect pregnant females. Only Human ES cells, however do not respond to LIF.a few strains of laboratory mice—notably 129,C57BL/6, and a hybrid strain—yield cultures of ES Second, after several days to a week, proliferatingcells. Even then, ES cells derived from C57BL/6 blasto- colonies of cells are removed and dispersed into newcysts do not behave as reliably as do ES cells from culture dishes, each of which also contains an MEFthe 129 strain of mice. The former are more difficult feeder layer. Under these in vitro conditions, the ESto propagate in vitro, generate chimeras less effi- cells aggregate to form colonies. Some coloniesciently than do ES cells from the 129 strain of mice, consist of dividing, nondifferentiated cells; in otherand infrequently contribute to the germ line [4]. colonies, cells may be differentiating. It is difficult to maintain human ES cells in dispersed cultures whereAnother influence on the efficiency with which ES cells cells do not aggregate, although mouse ES cells cancan be cultured from mouse blastocysts is the preg- be cultured this way. Depending on the culture con-nancy status of the female. Pregnant mice that are in ditions, it may also be difficult to prevent the spon-diapause tend to yield ES cells with greater success. taneous differentiation of mouse or human ES cells.Diapause occurs in female mice that have producedone litter and are still nursing when they become In the third major step required to generate ES cellpregnant again. Diapause is a naturally occurring lines, the individual, nondifferentiating colonies aredelay in the process of blastocyst implantation, which dissociated and replated into new dishes, a stepcauses an arrest in embryonic development and a called passage. This replating process establishes asmall increase in the number of epiblast cells [28]. “line” of ES cells. The line of cells is “clonal” if a singleThese findings have led to the notion that genetic ES cell generates it. Following some version of thisfactors that are peculiar to specific strains of inbred fundamental procedure, human and mouse ES cellsmice, and other in vivo influences such as diapause, can be grown and passaged for two or more years, B-1
  • Appendix B: Mouse Embryonic Stem Cells Embryonic Stem Cells Embryonic Germ Cells Embryonic Carcinoma Cells E10.5 Inner mouse Testicular cell embryo teratocarcinoma mass Primordial germ cells Mouse blastocyst Transfer of stem cells to mouse blastocyst Normal Normal Normal blastocyst blastocyst blastocyst© 2001 Terese Winslow Figure B.1. Origins of Mouse Pluripotent Stem Cells. through hundreds of population doublings, and still feeder layers of MEF cells. An alternative to culture on maintain a normal complement of chromosomes, feeder layers is the addition of leukemia inhibitory called a karyotype [31, 35]. factor (LIF) to the growth medium [31, 39]. LIF is pro- duced by feeder cells and, in their absence, allows MAINTAINING MOUSE EMBRYONIC mouse ES cells in vitro to continue proliferating without STEM CELLS IN THEIR differentiating [20]. LIF exerts its effects by binding to a two-part receptor complex that consists of the LIF UNDIFFERENTIATED STATE receptor and the gp130 receptor. The binding of LIF Leukemia Inhibitory Factor and STAT3 Activation triggers the activation of the latent transcription factor Mouse ES cells can be maintained in a proliferative, STAT3, a necessary event in vitro for the continued undifferentiated state in vitro by growing them on proliferation of mouse ES cells [5, 12, 14]. Recent B-2
  • Appendix B: Mouse Embryonic Stem Cellsevidence indicates that two transcription factors,STAT3 and Oct-4, may interact and perhaps affectthe function of a common set of target genes [15]. BlastocystIn vivo, signaling through the gp130 receptor is not Inner cellnecessary for normal, early embryonic development massbut is required to maintain the epiblast duringdiapause. After gastrulation, LIF signaling and STAT3 Embryonic stem cellsactivation promote the differentiation of specific celllineages such as the myeloid cells of the hemato- Cell receptorspoietic system or the astrocyte precursor cells in thecentral nervous system [9]. Plasma membrane gp130The self-renewal of mouse ES cells also appears to LIFRbe influenced by SHP-2 and ERK activity. SHP-2 is a JAKtyrosine phosphatase, an enzyme that removes JAKphosphate groups to the tyrosine residues of various SHP2 Gabproteins. SHP-2 interacts with the intracellular (amino Transcription STAT3 ERKterminus) domain of the gp130 receptor. ERK (which factor activationstands for extracellular regulated kinase) is one of sev- STAT Signal activation Transductioneral kinds of enzymes that becomes activated when Pathwaythe gp130 receptor and other cell-surface receptors STAT3are stimulated. Both ERK and SHP-2 are components STAT3 ERK 1/2of a signal-transduction pathway that counteracts theproliferative effects of STAT3 activation. Therefore, ifERK and SHP-2 are active, they inhibit ES cell self- Self-Renewal Blocks Self-Renewal © 2001 Terese Winslow, Lydia Kibiukrenewal [5] (see Figure B.2. The LIF-STAT3 SignalingPathway Promotes Embryonic Stem Cell Self-Renewal).It is possible that some of the components of signal-ing pathways in cultured mouse ES cells are uniqueto these cells. For example, mouse ES cells in vitroexpress high amounts of a modified version of anadapter protein, Gab1. The unusual form of Gab1 Figure B.2. The LIF-STAT3 Signaling Pathway Promotesthat occurs in ES cells may suppress interactions of Embryonic Stem Cell Self-Renewal.specific receptors to the Ras-ERK signaling pathway[31]. Further, the expression of this altered form of called Oct-3 or Oct-3/4). Oct-4 is present in theGab1 may be promoted by the transcription factor mouse zygote, and is required throughout blastocystOct-4. In mouse ES cells, Oct-4 expression and development to establish [13] and maintain [15] theincreased synthesis of Gab1 may help suppress pluripotency of the inner cell mass and the epiblast.induction of differentiation [30]. Oct-4 is also expressed in the primordial germ cells of mice and in mature germ cells [19, 23, 26].Thus, the emerging picture is that the effects ofvarious signaling pathways must be balanced in a Mouse ES cells in vitro can replicate indefinitely andparticular way for ES cells to remain in a self-renewing produce 109 to 1010 (1 to 10 billion) cells withoutstate. If the balance shifts, ES cells begin to differen- differentiating. In vitro, undifferentiated, proliferatingtiate [29, 30]. mouse [18] and human [21] ES cells express Oct-4. Studies of Oct-4 expression and function in humanExpression of Oct-4 in Undifferentiated, cells are incomplete, however, and most of thePluripotent Cells information about Oct-4 comes from the study ofOne of the hallmarks of an undifferentiated, pluri- mouse ES cells in vitro.potent cell is the expression of the Pou5f1 gene,which encodes the transcription factor Oct-4 (also B-3
  • Appendix B: Mouse Embryonic Stem CellsAs is the case with inner cell mass and epiblast cells human ES and EC cells do not. But human ES and ECin vivo, Oct-4 expression in vitro is required to main- cells express SSEA-3 and SSEA-4, whereas mouse EStain the pluripotent, undifferentiated state of ES cells. and EC cells do not [21, 35].If Oct-4 expression is inhibited in cultured mouse ES Human EG cells, which are derived from primordialcells, the cells generate trophectoderm. If Oct-4 germ cells, express all three markers: SSEA-1, SSEA-3,expression is artificially increased, mouse ES cells dif- and SSEA-4. The biological significance of the expres-ferentiate into primitive endoderm and mesoderm. sion patterns of these surface antigens is unclear, butTherefore, the level of Oct-4 expression dictates a SSEA-1 expression may be related to the growth char-significant aspect of the developmental program of acteristic of the cells in vitro. Undifferentiated humanmouse ES cells, making the protein a candidate ES and EC cells tend to grow in flat, relatively loose“master regulator” of ES cell pluripotency [15]. colonies. In contrast, mouse ES and EC colonies tendHow and why the Oct-4 transcription factor plays such to be multilayered and compact [27]. Alternatively, thean important role in early embryogenesis depend on surface expression of various SSEAs may reflect a differ-the genes it regulates. Seven to eight target genes for ence in the developmental stages of the cells [17].Oct-4 have been identified to date; it activates some Other markers used to identify ES cells are the surfaceand represses others. In fact, the overall impact of antigens TRA1-60, TRA1-81, and the enzyme alkalineOct-4 may be to prevent the expression of genes that phosphatase. All occur in human ES [3, 27, 35], asare required for differentiation [19]. they do in their mouse counterparts.The Cell Cycle of Mouse Embryonic Stem Cells: Its Genomic Imprinting in Embryonic Stem CellsRole in Preventing Differentiation It is known that if genomic imprinting patterns areLike the cells of the epiblast in the preimplantation disturbed before blastocyst implantation in vivo, fetalmouse embryo, mouse ES cells in vitro have an abnormalities may result. In genomic imprinting,unusual cell cycle. Specifically, the G1 checkpoint DNA methylation marks certain genes, depending ondoes not appear to operate in proliferating epiblast whether they are inherited from the mother or theand ES cells [25, 38]. This may explain why it has not father. The marked genes are turned on or off in abeen possible to induce quiescence—withdrawal non-random pattern that is determined by parentalfrom the cell cycle to a G1 or G0 state—in origin. Imprinting marks are erased in the primordialundifferentiated ES cells [29]. germ cells and then reestablished during theHowever, if ES cells begin to differentiate by forming formation of eggs and sperm.embryoid bodies, cyclin D expression increases, the However, when embryonic development occursG1 phase of the cell cycle becomes longer, and the in vitro or when ES cells are grown in tissue culture,rate of cell division slows [25]. This can occur if LIF or normal patterns of genomic imprinting may befeeder layers are withdrawn from mouse ES cell cul- disturbed. For example, mouse embryos that weretures. Then, cell division continues for only a few days grown in vitro in the presence of fetal calf serum—as the process of differentiation begins [29]. Perhaps a very different environment than the oviduct—andconstant cell proliferation somehow inhibits cell then allowed to develop in vivo, showed abnormaldifferentiation, and once the signals for cell division genomic imprinting patterns and abnormal develop-are removed, differentiation can occur [37]. ment. Apparently, the presence of fetal calf serum,Markers of Undifferentiated Embryonic Stem Cells a common ingredient in mouse and human ESES and EC cells, as well as cells of the inner cell mass cultures, decreases the expression of certainof mouse blastocysts, express a panel of surface imprinted genes [8].markers that are used to characterize undifferen- How or whether the use of fetal calf serum fortiated, pluripotent embryonic cells. (see Table B.1. culturing mouse or human ES cells affects genomicComparison of Mouse, Monkey, and Human imprinting and the behavior of ES cells in vitro is notPluripotent Stem Cells). The markers also distinguish known. But for mouse ES cells, the parental imprintingmouse ES and EC cells from human ES and EC cells. pattern apparently persists in vitro [16, 22]. TheFor example mouse ES and EC cells express the imprinting pattern of human ES cells in vitro has notstage-specific embryonic antigen SSEA-1, whereas yet been determined. B-4
  • Appendix B: Mouse Embryonic Stem Cells Table B.1. Comparison of Mouse, Monkey, and Human Pluripotent Stem Cells Marker Mouse EC/ Monkey Human Human Human Name ES/EG cells ES cells ES cells EG cells EC cells SSEA-1 + – – + – SSEA-3 – + + + + SEA-4 – + + + + TRA-1-60 – + + + + TRA-1-81 – + + + + Alkaline + + + + + phosphatase Oct-4 + + + Unknown + Telomerase activity + ES, EC Unknown + Unknown + Feeder-cell ES, EG, Yes Yes Yes Some; relatively low dependent some EC clonal efficiency Factors which aid LIF and other Co-culture with Feeder cells + LIF, bFGF, Unknown; in stem cell factors that act feeder cells; other serum; feeder forskolin low proliferative self-renewal through gp130 promoting factors layer + capacity receptor and can have not been serum-free substitute for identified medium + bFGF feeder layer Growth Form tight, Form flat, loose Form flat, loose Form rounded, Form flat, loose characteristics rounded, aggregates; aggregates; multi-layer clumps; aggregates; in vitro multi-layer clumps; can form EBs can form EBs can form EBs can form EBs can form EBs Teratoma + + + – + formation in vivo Chimera + Unknown + – + formation KEY ES cell = Embryonic stem cell TRA = Tumor rejection antigen-1 EG cell = Embryonic germ cell LIF = Leukemia inhibitory factor EC cell = Embryonal carcinoma cell bFGF = Basic fibroblast growth factor SSEA = Stage-specific embryonic antigen EB = Embryoid bodiesTargeted Differentiation of Mouse Embryonic similar conditions are used to direct the differentiationStem Cells. of mouse ES cells to yield pancreatic islet cells thatOutlined here are three different ways to direct secrete insulin.mouse ES cell differentiation in vitro. In the first exam- Making Vascular Progenitors from Mouse Embryonicple, mouse ES cells are directed to generate primitive Stem Cellsblood vessels. In the second, mouse ES cells aredirected to become neurons that release the trans- In the mouse embryo, blood cells and blood vesselsmitters dopamine and serotonin. And in the third— are formed at roughly the same time, when blooda series of experiments conducted by the same lab islands first appear in the wall of the yolk sac. A pre-group that generated dopamine neurons—very vailing idea is that blood cells and blood vessels arise B-5
  • Appendix B: Mouse Embryonic Stem Cellsfrom a common precursor cell derived from in vitro. To test that hypothesis, single Flk1+ cells weremesoderm, the hemangioblast. After hemangioblasts cultured. The individual ES cells generated three kindsdifferentiate from the mesoderm, they aggregate to of colonies: pure mural cells (SMA+), pure endothelialform blood islands. The inner cells of the blood islands cells (PECAM1+), and mixed mural and endothelialbecome hematopoietic stem cells, or blood-forming cells. That result indicated that ES cells can give risecells. The outer cells of the blood islands become to Flk1+ cells that are precursors for both mural andangioblasts, which give rise to the blood vessels. endothelial cells.A recent study showed that mouse ES cells in vitro The next test was to see whether the mural cells andcould be induced to follow this in vivo develop- endothelial cells generated from Flk1+ precursorsmental pathway. could assemble into primitive blood vessels in vitro.In vivo, blood vessel formation occurs in two ways: They did. By growing hundreds of Flk1+ cells in colla-by vasculogenesis and angiogenesis. Vasculogenesis gen gel suspensions with fetal calf serum and VEGF,helps establish the blood islands and the capillary tube-like structures formed within three to five days.network that connects them. During angiogenesis, This change in the culture conditions allowed the ESnew blood vessels form by remodeling or adding to cells to grow in suspension and interact with eachexisting vessels. Both vasculogenesis and angio- other. As a result, the cells spontaneously organizedgenesis are regulated by the actions of a series of themselves into tube-like structures that resembleparacrine growth factors, which include fibroblast blood vessels in vivo. The tubes were composed ofgrowth factor–2 (FGF-2), vascular endothelial growth endothelial (PECAM1+) cells and mural (SMA+) cells.factor (VEGF), and later (in the adult) platelet-derived Occasionally, they formed branching structures,growth factor (PDGF) and transforming growth factor which is typical of the organization of blood vesselsbeta (TGFß). Each of these growth factors binds to in vivo. Also, blood cells (bearing the markers CD45specific receptors. VEGF, for instance, binds to two and Ter119) formed inside the tubes, which alsodifferent receptors: VEGF-R1, also known as Flt1, and mimicked the organization of blood islands in theVEGF-R2, also known as Flk1 [7]. early embryo in vivo.To make vascular progenitors from mouse ES cells, The final test was to see whether the Flk1+ cells gener-Shin-Ichi Nishikawa of Kyoto University Graduate ated from mouse ES cells in vitro would differentiateSchool of Medicine in Japan and his colleagues tried into endothelial cells and mural cells in vivo. Again,to mimic this in vivo pathway for blood vessel forma- they did. Flk1+ cells were engineered to express LacZtion [40]. They grew undifferentiated ES cells on (which allows the cells to be tracked visually) andcollagen-coated dishes in medium containing fetal injected into the developing hearts of stage 16-17calf serum but no leukemia inhibitory factor (LIF). This chick embryos. The donor mouse cells populatedinduced the generation of cells that express Flk1, a blood vessels in the chicks’ head, yolk sac, heart, andreceptor for VEGF. Several days later, the cells began regions between the somites, forming endothelialto differentiate. Nearly all the mouse ES cells cells and mural cells in those regions.expressed Ȋ-smooth muscle actin (SMA), a marker for Thus, undifferentiated mouse ES cells can bemural cells. (Mural cells, which include pericytes and directed to differentiate into Flk1+ precursors thatsmooth muscle cells, normally interact in vivo with give rise to endothelial cells and mural cells in vitroendothelial cells to make blood vessels.) When VEGF and in vivo. Further, the differentiated cells can formwas added to the culture medium, sheets of tube-like vascular structures in vitro. The experimentsendothelial cells formed that expressed platelet- not only demonstrate the power of directed differen-endothelial cell adhesion molecule (PECAM1) and tiation of ES cells into individual cell types, they alsoother endothelial cell markers. At this point, the culture show that ES cells can generate multiple cell typescontained two differentiating cell types, endothelial that then spontaneously organize themselves intocells and mural cells. tissues that resemble those in vivo. In addition, theTherefore, it appeared that the mouse ES cells had experiments by Nishikawa and his co-workers [40]differentiated into Flk1+ precursor cells, which then reveal that Flk1+ cells are important for generatinggave rise to both mural cells and endothelial cells blood vessels in vivo. B-6
  • Appendix B: Mouse Embryonic Stem CellsMaking Dopamine Neurons from Mouse En1, and Nurr1 help control the differentiation ofEmbryonic Stem Cells neurons that release the transmitters dopamine andA second example of the directed differentiation of serotonin [24, 33]. Furthermore, when the proteinsmouse ES cells in vitro yielded the formation of sonic hedgehog (SHH) and fibroblast growth factor-8particular kinds of neurons that normally occur in the (FGF-8) are added to explant cultures (small chunksmammalian midbrain and hindbrain. For a long time, of tissue maintained in vitro) of neural plate, thethe goal of efficiently inducing the formation of these development of midbrain neurons is enhanced [41].neurons—which release the neurotransmitters Taking into account these and other findings, McKaydopamine and serotonin, respectively— was highly and his coworkers devised an in vitro system fordesired, but elusive. In Parkinson’s Disease, a key controlling the differentiation of mouse ES cells intopopulation of midbrain neurons that releases midbrain neurons that release dopamine and hind-dopamine dies. So finding a way to grow large brain neurons that release serotonin [11]. The culturequantities of nerve cells in vitro that might be able conditions they used differ from those devised byto replace lost dopamine neurons in vivo is a clinical Nishikawa and his colleagues (described above), butpriority (see Chapter 8. Rebuilding the Nervous System the starting material—undifferentiated, proliferatingwith Stem Cells). mouse ES cells—was the same in both experiments.Last year, Ron McKay and his colleagues reported an McKay and his colleagues cultured mouse ES cells inefficient technique for inducing mouse ES cells in vitro five distinct stages, each of which they identified byto differentiate into dopamine neurons of the mid- the changes in culture conditions and the behaviorbrain and serotonin neurons, which normally populate of the cells (see Figure B.3. Directed Differentiation ofthe hindbrain. Like Nishikawa and his colleagues [40], Mouse Embryonic Stem Cells Into Neurons orMcKay and his collaborators [10] triggered the differ- Pancreatic Islet-Like Clusters).entiation of mouse ES cells in vitro at various stages In stage 1, undifferentiated mouse ES cells were dis-by changing the growth conditions to mimic, in part, sociated into single cells and plated at low density.those that occur during embryogenesis in vivo. The They proliferated in plastic culture dishes coated withresulting differentiated nerve cells look and function gelatin. The growth media contained LIF and fetallike their in vivo counterparts. calf serum and was supplemented with amino acids,During embryogenesis, central nervous system (CNS) conditions that promoted the proliferation of undiffer-development is a long, complex process that entiated ES cells. In stage 2, the cells were induceddepends on a highly coordinated series of cellular to form embryoid bodies by dissociating them andand molecular events. Different signals direct the replating at a higher density on a nonadherent sur-formation of the neurectoderm from the epiblast, a face. These conditions allowed the cells to aggre-process that ultimately results in the formation of the gate and begin the process of differentiation. AfterCNS, the brain and spinal cord. Later, other signals four days, the cells were replated on an adherentregulate the development of different parts of the substrate in the original (stage 1) growth medium.brain. For example, early in the formation of the Twenty-four hours later, the growth medium wasbrain, the homeobox genes OTX1 and OTX2 are replaced with serum-free insulin/transferrin/selenium/expressed [28]. Cells of the epiblast express OTX2 fibronectin (ITSFn) medium. This switch to a serum-freebefore the onset of gastrulation. Then, during gastru- medium (one lacking fetal calf serum) caused manylation, OTX2 is expressed in the anterior neurecto- cells to die but allowed the survival of cells thatderm, where it is necessary for the formation of the express nestin. This intermediate filament protein ismidbrain and forebrain. Meanwhile, OTX1 expression used as a marker to identify CNS stem cells in vivooccurs in the region of the neurectoderm that gives and in vitro, although it is also expressed by other cellrise to the dorsal forebrain. Interactions between OTX1 types. Stage 1 neurons expressed high levels of OXT2,and OTX2 are thought to help shape the midbrain which decreased in stages 2 and 3. OXT1 was notand hindbrain [1]. expressed until the cells reached stage 3.Once these major brain structures form, various Guiding the mouse ES cells through stages 4 and 5genes control the development of individual nerve of in vitro development yielded fully differentiatedcell types. For example, the genes Pax2, Pax5, Wnt1, dopaminergic and serotoninergic neurons. After 6 to B-7
  • Appendix B: Mouse Embryonic Stem Cells Undifferentiated embryonic stem cells Inner cell mass of blastocyst EMBRYOID BODIES ITFSn medium (insulin/transferrin/ fibronectin/selenium) Adherent substrate SELECTION OF NESTIN-POSITIVE CELLS N2 medium/bFGF/ N2 medium/bFGF/laminin B27 media supplement Expansion Phase NESTIN-POSITIVE NEURONAL NESTIN-POSITIVE PANCREATIC PRECURSOR CELLS PROGENITOR CELLS Remove bFGF Remove bFGF Differentiation Add nicotinamide Phase DOPAMINE- AND SEROTONIN- INSULIN-SECRETING PANCREATIC SECRETING NEURONS ISLET-LIKE CLUSTERS TYROSINE HYDROXYLASE/SEROTONIN INSULIN/GLUCAGON© 2001 Terese Winslow, Caitlin Duckwall Reproduced with permission Reproduced with permission from Nature Biotechnology from Science Figure B.3. Directed Differentiation of Mouse Embryonic Stem Cells Into Neurons or Pancreatic Islet-Like Clusters. B-8
  • Appendix B: Mouse Embryonic Stem Cells10 days in the medium that selects for cells that “selects” for nestin-positive cells (stage 3). As before,express nestin, the cells were dissociated and cells that express nestin are expanded by adding theinduced to divide in another medium, N2, supple- mitogen basic FGF to the serum-free medium (stagemented with laminin and basic FGF, a growth factor 4). When basic FGF is withdrawn, the cells stopthat induces proliferation. Other critical additives to dividing and begin their final stages of differentiation.yield stage 4 cells were SHH and FGF-8. Cells at It is at this point that the techniques for generatingstages 3 and 4 express genes that, in vivo, trigger the neurons that release dopamine and pancreatic isletdevelopment of dopaminergic and serotinergic cells that release insulin diverge.neurons—namely, Pax2, Pax5, Wnt1, En1, and Nurr1. To generate neurons that release dopamine, ES-Stage 5, the final stage of differentiation, was derived cells were cultured in medium that containedachieved by removing basic FGF from the growth SHH and FGF-8 and later, an N2 medium supple-medium (which made the cells stop dividing) and mented with laminin and ascorbic acid [10]. Togrowing the cells for 6 to 15 days in N2 medium generate pancreatic islet cells, however, B27 culturesupplemented with laminin and ascorbic acid, a medium was used for stage 4, and nicotinamide wascombination of additives that induces the differen- added to stage 5 cultures. Another change in thetiation of serotonin neurons. pancreatic islet culture system was to co-cultureThis complex, multistage differentiation process yield- individual stage 4 or 5 ES cells, which were taggeded a higher percentage (30 percent) of neurons that with the marker green fluorescent protein (GFP), withexpress tyrosine hydroxylase (TH, the rate-limiting nontagged ES cells. This meant that an individual,enzyme in the synthesis of dopamine) than any other tagged ES cell could be followed so its develop-reported in vivo or in vitro technique. The cells were mental fate could be traced, a technique that madeconfirmed to be true dopamine neurons by several possible the clonal analysis of the labeled ES cell andfunctional assays. The neurons secreted dopamine its progeny. Tagged, GFP-expressing ES cells gave riseinto the culture medium, showed the electrical to both pancreatic islet cells and neurons, indicatingactivity typical of neurons, and responded to the that the same cell acted as the precursor for bothaddition of a high concentration of potassium ions differentiated cell types (see Figure B.3. Directed(via the addition of potassium chloride) by releasing Differentiation of Mouse Embryonic Stem Cells Intomore dopamine, much as they would in vivo. A sep- Neurons or Pancreatic Islet-Like Clusters).arate population of neurons in the mouse ES cell cul- The tests that identified the differentiated cells typestures stained positive for serotonin. The differentiation as pancreatic islet cells and neurons included assaysof serotoninergic neurons could be induced by adding of various markers. The ES cells at stages 1 and 5only SHH to the culture medium; addition of FGF-8 expressed GATA-4 and HNFb, markers for embryonicwas not required. Thus, mouse ES cells in vitro can be endoderm and extra-embryonic endoderm. This indi-directed to differentiate at a high efficiency into cates that endodermal markers are present in undif-neurons that release either dopamine or serotonin. ferentiated ES cells. But stage 5 cells express addition-Making Pancreatic Islet Cells from Mouse al markers that are characteristic of endocrine pan-Embryonic Stem Cells creatic islet cells: mouse insulin I and II, islet amyloidThe experimental strategy is similar to that described polypeptide, and the glucose transporter GLUT-2.above [10] and is based on a five-stage, in vitro Other cells stained positive for glucagon, a hormonesystem. As before (to differentiate neurons that pro- produced by the alpha cells of the pancreas, andduce dopamine), undifferentiated mouse ES cells are somatostatin, a peptide hormone produced by pan-induced to proliferate in LIF-supplemented medium creatic endocrine cells in vivo. Nerve cells that sur-(stage 1). Then, the cells are induced to form embry- rounded the clusters of islet cells—a spontaneouslyoid bodies (EBs) in serum-free ITSFn medium without forming, in vitro arrangement of cell types that mim-LIF (stage 2). ES stage 1 cells expressed Oct-4, a tran- icked their arrangement in vivo—stained positive forscription factor that characterizes undifferentiated, neuron-specific tubulin. No cells stained positive forproliferating, pluripotent cells. Again, cells that express both pancreatic islet markers and neuronal markers,nestin survive in serum-free medium, whereas other indicating that the two cell types had differentiatedcell types do not, thus creating an environment that completely from a common precursor cell. B-9
  • Appendix B: Mouse Embryonic Stem CellsOther tests demonstrated the functional properties of cells – form the basis of an acid test for all claims ofthe pancreatic islet cells differentiated from mouse directed differentiation of either ES cells or of adultES cells. Adding glucose to the culture medium stem cells. Unfortunately, very few experiments meettriggered the release of insulin in a dose-dependent these criteria, which too often makes it impossible tomanner. Agonists and antagonists of insulin release assess whether a differentiated cell type resulted fromin vivo stimulated or blocked insulin release in vitro, the experimental manipulation that was reported.indicating that the pharmacological responses of theES-derived islet cells in vitro mirrored in vivo responses. REFERENCESFinally, when cell clusters of the cultured pancreatic 1. Acampora, D. and Simeone, A. (1999). The TINS Lecture.islets were grafted under the skin of diabetic mice Understanding the roles of Otx1 and Otx2 in the control of(whose diabetes was induced by treatment with brain morphogenesis. Trends. Neurosci. 22, 116-122.streptozocin), the grafts survived and became infil- 2. Andrews, P .W., Casper, J., Damjanov, I., Duggan-Keen, M.,trated with blood vessels. The ES-derived pancreatic Giwercman, A., Hata, J., von Keitz, A., Looijenga, L.H.,islets released only one-fiftieth the amount of insulin Millan, J.L., Oosterhuis, J.W., Pera, M., Sawada, M., Schmoll,they released as islet cells in vivo, however, the H.J., Skakkebaek, N.E., van Putten, W., and Stern, P (1996). . Comparative analysis of cell surface antigens expressed bydiabetic mice experienced no correction of their cell lines derived from human germ cell tumours. Int. J.hyperglycemia (see Chapter 7. Stem Cells and Cancer. 66, 806-816.Diabetes; and Figure 7.2. Development of Insulin 3. Andrews, P .W., Damjanov, I., Simon, D., Banting, G.S., Carlin,Secreting Pancreatic-Like Cells from Mouse C., Dracopoli, N.C., and Fogh, J. (1984). PluripotentEmbryonic Stem Cells). embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo andTaken together, the three studies show that the differ- in vitro. Lab. Invest. 50, 147-162.entiation of lines of mouse ES cells can be directed 4. Brook, F.A. and Gardner, R.L. (1997). The origin and efficientin vitro to yield vascular structures [40], neurons that derivation of embryonic stem cells in the mouse. Proc. Natl.release dopamine and serotonin [10], and endocrine Acad. Sci. U. S. A. 94, 5709-5712.pancreatic islet cells. In all three cases, proliferating, 5. Burdon, T., Chambers, I., Stracey, C., Niwa, H., and Smith,undifferentiated mouse ES cells provided the starting A. (1999). Signaling mechanisms regulating self-renewalmaterial and functional, differentiated cells were the and differentiation of pluripotent embryonic stem cells. Cells Tissues Organs. 165, 131-143.result. Also, in all three experiments, the onset ofmouse ES cell differentiation was triggered by with- 6. Evans, M.J. and Kaufman, M.H. (1981). Establishment indrawing the cytokine LIF, which promotes the division culture of pluripotential cells from mouse embryos. Nature. 292, 154-156.of undifferentiated mouse ES cells, but — inexplicably— does not have the same effect on human ES cells. 7. Gilbert, S.F. (2000). Developmental biology. (Sunderland, MA: Sinauer Associates).In addition, the ES cells in all three examples citedwere induced to aggregate, a change in their 8. Khosla, S., Dean, W., Brown, D., Reik, W., and Feil, R. (2001).three-dimensional environment that presumably Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. 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A.in vivo counterparts do. 78, 7634-7638.These two criteria – demonstrating that a single cell 12. Matsuda, T., Nakamura, T., Nakao, K., Arai, T., Katsuki, M.,can give rise to multiple cells types (clonal analysis), Heike, T., and Yokota, T. (1999). STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonicand that the functional properties of the differentiated stem cells. EMBO J. 18, 4261-4269. B-10
  • Appendix B: Mouse Embryonic Stem Cells13. Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe- 28. Simeone, A. (1998). Otx1 and Otx2 in the development Nebenius, D., Chambers, I., Scholer, H., and Smith, A. and evolution of the mammalian brain. EMBO J. 17, (1998). Formation of pluripotent stem cells in the 6790-6798. mammalian embryo depends on the POU transcription factor Oct4. Cell. 95, 379-391. 29. Smith, A.G. (2001). Embryonic stem cells. Marshak, D.R., Gardner, D.K., and Gottlieb, D. eds. (Cold Spring Harbor,14. Niwa, H., Burdon, T., Chambers, I., and Smith, A. (1998). New York: Cold Spring Harbor Laboratory Press). 205-230. Self-renewal of pluripotent embryonic stem cells is mediat- ed via activation of STAT3. Genes Dev. 12, 2048-2060. 30. Smith, A., personal communication.15. Niwa, H., Miyazaki, J., and Smith, A.G. (2000). Quantitative 31. Smith, A.G. (2001). Origins and properties of mouse expression of Oct-3/4 defines differentiation, dedifferen- embryonic stem cells. Annu. Rev. Cell. Dev. Biol. tiation or self-renewal of ES cells. Nat. Genet. 24, 372-376. 32. Stevens, L.C. (1970). The development of transplantable16. O’Shea, K.S. (1999). Embryonic stem cell models of teratocarcinomas from intratesticular grafts of pre-and development. Anat. Rec. 257, 32-41. postimplantation mouse embryos. Dev. Biol. 21, 364-382.17. Pera, M., personal communication. 33. Stoykova, A. and Gruss, P (1994). Roles of Pax-genes in . developing and adult brain as suggested by expression18. Pesce, M., Gross, M.K., and Scholer, H.R. (1998). In line with patterns. J. Neurosci. 14, 1395-1412. our ancestors: Oct-4 and the mammalian germ. Bioessays. 20, 722-732. 34. Thomson, J.A., Kalishman, J., Golos, T.G., Durning, M., Harris, C.P Becker, R.A., and Hearn, J.P (1995). Isolation of a ., .19. Pesce, M., Wang, X., Wolgemuth, D.J., and Scholer, H. primate embryonic stem cell line. Proc. Natl. Acad. Sci. (1998). Differential expression of the Oct-4 transcription U. S. A. 92, 7844-7848. factor during mouse germ cell differentiation. Mech. Dev. 71, 89-98. 35. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998).20. Rathjen, P.D., Toth, S., Willis, A., Heath, J.K., and Smith, A.G. Embryonic stem cell lines derived from human blastocysts. (1990). Differentiation inhibiting activity is produced in Science. 282, 1145-1147. matrix-associated and diffusible forms that are generated by alternate promoter usage. Cell. 62, 1105-1114. 36. Thomson, J.A. and Marshall, V.S. (1998). Primate embryonic stem cells. Curr. Top. Dev. Biol. 38, 133-165.21. Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A., and Bongso, A. (2000). Embryonic stem cell lines from human 37. Weissman, I.L. (2000). Stem cells: units of development, blastocysts: somatic differentiation in vitro. Nat. Biotechnol. units of regeneration, and units in evolution. Cell. 100, 18, 399-404. 157-168.22. Rohwedel, j., Sehlmeyer, U., Shan, J., Meister, A., and 38. Wianny, F., Real, F.X., Mummery, C.L., van Rooijen, M., Lahti, Wobus, A.M. (1996). Primordial germ cell-derived mouse J., Samarut, J., and Savatier, P (1998). G1-phase regulators, . embryonic germ (EG) cells in vitro resemble undifferen- Cyclin D1, Cyclin D2, and Cyclin D3: up-regulation at gas- tiated stem cells with respect to differentiation capacity trulation and dynamic expression during neurolation. Dev. and cell cycle distribution. Cell. Biol. Int. 20, 279-587. Dyn. 212, 49-62.23. Rosner, J.L. (1990). Reflections of science as a product. 39. Williams, R.L., Hilton, D.J., Pease, S., Willson, T.A., Stewart, Nature. 345, 108. C.L., Gearing, D.P Wagner, E.F., Metcalf, D., Nicola, N.A., ., and Gough, N.M. (1988). Myeloid leukaemia inhibitory24. Rowitch, D.H. and McMahon, A.P (1995). Pax-2 expression . factor maintains the developmental potential of in the murine neural plate precedes and encompasses the embryonic stem cells. Nature. 336, 684-687. expression domains of Wnt-1 and En-1. Mech. Dev. 52, 3-8. 40. Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M.,25. Savatier, P Lapillonne, H., van Grunsven, L.A., Rudkin, B.B., ., Nishikawa, S., Yurugi, T., Naito, M., Nakao, K., and Nishikawa, and Samarut, J. (1996). Withdrawal of differentiation S. (2000). Flk1-positive cells derived from embryonic stem inhibitory activity/leukemia inhibitory factor up-regulates cells serve as vascular progenitors. Nature. 408, 92-96. D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells. Oncogene. 12, 309-322. 41. Ye, W., Shimamura, K., Rubenstein, J.L., Hynes, M.A., and Rosenthal, A. (1998). FGF and Shh signals control26. Schöler, H.R., Ruppert, S., Suzuki, N., Chowdhury, K., and dopaminergic and serotonergic cell fate in the anterior Gruss, P (1990). New type of POU domain in germ line- . neural plate. Cell. 93, 755-766. specific protein Oct-4. Nature. 344, 435-439.27. Shamblott, M.J., Axelman, J., Wang, S., Bugg, E.M., Littlefield, J.W., Donovan, P Blumenthal, P .J., .D., Huggins, G.R., and Gearhart, J.D. (1998). Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl. Acad. Sci. U. S. A. 95, 13726-13731. B-11
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  • APPENDIX C: HUMAN EMBRYONIC STEM CELLS AND HUMAN EMBRYONIC GERM CELLSMETHODS FOR GROWING HUMAN single cell and are, therefore, genetically identical)EMBRYONIC STEM CELLS IN VITRO [35] (see Figure C.1. Techniques for Generating Embryonic Stem Cell Cultures).To grow cultures of human ES cells, Thomson and hiscollaborators used 36 fresh or frozen embryos gener- The five original human ES cell lines continued toated in IVF laboratories from couples undergoing divide without differentiating for 5 to 6 months [35].treatment for infertility. From the 14 embryos that Since then, the H9 line has divided for nearly twodeveloped to the blastocyst stage, they established years in vitro, for more than 300 population doublings5 human ES cell lines—H1, H7, H9, H13 and H14 [35]. and has yielded two subclones, H9.1 and H9.2 [1] 1.Four of the 5 lines were derived from frozen embryos All the ES cell lines express high levels of telomeraseprovided to Thomson’s laboratory by Josef Itskovitz- [1, 36], the enzyme that helps maintain telomeresEldor, of the Rambam Medical Center in Haifa, Israel. which protect the ends of chromosomes. TelomeraseThe ES cell line from the fifth, fresh embryo was activity and long telomeres are characteristic ofderived from an embryo donated in Wisconsin. proliferating cells in embryonic tissues and of germ cells. Human somatic cells, however, do not showTo generate human ES cell cultures, cells from the telomerase activity and their telomeres are consider-inner cell mass of a human blastocyst were cultured ably shorter. Unlike ES cells, differentiated somaticin a multi-step process. The pluripotent cells of the cells also stop dividing in culture—a phenomenoninner cell mass were separated from the surrounding called replicative senescence (see Figure C.2.trophectoderm by immunosurgery, the antibody- Telomeres and Telomerase).mediated dissolution of the trophectoderm. The innercell masses were plated in culture dishes containing Three of the human ES cell lines generated bygrowth medium supplemented with fetal bovine Thomson were XY (male) and two were XX (female);serum on feeder layers of mouse embryonic fibro- all maintained a normal karyotype. Like monkey ESblasts that had been gamma-irradiated to prevent cells [34], human ES cells express a panel of surfacetheir replication. After 9 to 15 days, when inner cell makers that include the stage-specific embryonicmasses had divided and formed clumps of cells, antigens SSEA-3 and SSEA-4, as well as TRA-1-60,cells from the periphery of the clumps were chemi- TRA-1-81, and alkaline phosphatase [14, 25, 26, 35].cally or mechanically dissociated and replated in Mouse ES cells do not express SSEA-3 or SSEA-4; theythe same culture conditions. Colonies of apparently express SSEA-1, which human and monkey ES cellshomogeneous cells were selectively removed, do not. Human ES cells also express the transcriptionmechanically dissociated, and replated. These were factor Oct-4 [26], as mouse ES cells do.expanded and passaged, thus creating a cell line. A somewhat different technique for deriving andNone of the initial 5 human ES cell lines generated in culturing human ES cells has now been reported bythis manner was derived clonally (cloned from a1 The H9.1 and H9.2 clonal cell lines were produced by first plating 105 of the parent H9 cells per well in tissue-culture plates. The culture medium contained KnockOut Dulbecco’s modified minimal essential medium (a serum-free substitute for the 20% fetal bovine serum used in the 1998 experiments), and basic FGF, which is necessary to maintain cell proliferation and prevent differentiation. To generate clonal cell lines from individual H9 ES cells, 384 single cells were removed from these cultures and transferred individually to the wells of larger plates that contained non-dividing mouse embryonic fibroblasts (MEF) as feeder layers. The single ES cells proliferated and, every 7 days, were dissociated and replated, a process that generate two clonal cell lines, H9.1 and H9.2. C-1
  • Appendix C: Human Embryonic Stem Cells and Human Embryonic Germ Cells Cleavage stage embryo Cultured blastocyst Isolated inner cell mass Cells dissociated Irradiated mouse and replated fibroblast feeder cells New feeder cells© 2001 Terese Winslow, Caitlin Duckwall Established embryonic stem cell cell cultures Figure C.1. Techniques for Generating Embryonic Stem Cell Cultures. C-2
  • Appendix C: Human Embryonic Stem Cells and Human Embryonic Germ Cells TELOMERES EXTENDING THE LENGTH OF A TELOMERE Embryonic Adult Stem Stem Cells Cells Before Nucleus Chromosome Short end of DNA Telomerase RNA template New DNA Telomere Telomere long short