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  • 1.
    • Summer 2007 Workshop
    • in Biology and Multimedia
    • for High School Teachers
  • 2. Stem Cell Research Overview Mountainous Path
  • 3. Outline
    • What are Stem Cells?
    • Potential Uses
    • Claims Against Using Stem Cells
    • Cultivation Process
    • Stem Cells and Cloning
    • Stem Cell Theory of Cancer
    • Worldwide Status
  • 4. What are stem cells?
    • Stem cells are undifferentiated cells that have many potential scientific uses:
      • Cell based therapies
        • Often referred to as regenerative or reparative medicine
      • Therapeutic cloning
      • Gene therapy
      • Cancer research
      • Basic research
  • 5. Two types of stem cells
    • Embryonic Stem Cells (ESC): received from:
      • Embryos created in vitro fertilization
      • Aborted embryos
    • Adult Stem Cells (ASC): can be received from:
      • Limited tissues (bone marrow, muscle, brain)
        • Discrete populations of adult stem cells generate replacements for cells that are lost through normal wear and tear, injury or disease
      • Placental cord
      • Baby teeth
  • 6. Source of ESC
    • Blastocyst
      • “ ball of cells”
      • 3-5 day old embryo
      • Stem cells give rise to multiple specialized cell types that make up the heart, lung, skin, and other tissues
    • Human ESC were only studied since 1998
      • It took scientists 20 years to learn how to grow human ESC following studies with mouse ESC
  • 7. How are embryonic stem cells harvested?
    • Human ES cells are derived from 4-5 day old blastocyst
    • Blastocyst structures include:
      • Trophoblast : outer layer of cells that surrounds the blastocyst & forms the placenta
      • Blastocoel : (“blastoseel”) the hollow cavity inside the blastocyst that will form body cavity
      • Inner cell mass : a group of approx. 30 cells at one end of the blastocoel:
        • Forms 3 germ layers that form all embryonic tissues (endoderm, mesoderm, ectoderm)
  • 8. Blastocyst http://www. ivf -infertility.com /infertility/infertility5. php
  • 9. Unique characteristics of Stem Cells
    • Stem cells can regenerate
      • Unlimited self renewal through cell division
    • Stem cells can specialize
      • Under certain physiologic or experimental conditions
      • Stem cells then become cells with special functions such as:
        • Beating cells of the heart muscle
        • Insulin-producing cells of the pancreas
  • 10. Unspecialization
    • Stem Cells are unspecialized
      • They do not have any tissue-specific structures that allow for specialized function
      • Stem cells cannot work with its neighbors to pump blood through the body (like heart muscle cells)
      • They cannot carry molecules of oxygen through the bloodstream (like RBCs)
      • They cannot fire electrochemical signals to other cells that allow the body to move or speak (like nerve cells)
  • 11. Self - Renewal (Regeneration)
    • Stem cells are capable of dividing & renewing themselves for long periods
      • This is unlike muscle, blood or nerve cells – which do not normally replicate themselves
      • In the lab, a starting population of SCs that proliferate for many months yields millions of cells that continue to be unspecialized
        • These cells are capable of long-term self-renewal
  • 12. Specialization of Stem Cells: Differentiation
    • Differentiation : unspecialized stem cells give rise to specialized (differentiated) cells in response to external and internal chemical signals
      • Internal signals : turn on specific genes causing differential gene expression
      • External signals include:
        • Chemicals secreted by other cells such as growth factors, cytokines, etc.
        • Physical contact with neighboring cells
  • 13. Differentiation
    • Why do your body cells look different although they all carry the same DNA, which was derived from one fertilized egg?
    • Differentiation example (http://learn.genetics.utah.edu/units/biotech/microarray/)
  • 14. Potential of Stem Cells (vocab)
    • Totipotent (total):
      • Total potential to differentiate into any adult cell type
      • Total potential to form specialized tissue needed for embryonic development
    • Pluripotent (plural):
      • Potential to form most or all 210 differentiated adult cell types
    • Multipotent (multiple):
      • Limited potential
      • Forms only multiple adult cell types
        • Oligodendrocytes
        • Neurons
  • 15. Adult Stem Cells
    • Adult or somatic stem cells have unknown origin in mature tissues
      • Unlike embryonic stem cells, which are defined by their origin (inner cell mass of the blastocyst)
  • 16. http://www.stemcellresearch.org/testimony/20040929prentice.htm Reprinted with permission of Do No Harm.
  • 17. Adult stem cells continued
    • Adult stem cells typically generate the cell types of the tissue in which they reside
      • Stem cells that reside in bone marrow give rise to RBC, WBC and platelets
      • Recent experiments have raised the possibility that stem cells from one tissue can give rise to other cell types
        • This is known as PLASTICITY
  • 18. Adult Stem Cell Plasticity Examples
    • Blood cells becoming neurons
    • Liver cells stimulated to produce insulin
    • Hematopoietic (blood cell producing) stem cells that become heart cells
    • CONCLUSION: Exploring the use of adult stem cells for cell-based therapies has become a very important (and rapidly increasing) area of investigation by research scientists!
  • 19. Adult stem cells: A brief history
    • Adult stem cell research began about 40 years ago
    • Stem cell discoveries in 1960s:
      • Bone marrow contains 2 populations of stem cells
        • Hematopoietic stem cells – forms all blood cell types
        • Bone marrow stromal cells – mixed cell population that generates bone, cartilage, fat and fibrous connective tissue
      • Rat brain contains two regions of dividing cells, which become nerve cells
  • 20. History Cont.
    • Stem Cell Discoveries in the 1990s
      • Neural stem cells in brain are able to generate the brain’s three major cell types
        • Astrocytes
        • Oligodendroglial cells
        • Neurons
    http://www.alsa.org/images/cms/Research/Topics/cell_targets.jpg
  • 21. Adult Stem Cell Facts
    • Adult stem cells were found in many more tissues than expected
    • Some may be able to differentiate into a number of different cell types, given the right conditions
    • General consensus among scientist:
      • Adult stem cells DO NOT have as much potential as embryonic stem cells
    • CLARIFICATION : not all new adult cells arise from stem cells
      • Most arise by MITOSIS of differentiated cells
  • 22. Potential Uses of Stem Cells
    • Basic research – clarification of complex events that occur during human development & understanding molecular basis of cancer
      • Molecular mechanisms for gene control
      • Role of signals in gene expression & differentiation of the stem cell
      • Stem cell theory of cancer
  • 23. Potential uses cont.
    • Biotechnology (drug discovery & development) – stem cells can provide specific cell types to test new drugs
      • Safety testing of new drugs on differentiated cell lines
      • Screening of potential drugs
        • Cancer cell lines are already being used to screen potential anti-tumor drugs
        • Availability of pluripotent stem cells would allow drug testing in a wider range of cell types & to reduce animal testing
  • 24. Potential uses cont.
    • Cell based therapies :
      • Regenerative therapy to treat Parkinson’s, Alzheimer’s, ALS, spinal cord injury, stroke, severe burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis
      • Stem cells in gene therapy
        • Stem cells as vehicles after they have been genetically manipulated
      • Stem cells in therapeutic cloning
      • Stem cells in cancer
  • 25. Embryonic vs Adult Stem Cells
    • Totipotent
      • Differentiation into ANY cell type
    • Known Source
    • Large numbers can be harvested from embryos
    • May cause immune rejection
      • Rejection of ES cells by recipient has not been shown yet
    • Multi or pluripotent
      • Differentiation into some cell types, limited outcomes
    • Unknown source
    • Limited numbers, more difficult to isolate
    • Less likely to cause immune rejection, since the patient’s own cells can be used
  • 26. Claims against ESC (unsubstantiated thus far!)
    • Difficult to establish and maintain *
    • Difficulty in obtaining pure cultures from dish*
    • Potential for tumor formation and tissue* destruction
    • Questions regarding functional differentiation
    • Immune rejection
    • Genome instability
    • Few & modest results in animals, no clinical treatments
    • Ethically contentious
    * = same problem with ASC
  • 27. Cell Culture Techniques for ESC
    • Isolate & transfer of inner cell mass into plastic culture dish that contains culture medium
    • Cells divide and spread over the dish
    • Inner surface of culture dish is typically coated with mouse embryonic skin cells that have been treated so they will not divide
  • 28.
    • This coating is called a FEEDER LAYER
      • Feeder cells provide ES cells with a sticky surface for attachment
      • Feeder cells release nutrients
    • Recent discovery: methods for growing embryonic stem cells without mouse feeder cells
      • Significance – eliminate infection by viruses or other mouse molecules
    • ES cells are removed gently and plated into several different culture plates before crowding occurs
  • 29. http://www.news.wisc.edu/packages/stemcells/illustration.html Images depict stem cell research at the University of Wisconsin Madison.
  • 30. Cloning of whole organisms
    • Purpose:
      • Reproductive cloning in animals
      • Therapeutic cloning in animals
      • Breeding animals or plants with favorable traits
      • Producing TRANSGENIC animals that:
        • Make a therapeutic product (vaccine, human protein etc)
        • Act as animal models for human disease
        • Deliver organs that will not be rejected (cells lacking cell surface markers that cause immune rejection)
      • Vaccines in biotech industry: steps in cloning a gene
  • 31. SCNT: Somatic Cell Nuclear Transfer
    • SCNT is a method used for:
      • Reproductive cloning such as cloning an embryo
      • Regenerative cloning to produce “customized” stem cells & overcome immune rejection
    • Blastula stage cannot continue to develop in vitro
      • It must be implanted into surrogate mom
      • Surrogate mom is just a container that provides protection & chemical signals necessary for development
  • 32. http://www. kumc . edu / stemcell /early.html Reprinted with permission from the University of Kansas Medical Center.
  • 33. http://www.stemcellresearch.org/testimony/20040929prentice.htm Reprinted with permission of Do No Harm.
  • 34. Challenges of Reproductive Cloning
    • Many animals were cloned after Dolly
      • Cats, pigs, mice, goats, cattle, rabbits
    • Obstacles:
      • Very inefficient process
      • Most clones have deleterious effects & die early
      • Surviving clones show premature aging signs
      • Signs of abnormal embryonic development:
        • Clones & their placentas grow much faster than expected in surrogate mom
  • 35. Therapeutic Cloning
    • 3 goals of therapeutic cloning by SCNT in humans:
      • Use embryo as source for ES cells
      • Use ES cells to generate an organ
        • In this case the organ generated will carry cells with the same genetic markers as the patient (recipient)
      • Correct genetic error in ESC in blastula stage
  • 36. Pitfalls of therapeutic cloning (1) Some immune rejection may occur- WHY?
      • About 1% of the DNA in the clone will NOT be identical to donor cell (patient)
      • It will be identical to egg cell used in SCNT
      • REASON: mitochonrial DNA in eggs
        • Human mitochondria carry about 13 genes, some of which code for surface proteins
  • 37. Pitfalls of therapeutic cloning (2)
    • Large number of eggs needed for SCNT
    • To harvest large number of eggs:
      • Excessive hormone treatment of females to induce high rate of ovulation
      • Surgery to retrieve eggs
        • Both can be harmful to female human
        • Cow/pig females may be used
      • Cow/pig eggs will carry species-specific mitochondrial genes
        • Mixing species is reason for concern!
  • 38. Common Opinions
    • Reproductive cloning is a criminal offense (it is ILLEGAL worldwide!)
    • Therapeutic cloning is acceptable, however there is still significant controversy over whether:
      • the clone is implanted into the uterus of surrogate mom? OR
      • the clone is explanted into culture dish to generate ES cells
  • 39. Stem Cell Theory of Cancer
    • 1855: Rudolf Virchow developed the Embryonal- Rest Hypothesis
      • Microscopic examination of tumor samples revealed many morphological (structural & functional) resemblances to ESC in a developing fetus
    • Isolation of teratoma: nonmalignant tumors
      • Teratoma represents a ball of almost all cell types
      • This indicates that teratoma may originate from unregulated stem cells that can give rise to almost all tissues
  • 40. Teratoma
    • Ovarian Teratoma
      • You can see teeth!
    http://home.earthlink.net/~radiologist/ tf /040802. htm Image courtesy of Leonard J. Tyminski, M.D., Radiologist at earthlink.net
  • 41. Current Efforts with SC and Cancer
    • Determine difference between cancer & normal stem cells
    • Identify potential points in pathways critical for the survival of cancer SCs
    • Develop therapies that specifically target cancer SC
    • Duke University Explanation
    Drawn by Christine Rodriguez Tumor stem cell Tumor cell
  • 42. Status of SC research in other countries
    • Great Britain
      • Very liberal policies on research
      • Therapeutic cloning allowed, use of excess embryos & creation of embryos allowed
      • Stem cell research allowed
    • France
      • Less liberal politics
      • Use of excess embryos from IVF allowed
      • Reproductive AND therapeutic cloning banned
    • Germany
      • Very strict policies
      • Use of excess embryos and creation of embryos banned
      • Scientists can IMPORT embryos
  • 43. Debate in US
    • Federal funding available for research using the Bush lines only:
      • ES cell lines that were already in existence by 8/9/01
    • Disadvantage of Bush stem cell lines:
      • May have lost regenerative ability
      • May have accumulated mutations or infections
    • Private companies continue to pursue stem cell research
      • Use of human embryos for IVF & therapeutic cloning is legal in most states
        • No federal funding
      • Some states are considering banning both
  • 44. Global Status
    • Ongoing debate regarding use of embryos
    • United Nations: proposal for a global policy to ban reproductive cloning only
  • 45. References
    • Stem cells & Cloning Stem cells & Cloning; David A. Prentice, Benjamin Cummings, 2003
    • http://www.pbs.org/wgbh/nova/sciencenow/3302/06.html
    • http://www.stemcellresearch.org
    • http://www.stemcells.nig.gov/info/nasics/nasics7.asp
    • http://www.stemcells.nig.gov/info/scireport/2006report.htm
    • http://www.whitehouse.gov/news/re;eases/2001/08/20010809-2.html
    • Stem cells in class; Badran, Shahira; Bunker Hill Community College, 2007, Boston Museum of Science Biotechnology Symposium
    • Harvard Stem Cell Institute