1. Lisheng WANG Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine Lecture 9. Differentiation of human pluripotent stem cells into a given cell type for potential application – current progress & Review previous Quiz

2. The challenges for clinical application of hESC and human iPSC derivatives Expansion of hESCs Appropriate differentiation Functional integration Risk of teratoma or tumor formation Immune rejection

3. Cell replacement therapy Drugs, growth factors, gene transfer, … Stem cells (derived from hESCs and iPSCs) replacement therapy Organ transplantation

4. Paul J. Fairchild Immunological aspects in potential hESC therapy Patient-tailored pluriptent stem cells (iPSC, SCNT,…) Differentiation Modified from MSC

5.  Neuron can be efficiently generated from hESCs and iPSCs (Proc Natl Acad Sci U S A. 2010 Sep 7;107(36):15921-15926. Swistowski A, et al. Stem Cells. 2010 Aug 16 [Epub ahead of print]. Weick JP, et al. Stem Cells. 2010 Sep 8. [Epub ahead of print]).  MicroRNA-mediated conversion of human fibroblasts to neurons (Yoo et al, Nature 2011; doi:10.1038/nature10323)  Direct generation of functional dopaminergic neurons from human fibroblasts (Caiazzo et al, Nature 2011; doi:10.1038/nature10284) Isolation and directed differentiation of functional neuron from hESCs and iPSCs

6.  Differentiation of motor neurons (~50%) by a simple sequential application of retinoid acid (RA) and sonic hedgehog (SHH) in a chemically defined suspension culture. Ann N Y Acad Sci. 2010 Jun;1198:192-200 (Review) Li XJ, Hu B, Jones SA.. et al, Stem Cells 2008; 26(4):886-93 Differentiation of Ventral Spinal Progenitors and Motor Neurons from hESC by Small Molecules http://www.etsu.edu/cpah/hsci/forsman/Histology%2 0of%20musclefor%20web_files/image015.jpg

7. Toward the generation of rod and cone photoreceptors from mouse, monkey and human ES cells (NATURE BIOTECHNOLOGY 2008; 26: 215-224) Photoreceptors rodcorn Pigment epithelium Image from: http://www.usc.edu/dept/gero/AgeWorks/core_courses/gero500_core/biology_b_lect/index_a.htmhttp://www.usc.edu/dept/gero/AgeWorks/core_courses/gero500_core/biology_b_lect/index_a.htm http://thebrain.mcgill.ca/flash/i/i_02/i_02_cl/i_02_cl_vis/i_02_cl_vis.html

8. Toward the generation of rod and cone photoreceptors from mouse, monkey and hESCs NATURE BIOTECHNOLOGY 2008; 26: 215-224  Feeder- and serum-free suspension culture combined with Wnt and Nodal inhibitors induced differentiation of Rx+ (retinal homeobox) or Mitf+ (retinal pigment epithelial progenitors) retinal progenitors, which produced retinal pigment epithelial cells.  Subsequent treatment with retinoic acid and taurine induced photoreceptor differentiation.  These findings may facilitate the development of hESC– based transplantation therapies for retinal diseases. Generation of functional retinal pigment epithelium cells from hESCs (Cell Stem Cell. 2009 Oct 2;5(4):396-408)

9. Generation of Mechanosensitive Hair Cell-like Cells from mouse ESCs and iPSCs Cell 2010 May 14; 141: 704–716 The inability of the mammalian inner ear to regenerate lost hair cells is the major reason for the permanence of hearing loss and certain balance disorders. Scanning electron microscopic views of Hair Bundle-like protrusions of ESC- and iPSC-derived cells

10.  Generation of functional hepatocytes from hESCs under chemically defined conditions that recapitulate liver development. hESC-derived hepatocytes are able to carry out a range of hepatocyte functions (Touboul T, et al. Hepatology. 2010 May;51(5):1754-1765. Hay, DC. et al, Stem Cells 2008; 26(4):894-902. Methods Mol Biol. 2010;640:237-46.)  Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. (J Clin Invest. 2010 Sep 1;120(9):3127-3136) hESC-derived Hepatocytes & modeling liver disease with iPSCs http://www.transplant.bc.ca/pre_livingliver.htm

11. Production of pancreatic hormone– expressing endocrine cells from hESCs Stem Cell Res. 2009 Sep-Nov;3(2-3):73-87 (Review) D’Amour KA. et al, Nature Biotechnology 2006; 24: 1392 Kroon E, et al. Nat Biotechnol. 2008 Apr;26(4):443-452.  D’Amour et al. developed a differentiation process that converts hESCs to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin.  This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor—en route to cells that express endocrine hormones.  The hESC–derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal β-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose.  Pancreatic endoderm derived from hESCs generates glucose- responsive insulin-secreting cells in vivo.

12. Cardiomyocytes derived from hESCs in pro-survival factors enhance function of infarcted rat hearts  The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function after myocardial infarction compared with controls (Laflamme MA. et al, Nature Biotechnology. 2007 Sep;25(9):1015-1024)  Nongenetic method for purifying pluripotent stem cell- derived cardiomyocytes (>99% purity) (Nat Methods. 2010 Jan;7(1):61-66)  Generation of highly purified human cardiomyocytes by differentiation of hESCs in the presence of optimized concentration of Activin A and BMP4 (Kattman et al, Cell Stem Cell 2011; 8: 228-240).

13. Derivation of engraftable skeletal myoblasts from hESCs  Selective culture conditions and fluorescence-activated cell sorting (FACS)-mediated purification yielded multipotent mesenchymal precursors and skeletal myoblasts.  Skeletal muscle cells undergo in vitro maturation, resulting in myotube formation and spontaneous twitching.  hESC-derived skeletal myoblasts were viable after transplantation into the tibialis anterior muscle of SCID/Beige mice, as assessed by bioluminescence imaging.  Lack of teratoma formation and evidence of long-term myoblast engraftment suggests considerable potential for future therapeutic applications. Barberi T. et al, NATURE MEDICINE 2007; 13: 642

14. Derivation of endothelial and hematopoietic cells from hESCs Successful generation of Endothelial cells and progenitors Hematopoietic progenitors

15. Derivation of endothelial cells from hESCs (David T Scadden group: Nature Biotechnology 2007; 25:37) In vitro network In vivo functional blood vessel formation (green: hESC-derived endothelial cells; red: Red blood cells (Immunity, Vol. 21, 31–41, July, 2004) Endothelial markers: VE-cadherin+ PECAM-1/CD31+ LDL+ HUVECMEFhESC-derived Endo

16. 15d-EB, 14d-CFUx100, Hoffman Development of hESCs Into Blood (hematopoiesis) and endothelial cells

17. In vitro examine colony size and composition of hematopoietic progenitor cells derived from hESCs CFU-GM (Granulocyte/ Macrophage) CFU-GEMM ( Colony-Forming Unit- Granulocyte/ Erythroid/Monocyte/ Megakaryocyte ) BFU-E (Burst-Forming Unit-Erythroid) CFU-E (Colony-Forming Unit-Erythroid) 125x Multipotent UnipotentBipotent A “Unit” is now known to be an individual cell CFU-M (Macrophage) CFU-G (Granulocyte) Development of hESCs Into Blood (hematopoiesis) and endothelial cells

18. (Immunity 2007;26:669) CFU-s, colony-forming unit-spleen; CMP, common myeloid progenitor; CLP, common lymphoid progenitor, MEP, megakaryocyte- erythroid progenitor; GMP, granulocyte- macrophage progenitor; BFU-E, burst- forming unit-erythroid; Meg, megakaryocyte; Eo, eosinophil; G, granulocyte; and M, macrophage. Hierarchy of adult hematopoiesis Hematopoiesis - after born

19. Repopulating capacity of hESC-derived hematopoietic cells in NOD/SCID mice or fetal sheep? Do hESC-derived hematopoietic cells have hematopoietic stem cell potential?  A few research groups have currently reported that hESC- derived hematopoietic cells showed limited some hematopoietic stem cell properties after transplantation into immunodeficient mice or fetal sheep (J. Exp. Med. 2005; 201, 1603–1614. Stem Cells 2006; 24, 1370–1380. Blood 2006; 107, 2180–2183; 1: Cell Stem Cell. 2008 Jul 3;3(1):85-98)  The ability to generate transplantable hematopoietic stem cells from hESCs remains challenging.

20. Review previous Quiz