Biology stem cells-nih-2001!!

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Biology stem cells-nih-2001!!

  1. 1. Stem Cells: Scientific Progress and Future Research Directions© 2001 Terese Winslow
  2. 2. STEM CELLS:SCIENTIFIC PROGRESS AND FUTURERESEARCH DIRECTIONS June 2001
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  4. 4. 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
  5. 5. 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
  6. 6. 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
  7. 7. REPORT PREPARED BY THE NATIONAL INSTITUTES OF HEALTH Ruth Kirschstein, M.D. Acting Director Office of Science Policy Lana R. Skirboll, Ph.D. Director
  8. 8. 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
  9. 9. 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
  10. 10. 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
  11. 11. 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
  12. 12. 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
  13. 13. 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
  14. 14. 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
  15. 15. 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
  16. 16. 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
  17. 17. 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
  18. 18. 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
  19. 19. 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
  20. 20. 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
  21. 21. 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
  22. 22. 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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  24. 24. 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
  25. 25. 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
  26. 26. 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

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