Stem Cells: Minireview Presentation

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PowerPoint giving a summary on research in stem cells (brief historical overview), and the explanatory component of the papers which changed the game of stem cell research Yamanka's Nuclear Reprogramming.

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  • Stem Cells: Minireview Presentation

    1. 1. Stem Cells Dr. Tai-ping Sun Biology 280S: Biotechnology & Genetic Engineering Jon Martin Joshua Mendoza-Elias Fall 2008
    2. 2. Outline: <ul><li>I. Introduction </li></ul><ul><li>A. Definition of Stem Cell </li></ul><ul><li>B. History and Discovery of SCs </li></ul><ul><li>II. Therapeutic Applications of SCs </li></ul><ul><li>A. Methods for studying Stem Cells </li></ul><ul><li>B. Problems with cloning </li></ul><ul><li>C. Alternatives to cloning </li></ul><ul><li>III. Policy </li></ul><ul><li>IV. Nuclear Reprogramming </li></ul>
    3. 3. Properties of Stem Cells <ul><li>Self-renewal </li></ul><ul><li>Potency </li></ul><ul><li>・ Totipotent : Includes fertilized zygote and first few divisions of the fertilized egg. These cells can differentiate into embryonic and extraembryonic cell types. </li></ul><ul><li>・ Pluripotent: SC’s are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. </li></ul><ul><li>・ Multipotent: SC’s can produce only cells of a closely related family of cells (e.g. hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). </li></ul><ul><li>・ Unipotent : SC’s cells can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem cells (e.g. muscle stem cells). </li></ul>
    4. 4. How do SC arise?
    5. 5. Where do SC come from? Embryonic Fetal Adult Berashis Stem Cells
    6. 6. When do SC arise?
    7. 7. For the love of cloning <ul><li>Nuclear transfer technique: </li></ul><ul><li>Donor egg: oocyte from female denucleated </li></ul><ul><li>Cloned: Nucleus from Blastula of albino frog </li></ul><ul><li>transferred to donor egg. </li></ul>J.B. Gurdon (1962): S. African clawed frog ( Xenopus laevis ) Evidence from EvoDevoBio: The development of cloning techniques revealed organisms could be grown from one cell.
    8. 8. One of these things is not like the other. <ul><li>Evidence from immunology </li></ul><ul><li>suggests that a cell type </li></ul><ul><li>possesses similar totipotent </li></ul><ul><li>Qualities of ES cells in adult cells. </li></ul>Proof of Concept: Clinical Procedure:
    9. 9. Mechanism of progenitor cells in the first steps of B-cell maturation. Receptors and ligands look familiar? ( c-kit , SCF )
    10. 10. Techniques for studying ES cells The more things change, the more the stay the same. <ul><li>Experimental techniques focus on determining </li></ul><ul><li>“ uniqueness” of ES cells in the hopes of </li></ul><ul><li>imitating it. </li></ul><ul><li>-genetic expression profiles </li></ul><ul><li>-Epigenetic state of genome </li></ul><ul><li>-juxtracrine/endocrine signaling </li></ul><ul><li>-transplantation effects </li></ul><ul><li>-TF’s </li></ul>
    11. 11. II. Techniques in Stem Cell Therapy
    12. 12. Therapeutic Cloning <ul><li>Nuclear Transfer: Method is the same as was used to clone Dolly the sheep in February 1997 </li></ul>
    13. 13. Therapeutic Cloning
    14. 14. Problems with Therapeutic Cloning <ul><li>Cost </li></ul><ul><li>Destruction of embryos </li></ul><ul><li>Inefficiency – 277 cells taken from Dolly’s “mother,” but only 30 became blastocysts. (13.21%) </li></ul>
    15. 15. Alternative Therapeutic Methods <ul><li>Immunosuppression </li></ul><ul><ul><li>Associated risks </li></ul></ul><ul><li>“ Invisible” cells </li></ul><ul><ul><li>Notch protein </li></ul></ul><ul><ul><li>MHC replacement </li></ul></ul>
    16. 16. Notch Protein
    17. 17. Major Histocompatablility Complex
    18. 19. Other Alternative Methods <ul><li>Use of haematopoietic stem cells (HSCs) </li></ul><ul><li>Own bone marrow </li></ul>
    19. 20. The Heart <ul><li>Bone marrow stem cells effective in trials to restore lost tissue </li></ul><ul><li>Thigh tissue has also been used, though problems have arisen </li></ul><ul><li>Use of mesenchymal stem cells and G-SCF </li></ul>
    20. 21. Study: Orlic et al. <ul><li>Studied effects of using bone marrow stem cells to treat mice that suffered heart attacks </li></ul><ul><li>Sorted bone marrow cells according to whether they expressed c-kit , a surface protein found on HSCs </li></ul><ul><li>Transplanted into heart </li></ul>
    21. 22. Orlic Study Results <ul><li>Arrows indicate regenerating myocardium </li></ul><ul><li>VM: viable myocardium </li></ul><ul><li>MI: myocardial infarcted </li></ul><ul><li>Red = myosin. There is less in the infarcted region. </li></ul><ul><li>Green = Nuclei </li></ul><ul><li>Areas of growth indicated by arrows </li></ul>Figure 1(a) Magnification: 12x
    22. 23. Study Results, cont’d <ul><li>68% of the space affected by heart attack was replaced by new myocardium </li></ul><ul><li>New tissue had myocytes and vascular structures </li></ul><ul><li>Improved haemodynamics over untreated heart ( c-kit negative) </li></ul>
    23. 24. III. Policy Issues <ul><li>No federal law criminalizing destruction of embryos, though some states have these laws </li></ul><ul><li>Proposition 71 in California – explicitly permits research using somatic cell nuclear transfer </li></ul><ul><li>2008 presidential election could bring about a significant change in current policy </li></ul><ul><li>What’s your opinion? </li></ul><ul><li>Current policy only allows federal funding for existing embryonic stem cell lines (21 in all), not new ones </li></ul>
    24. 25. Stem Cell Viddeo http: //youtube .com/watch? v=mUcE1Y_bOQE
    25. 26. IV. A Transcriptional Logic for Nuclear Reprogramming Takahashi & Yamanaka/ Kit T. Rodolfa and Kevin Eggan Cell (2006) 126: 652-655 <ul><li> . Background: </li></ul><ul><li>-Pluripotency: nuclear transfer or fusion with ES. </li></ul><ul><li>-Factors for reprogramming unknown. </li></ul><ul><li>Questions: </li></ul><ul><li>(i.) Minimal factors? </li></ul><ul><li>(ii.) Can we use make ES-like cells? </li></ul><ul><li>(iii.) How do these cells compare to ES cells? </li></ul>
    26. 27. Trans Reprogramming continued <ul><li>Hypothesis : </li></ul><ul><li>(i.) We can find factors. </li></ul><ul><li>(ii.) We can build pluripotent cells. </li></ul><ul><li>Experimental Design: </li></ul><ul><li>-MEF and Tail Tip Fibroblasts (TTF) introduce factors via retrovirus </li></ul><ul><li>-Test cell cultures. </li></ul>
    27. 28. Nuclear Reprogramming Continued <ul><li>The Experiment: </li></ul>Figure 1: Reprogramming Differentiated Somatic Cells Individual factors insufficient to trigger embryonic reporter (Fbx15:  -geo). 24 previously identified genes: Self-renewal : Oct3 /4, Sox2 , Nanog Pluripotency: c-myc , Eras , Klf4 Q: What’s the magic formula? Results: Iterate through combinations. Narrowed pool down to 4 cDNAs (all above except Nanog).
    28. 29. Comparison of Methods
    29. 30. Nuclear Reprogramming cont’d: Results & Discussion <ul><li>Q3: How do iPS cells compare to ES cells? </li></ul><ul><li> iPS-TFF chimeric mice </li></ul><ul><li>X No chimerism post natal animals. </li></ul><ul><li>Clone-to-clone in expression variation. Certain genes “lost” </li></ul><ul><li>Epigenetics: intermediate to somatic & ES cells. </li></ul><ul><li> . Future Directions: </li></ul><ul><li>iPS cells different. Can additional reprogramming fix this? </li></ul>
    30. 31. Generation of germline-competent induced pluripotent stem cells Keisuke Okita, Tomoko Ichisaka, & Shinya Yamanaka Nature 448 : 313-317 <ul><li> . Previously . . . </li></ul><ul><li>-Retroviral introduction Oct3/4 , Sox2 , c-Myc , and Klf4 </li></ul><ul><li>-Fbx15 IPS cells similar to ES cells, but different: </li></ul><ul><li>gene expression profile, epigenetics, & NO adult chimeras. </li></ul><ul><li>Q: Can we build better ES-mimetic cells called Nanog iPS cell clones? </li></ul><ul><li>H: You betcha! Using Nanog should make them germline-competent. </li></ul><ul><li>E: Same idea but new trick. New GFP marker used to indicate pluripotency. Then, add the four previously identified factors. Screen/culture/test. </li></ul>
    31. 32. Generation of germline. . . continued <ul><li>The Experiment: </li></ul>Construct : Enhanced GFP (EGFP)-internal ribosome entry site (IRES)-puromycin resistance (Pur R ) into 5’ UTR <ul><li>Results: </li></ul><ul><li>Nanog iPS cells found in blastocyst, migrating primordial germ cells (9.5 dpc), & genital ridges (13.5 dpc). </li></ul><ul><li>Teratomas generated with 3 germ layers. </li></ul>~5% were were GFP positive - Cells indistinguishable form ES cells (morphology/proliferation*) Nanog iPS, Fbx15 iPS, and ES cells were then characterized.
    32. 33. Results <ul><li>Q: How sim/diff are Nanog iPS cells to Fbx15 iPS cells and ES cells? </li></ul><ul><li>RT-PCR data: Nanog iPS cells expression profile closer to ES cell profile </li></ul>Epigenetics: Methylation signature closer to ES cells SSLP (single sequence length polymorphism): Unique signature* Induction Efficiency: Nanog iPS cells: 0.001-0.03% Fbx15 iPS cells: 0.01-0.5% Differentiation in LIF & RA of Nanog iPS more closely resembles ES cells.
    33. 34. Discussion: <ul><li>Nanog iPS cells were “more ES-like” </li></ul><ul><li>Germline competism: </li></ul><ul><li>Chimerism: 10%-90% </li></ul><ul><li>Cross: </li></ul><ul><li>-Male mice had small testes and aspermatogenesis </li></ul><ul><li>-No Nanog iPS cell in mature sperm </li></ul><ul><li>-F 1 confirmed transmission of reporter </li></ul><ul><li>• Germline competency </li></ul><ul><li>Clinical Applications: </li></ul><ul><li>- c-myc lead to tumour reactivation </li></ul><ul><li>• Advise transient expression system </li></ul><ul><li>Low induction efficiency: </li></ul><ul><li>-Rare SC coexisting with MEF culture? </li></ul><ul><li>• Other determinants </li></ul>
    34. 35. Conclusion <ul><li>ES cell analogues are possible </li></ul><ul><li>Epigenetics </li></ul><ul><li>Further refinement </li></ul><ul><li>Identification of Factors </li></ul><ul><li>Delivery system </li></ul>
    35. 36. References: <ul><li>[1] Aldhous, P. Can they rebuild us? Nature (2001) 410 : 622-625. </li></ul><ul><li>[2] Wadman, M. Stem-cell issue moves up the US agenda. Nature (2007) 446 : 842. </li></ul><ul><li>[3] Holden, C. California's proposition 71 launches stem cell gold rush. Science (2004) 306 : 1111. </li></ul><ul><li>[4] Couzin, J. and Vogel, G. Renovating the heart. Science (2004) 304 : 192-194. </li></ul><ul><li>[5] Orlic, D., Kajstura, J., Chimenti, Stefano. Jakoniuk, I., Anderson, S.M., Baosheng, L., Pickel, J., McKay, R., Nadal-Ginard, B., Bodline, D.M., Leri, A., and Anversa, P. Bone marrow cells regenerate infarcted myocardium. Nature (2001) 410 : 701-705. </li></ul><ul><li>[6] Rodolfa, K.T., and Eggan, K. (2006) A transcriptional logic for nuclear reprogramming. Cell 126 : 652-655. </li></ul><ul><li>[7] Okita, K., Ichisaka, T., and Yamanaka, S.. Generation of germline-competent induced pluripotent stem cells. Nature (2007) 448: , 313-317. </li></ul>

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