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Overview Mouse embryonic stem cells Human embryonic stem cells Pluripotency genes and network Long-term self-renewal Dir

Overview Mouse embryonic stem cells Human embryonic stem cells Pluripotency genes and network Long-term self-renewal Directed differentiation Induced pluripotent stem cells . Stem cells, pluripotency and differentiation. Two major types of stem cells Adult and embryonic stem cells.

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Overview Mouse embryonic stem cells Human embryonic stem cells Pluripotency genes and network Long-term self-renewal Dir

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  1. Overview • Mouse embryonic stem cells • Human embryonic stem cells • Pluripotency genes and network • Long-term self-renewal • Directed differentiation • Induced pluripotent stem cells

  2. Stem cells, pluripotency and differentiation Two major types of stem cells Adult and embryonic stem cells Self-renewal The ability to undergo symmetrical divisions without differentiation Pluripotency The ability to give rise to differentiated cell types derived from all three primary germ layers of the embryo: endoderm, mesoderm, and ectoderm Induced pluripotent stem (iPS) cells induction of pluripotent stem cells from differentiated cells

  3. Differentiation of human tissues

  4. Generation of embryonic stem cells Two prominent features of ESCs: long-term self-renewal and pluripotency

  5. Inner cell mass (ICM): a cluster of cells at the blastocyst stage A blastocyst cultured on a petri dish Day 1 Day 2 Day 3 Day 4 DAPI Alkaline phosphatase positive

  6. Isolation of ICM cells Mouse embryos Rabbit Anti-mouse serum Pipetting Outer cells are lysed.

  7. Derivation of embryonic stem cells from mouse embryos Martin Evans 2007 Nobel Prize Karyotype is normal Evans, M.J. & Kaufman, M.H. Nature292, 154-156, 1981

  8. Feeders provide factors that maintain embryonic stem cell growth Day 13 mouse embryos MEFs: mouse embryonic fibroblasts Remove heads and internal organs MEFs Treat with trypsin and plate cells into a dish irradiated to stop MEF growth

  9. Embryonic stem cells are pluripotent Embryoid bodies Cells of three germ layers Lowattachment ESCs (mixture of differentiated cells) Teratomas Mouse injection

  10. Derivation of embryonic stem cells from human embryos Jamie Thomson Univ. of Wisconsin H9 cell line ICM-derived Critical factors: MEFs, basic FGF Differentiating cells Thomson, et al., Science, 1998

  11. What are the promises? • Understand early human development (infertility, birth defects) and control of cell division (cancer) • Cell-based therapy • Reduce need for organ and tissue donors/transplants • Replace mutant or damaged cells for treatment of diseases such as Parkinson’s disease, spinal cord injury, muscular dystrophy, heart disease, liver dysfunction, osteoporosis, vision and hearing loss • A short-cut for drug discovery and testing

  12. Transcription factors required for pluripotency Austin Smith Oct4 -/- embryo lack inner cell mass Oct4 -/- cells are not pluripotent Other important transcription factors: Sox2 and Nanog Inner cell mass

  13. Core ES cell regulatory circuitry Jaenisch and Young, Cell. 2008

  14. Regulation of long-term self renewal Mouse ESCs LIF (Smith et al., Nature, 1988) BMP (or serum) (Ying et al, Cell, 2003) 3i (Ying et al, Nature, 2008) (Buehr et al, Cell, 2008) LIF and BMP act on downstream differentiation signals of MAPK He S et al. 2009. Annu Rev Cell Dev Biol;

  15. Directed ES cell differentiation Transcription factor landscape Graf T and Enver T, 2009, Nature

  16. What would be an ideal method for directed differentiation? Rapid Simple Cheap Mimic development

  17. Conditions for directed differentiation 1. EBs OP9 co-culture Expansion EB medium EB digestion Hematopoietic stem cells EB formation Hematopoietic stem cells hESCs Neutrophils Progenitor Expansion medium 7d Terminal differentiation medium 6-7 d 18 d 2. Co-culture OP9 mouse stroma cells – hematopoietic differentiation PA6 or MS5 – neural differentiation 3. Monolayer cultures

  18. Hypothesis Differentiated somatic cells can be re-programmed into pluripotent stem (ESC-like) cells with gene(s) important for ESC identity (pluripotency and self-renewal) Shinya Yamanaka Kyoto University These cells would • Bypass ethical issues • Create patient-specific pluripotent stem cells

  19. 24 candidate genes Dppa2 b-catenin Oct4 Dppa3/ Stella Dnmt3l Rex1 Dppa4 Fthl17 Sall4 Dppa5/ Esg1 Grb2 Utf1 Ecat1 Sox2 Ecat3/ Fbx15 Sox15 Klf4 Ecat5/ Eras Tcl1 Myc Ecat8 Nanog Ecat9/ Gdf3 Stat3 Gene delivery: Retrovirus allowing gene integration into the host genome Takahashi and Yamanaka (2006) Cell126, 663-676

  20. Putting all 24 genes into MEFs “reprograms” FBX15: an ESC-specific gene; only expressed in ESCs bgeo:G418 (an antibiotics that kills the cells) resistance gene So, cells can survive only when they become ESC-like cells Viral promoter Takahashi and Yamanaka (2006) Cell126, 663-676

  21. Narrowing down the candidates Oct4 (14) Sox2 (15) Klf4 (20) Myc (22) Takahashi and Yamanaka (2006) Cell126, 663-676

  22. iPS cells are pluripotent Pluripotency markers EB formation Teratoma formation - Saw the same thing with tail-tip fibroblasts Takahashi and Yamanaka (2006) Cell126, 663-676

  23. Takahashi K., et al. (2007) “Induction of pluripotent stem cells from adult human fibroblasts by defined factors.” Cell 131, 861-72. OCT4, SOX2, KLF4, MYC Yu J., et al. (2007) “Induced pluripotent stem cell lines derived from human somatic cells” Science 318, 1917-1920. OCT4, SOX2, NANOG, LIN28 Park I.H., et al. (2007) “Reprogramming of human somatic cells to pluripotency with defined factors” Nature 451, 141-146. OCT4, SOX2, KLF4, MYC How about human cells?

  24. Stem cell-based therapy Regenerative Medicine Stem Cell Biology Human somatic cells Translation Cellular therapies Derivation iPSCs • Scale Up • Quantitative, systematic • approaches • Quality control Propagation Differentiation Tissue morphogenesis “Personalized medicine” Adapted from: Gepstein. Circ Res 2002 & http://stemcells.nih.gov/info/media/DSC_1187.jpg

  25. Pitfalls with iPSCs <0.1% • Low efficiency of derivation • Use of C-myc • Transgene integration • Are they really the same as ESCs?

  26. Low efficiency of derivation - Are all four genes expressed in the same cells? Approach: Using a single retroviral or lentiviral vector instead of four vectors (2A peptide) Somers A, et al 2010, Stem Cells (STEMCCA Cre-Excisable lentivector) Staerk, J et al, 2010, Cell Stem Cell (T cells and myeloid cells) • Use of C-myc • Chemical complementation (e.g., with small molecules such as VPA) to replace C-Myc • Other compounds: Vitamin C, sodium butyrate, ALK5 inhibitor(*, mESC medium), Apigenin and Luteolin (E-cadherin enhancing) • Reprogramming with small molecules only?

  27. Transgene integration - integrating-free vectors • Episomal vectors followed by selection of integration free cells • Cre/loxP-recombination system to deliver followed by removal • with Cre- recombinase • Single-vector reprogramming system combined with a piggyBac transposon ,

  28. - Protein and mRNA-based • Delivery of OCT-4, SOX2, Myc and Klf4 mRNA or proteins, instead of genes, into somatic cells Protein: polyarginine tag Mouse, 30 days, the need for VPA. Human, 50 days, HEK293 cell extracts Synthetic mRNA: 17 days, 2% efficiency

  29. Are iPSCs as good as ESCs? Mouse iPSCs: Can contribute to embryonic development (Takahashi and Yamanaka, Cell, 2006) Produce adult chimera and are germ-line competent (Okita et al, Nature, 2007) Are capable of giving rise to every cell in the new born mice (Zhao et al., Nature, 2009) Journal of Molecular Cell Biology (2010), 2, 171–172

  30. Human iPSCs • Global gene expression profiling; • 2. Modifications of histone tails; • 3. The state of X chromosome inactivation • 4. Profiles of DNA methylation At least for some clones, iPSCs are similar if not indistinguishable from ESCs (Mikkelsen et al., Nature, 2008)

  31. Stem cell-based therapy Regenerative Medicine Stem Cell Biology Human somatic cells Translation Cellular therapies Derivation iPSCs • Scale Up • Quantitative, systematic • approaches • Quality control Propagation Differentiation Tissue morphogenesis “Personalized medicine” Adapted from: Gepstein. Circ Res 2002 & http://stemcells.nih.gov/info/media/DSC_1187.jpg

  32. Disease Modeling using iPSCs Disease-specific iPSCs Disease-related differentiated cells Lee, G., Papapetrou, E.P., Kim, H., Chambers, S.M., Tomishima, M.J., Fasano, C.A., Ganat, Y.M., Menon, J., Shimizu, F., Viale, A., Tabar, V., Sadelain, M., and Studer, L. (2009). Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461, 402-406. Marchetto, M.C.N., Carromeu, C., Acab, A., Yu, D., Yeo, G. W., Mu, Y., Chen, G., Gage, F.H., and Muotri, A.R. (2010). A Model for Neural Development and Treatment of Rett Syndrome Using Human Induced Pluripotent Stem Cells. Cell, 143, 527-539.

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