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  • Inman_Stem_Cells_05

    1. 1. Stem Cells in Research <ul><li>Promises and Pitfalls </li></ul>Denise Inman, PhD University of Washington Department of Neurosurgery
    2. 3. Overview <ul><li>What are stem cells? </li></ul><ul><ul><li>How do embryonic and ‘adult’ stem cells differ? </li></ul></ul><ul><ul><li>How are different types of stem cell lines created? </li></ul></ul><ul><li>Stem cells in research and medicine </li></ul><ul><li>Alternatives to the embryo </li></ul>
    3. 4. Early Development Fertilized egg Totipotent stem cells Totipotent : Can become any cell in body or placenta Fate Decision Pluripotent stem cells Blastocyst Pluripotent : Can become any cell in body Implantation Gastrulation Fate Decision Multipotent stem cells Primary Germ Cells Endoderm (inner) Mesoderm (middle) Ectoderm (outer) Multipotent : Can become any cell within a specific germ layer or cell lineage Embryonic stem cells come from inner cell mass of blastocyst.
    4. 5. Embryonic Stem Cell Characteristics <ul><li>Not committed to a specific fate </li></ul><ul><li>Pluripotent — can differentiate into specialized cell types </li></ul><ul><li>Self-renewing </li></ul>endoderm mesoderm ectoderm Courtesy of James Thomson, U. Wisconsin-Madison
    5. 6. Is that an Embryonic Stem Cell? Embryonic stem cells injected into a SCID mouse will grow into teratomas, tumors of the germ cell layers. Individual ESCs under the correct conditions will make many different cell types. The true potential of stem cells can only be assessed retrospectively SCID Mouse: Severe Combined ImmunoDeficiency
    6. 7. Stem Cells: From Embryonic to Adult Embryonic stem cells are those removed from the blastocyst before the fate decision from pluripotentiality to multipotentiality. Adult stem cells are those multipotential cells that persist in fully developed tissues. These cells never differentiated into the mature cell types of the tissues in which they reside.
    7. 8. Adult Stem Cells <ul><li>Multipotential </li></ul><ul><ul><li>Make cells within a specific lineage </li></ul></ul><ul><li>Not differentiated </li></ul><ul><li>Rare </li></ul><ul><li>Self-replicating </li></ul>Neural stem cells in culture. One cell is extending a process.
    8. 9. Adult Stem Cells – Bone Marrow NIH: info/basics/basics4.asp Major repository of adult stem cells -Hematopoeitic -Mesenchymal Give rise to immune system cells Constant turnover
    9. 10. Stem Cell Phenotype Fate dictated by environment… Shihabuddin, et al., J. Neuroscience 20(23) 8727-8735, 2000 Neural stem cells Neurons Astrocytes Oligodendrocytes Oligodendrocyte Progenitor Cells Stem cells placed in brain become neurons… Stem cells placed in spinal cord become glial cells…
    10. 11. Re-cap: What are stem cells? <ul><li>Embryonic and adult stem cells </li></ul><ul><ul><li>Obtained at different developmental stages </li></ul></ul><ul><ul><li>Different potential </li></ul></ul><ul><ul><ul><li>Pluripotent versus Multipotent </li></ul></ul></ul><ul><ul><li>Sensitive to environment </li></ul></ul>
    11. 12. Overview <ul><li>What are stem cells? </li></ul><ul><ul><li>How do embryonic and ‘adult’ stem cells differ? </li></ul></ul><ul><ul><li>How are different types of stem cell lines created? </li></ul></ul><ul><li>Stem cells in research and medicine </li></ul><ul><ul><li>How do scientists work with stem cells? </li></ul></ul><ul><ul><ul><li>In situ labeling </li></ul></ul></ul><ul><ul><ul><li>Primary culture </li></ul></ul></ul><ul><ul><ul><li>Cell lines </li></ul></ul></ul><ul><ul><li>Promises and perils of stem cells </li></ul></ul><ul><li>Alternatives to the embryo </li></ul>
    12. 13. Cell Lines <ul><li>Cells under propagation </li></ul><ul><li>All cells are genetically identical </li></ul><ul><li>Can be frozen and stored </li></ul>Plate Exponential Growth Remove from plate Replate at lower density
    13. 14. Culturing Embryonic Stem Cells Obtain stem cells from 1. Remove inner cell mass 2. Put cells in dish with feeder layer 3. Cells divide Somatic Cell Nuclear Transfer Oocyte without nucleus Inject nucleus from adult somatic cell Blastocyst Fertilized egg Blastocyst + Sperm Oocyte In Vitro Fertilization
    14. 15. Origins of ES Cell Lines <ul><li>Excess IVF embryos </li></ul><ul><li>Therapeutic Cloning (somatic cell nuclear transfer) </li></ul><ul><ul><li>Donor oocyte+somatic cell nucleus </li></ul></ul><ul><ul><li>Cells have characteristics of nuclear donor </li></ul></ul><ul><ul><li>Lines representing different diseases </li></ul></ul><ul><ul><li>Individualized lines: non-immunogenic to donor </li></ul></ul>New England Journal of Medicine, Wellcome Trust
    15. 16. Somatic Cell Nuclear Transfer <ul><li>Challenging: In cloned cell lines, about 4% of genes function abnormally, owing to departures from normal activation or expression of certain genes </li></ul><ul><ul><li>-Imprinting, methylation state </li></ul></ul>Limited success: ~25 percent of nuclear transfers led to a blastocyst; 35 percent of blastocysts led to establishment of cell lines Patient-specific embryonic stem cells derived from human SCNT blastocysts. Science 308(5729):1777-1783, 2005. Removing the egg nucleus before transferring a somatic cell nucleus Roslin Institute
    16. 17. hES Cell Lines in the US <ul><li>Most, if not all, of the stem cell lines are contaminated with mouse feeder layer proteins. </li></ul><ul><li>These cells will never be used in clinical application. </li></ul><ul><li>Considerable biological variability across cell lines. </li></ul><ul><li>Increased culturing can cause ES cells to accumulate epigenetic and genetic changes, altering their ability to form different types of cells. </li></ul>
    17. 18. Promises and Perils of Stem Cells <ul><li>Embryonic stem cells in therapy </li></ul><ul><li>Cloning </li></ul><ul><li>Adult stem cells in therapy </li></ul><ul><li>Beyond cell replacement </li></ul><ul><li>Beyond the embryo </li></ul>What’s at stake?
    18. 19. What can ESCs do for you? <ul><li>Theoretically </li></ul><ul><ul><li>Replace damaged, diseased cells </li></ul></ul><ul><ul><li>Gene therapy </li></ul></ul><ul><ul><ul><li>Genetically manipulated hES cells might serve as vectors to carry and express genes in target organs following transplantation in the course of gene therapy </li></ul></ul></ul>
    19. 20. Why Clone? <ul><li>Human protein production </li></ul><ul><ul><li>Produce human protein-based medicine in milk from transgenic cows </li></ul></ul><ul><ul><ul><li>α -1-antitrypsin for cystic fibrosis </li></ul></ul></ul><ul><li>Transplants without immune response </li></ul><ul><ul><li>Organ rejection or graft-vs-host disease </li></ul></ul>Therapeutic and Reproductive Cloning
    20. 21. Therapeutic Cloning
    21. 22. How Promising are Adult Stem Cells? <ul><li>Bone marrow transplants </li></ul><ul><ul><li>Hematopoeitic stem cell transfer </li></ul></ul><ul><li>Difficulty maintaining control once in vivo </li></ul><ul><ul><li>Niche dictates phenotype </li></ul></ul><ul><ul><li>Plasticity </li></ul></ul>
    22. 23. Adult Stem Cell Clinical Trials <ul><li>Bone marrow stem cells from self or allogeneic (sibling) transplant </li></ul><ul><ul><li>after chemotherapy for myeloma, glioma, leukemia, lymphoma, neuroblastoma, lung cancer </li></ul></ul><ul><ul><li>sickle cell anemia, liver disease, autoimmune disorders, vascular disease </li></ul></ul><ul><li>Mesenchymal stem cells for myocardial infarction </li></ul>
    23. 24. Potential Beyond Cell Replacement <ul><li>Exploring disease mechanisms </li></ul><ul><ul><li>study how basic cellular mechanisms are disrupted or changed by disease proteins </li></ul></ul><ul><li>Drug discovery </li></ul><ul><ul><li>High-throughput assays will identify targets. For example, using mouse ES cell-derived neural cells for an assay to screen Alzheimer's disease </li></ul></ul><ul><li>Genetic screening </li></ul><ul><li>Toxicology testing </li></ul>
    24. 25. Overview <ul><li>What are stem cells? </li></ul><ul><ul><li>How do embryonic and ‘adult’ stem cells differ? </li></ul></ul><ul><ul><li>How are different types of stem cell lines created? </li></ul></ul><ul><li>Stem cells in research and medicine </li></ul><ul><li>Alternatives to the embryo </li></ul>
    25. 26. Beyond the Embryo <ul><li>The President’s Council for Bioethics </li></ul><ul><ul><li>White Paper published May 2005 </li></ul></ul><ul><ul><li> </li></ul></ul>
    26. 27. ESCs without the E <ul><li>De-differentiation </li></ul><ul><ul><li>Requires aid of special cytoplasmic factors obtained from oocytes (or from pluripotent embryonic stem cells) </li></ul></ul><ul><li>Obtainable from any adult </li></ul><ul><li>Immunocompatible </li></ul><ul><li>Some success with muscle, liver, blood </li></ul>Issues: How far back can dedifferentiation go? Muscle cells Multipotent progenitors
    27. 28. ESCs without the E <ul><li>Remove single cell from 6-8 cell embryo </li></ul><ul><ul><li>Spin-off of preimplantation diagnosis </li></ul></ul>Issues: Is there harm in removing a cell? Could a cell line be established with one cell? Is cell at this stage totipotent? Remove cell Establish cell line Embryo
    28. 29. ESCs without the E <ul><li>Removal from dead embryo </li></ul><ul><ul><li>Early IVF embryos (roughly 4-8 cells) that have spontaneously died. Normal-appearing blastomeres in cleavage-arrested, mosaic embryos. </li></ul></ul>Issues: Can markers of organismic death be found? Can pluripotent stem cells be derived from dead embryos? If so, will they be chromosomally (and otherwise) normal?
    29. 30. Parthenogenesis <ul><li>Biochemically trick a human oocyte into thinking it has been fertilized. </li></ul><ul><li>Treated eggs divide to the blastocyst stage (50-100 cells), at which point stem cells can presumably be derived. </li></ul><ul><li>The “parthenogenetic” (that is, unfertilized but still developing) blastocyst-like entity is assumed by most to lack the potential for development as a human being. </li></ul>Oocyte Blastocyst Establish cell line
    30. 31. ESCs without the E <ul><li>Bio-engineered embryo-like artifacts </li></ul><ul><ul><li>Embryos engineered to lack the essential elements of embryogenesis but still capable of some cell division and growth </li></ul></ul>Altered Nuclear Transfer Embryo Remove altered nucleus to oocyte Somatic cell Oocyte Cell Division Blastocyst
    31. 32. ESCs without the E <ul><li>De-differentiation </li></ul><ul><li>Single cell removal from embryo </li></ul><ul><li>Removal from dead embryo </li></ul><ul><li>Parthenogenesis </li></ul><ul><li>Bio-engineered embryo-like artifact </li></ul>Creative thinking, possible solutions to an ethical dilemma. Research has yet to determine if one or more of these proposals are possible.
    32. 33. Recent Research <ul><li>RNAi was used to change expression of a gene in a hESC line. </li></ul><ul><ul><li>Stem Cells 23(3):299–305, 2005 </li></ul></ul><ul><li>hESCs driven to develop into motor neurons. </li></ul><ul><ul><li>Nature Biotechnology 23:215-221, 2005. </li></ul></ul>
    33. 34. Recent Research <ul><li>Mesenchymal stem cells injected into rat heart increased pumping capacity and vessel growth after heart attack. </li></ul><ul><ul><li>Journal of Clinical Investigation 115:326–338, 2005. </li></ul></ul><ul><li>“ Stembrids” were made — one ESC was enucleated and then given the nucleus from an adult somatic cell. </li></ul><ul><ul><li>Not shown that the resulting “stembrid” would be immunologically acceptable to the adult somatic cell donor. </li></ul></ul>
    34. 35. Summary <ul><li>Stem cells </li></ul><ul><ul><ul><li>Embryonic vs. Adult </li></ul></ul></ul><ul><ul><ul><li>IVF, SCNT </li></ul></ul></ul><ul><ul><ul><li>Therapeutic cloning and immune matching </li></ul></ul></ul><ul><li>Much scientific progress, but therapies are not yet directly translated from research </li></ul><ul><ul><ul><li>Greatest potential contribution from mechanistic studies in ESCs </li></ul></ul></ul><ul><ul><ul><li>Embryonic alternatives need more development </li></ul></ul></ul>
    35. 36. Conclusion <ul><li>Stem cells are complicated: scientifically, ethically, legally. The best way to approach them is with education. </li></ul><ul><li>Working with stem cells is one of the most important opportunities of our time. </li></ul>