Stem cell biology

1. --2 Jan-Kan Chen College of Medicine Chang Gung University Stem cell biology

2. Invariant asymmetry ？ Populational asymmetry 成體幹細胞之分裂與分化模式 JKC

3. Microenvironment mediate cell differentiation ES-feeder interactions Growth factor paracrine ES-matrix

4. In vitro differentiation of ES cells Physiol. Rev., 85, 635, 2005

5. Definition of Adult Stem Cells (5/50)

6. History of Adult Stem Cell Research • Since the 1970’s, bone marrow transplants have been used for treatment of immunodeficiencies and leukemias.

7. Mammalian epidermal stem cells Nature, 414, 98, 2001

8. Published Reports on Identification of Human Adult Stem Cells Sources of adult stem cells include bone marrow, blood, the cornea and the retina of the eye, brain, skeletal muscle, dental pulp, liver, skin, adipocyte, the lining of the gastrointestinal tract, and pancreas. unipotent ?

9. Adult Stem Cells

10. Plasticity • Plasticity is the ability of an adult stem cell from one tissue to generate the specialized cell type of another tissue. • Example: Adult stem cells from bone marrow generated cells that resemble neurons

11. Different steps in the transition of adult corneal epithelium into an epidermis Cornea epithelium Embryo dermis Epidermis Development 127, 5487-5495 (2000) (After 21Days)

12. Possible Roles of Bone Marrow–Derived and Circulating Stem Cells in the Repair of Solid-Organ Tissue N ENGL J MED., 349, 570, 2008

13. Potential Application of Stem Cell Technology

14. How Does Cell Therapy Work? • Bone marrow transplants are an example of cell therapy in which the stem cells in a donor's marrow are used to replace the blood cells of the victims of leukemia. • Cell therapy is also being used in experiments to graft new skincells to treat serious burn victims, and to grow new corneas for the sight-impaired. • In all of these uses, the goal is for the healthy cells to become integrated into the body and begin to function like the patient's own cells.

15. What Diseases Can be Cured by Stem Cell Therapies? • Any disease in which there is tissue degeneration can be a potential candidate for stem cell therapies • Type 1 diabetes mellitus - beta cells of the pancreas • Parkinson's disease - dopamine-secreting cells of the brain • Spinal cord injuries leading to paralysis of the skeletal muscles • Ischemic stroke where a blood clot in the brain has caused neurons to die from oxygen starvation • Multiple sclerosis - loss of myelin sheaths around axons • Myocardium infraction – death of cardiomyocytes

16. Differentiation Engraftment Stem cells Specific cell types Tissue or organ Stem cell research / Cell therapy Disease Diabetes Parkinson’s disease Spinal cord injury Blindness …

17. Persons in the United States affected by diseases that may be helped by human pluripotent stem cell research Data from the Patients' Coalition for Urgent Research, Washington, DC

18. PART2: The Embryonic Stem Cell Jan-Kan Chen College of Medicine Chang Gung University Stem cell biology

19. Embryonic stem cells Totipotent stem cell pluripotent stem cell multipotent stem cell

20. Early developement in humans • Day 0: Fertilization of the oozyte in the oviduct. • Zygote – totipotent • Day 4-5: (16 cells) – morula, soloid mass of cells • Day 6-7: Blastocyst formation - pluripotent • 3rd week: Gastrulation, i.e formation of the three germ layers.

21. Factors associated with early embryogenesis • Inner cell mass: FGF-4 (embryogenesis and differentiation of trophectoderm) • Trophectoderm: leptin and STAT3 (implantation) • Trophoblast (mouse): Mash 2 (placenta formation) • Epiblast: goosecoid, T, Evx-1, follistatin (primitive streak formation)

22. Regulation of body pattern and differentiation • GATA-4, -6: Early differentiation • Hox: Anterior-posterior polarity • Nodal and Lefty: Left-right symmetry • Hex: Anterior-posterior development • Mrg1: Heart formation • BMP-4: Differentiation of mesenchymal cell, primitive streak migration, CNS development • Wnt3: Formation of the primitive streak and the node • HNF-4, STAT-3: Visceral endoderm differentiation

23. Culture of human embryonic stem cells Thomson et al., (1998) Science 282 : 1145-1147

24. How Many Human Embryonic Stem Cell Lines are There? • The actual number of human embryonic stem cell lines is a matter of some debate. • To date, more than 100 human embryonic stem cell lines have been derived worldwide. • However, most of those lines have not adequately characterized yet. • Only 22 cell lines are eligible for federal funding in the USA.

25. Comparison of some properties of mouse and human embryonic stem cells MEF, mouse embryonic fibroblasts; EB, embryoid body.

26. Maintaining mouse embryonic stem cells in their undifferentiated state • LIF, either produced by feeder cells or added exogenously, allows mouse ES cells to proliferate without differentiation in vitro • LIFR and gp130 are required for LIF binding, which in turn activates STAT3, which is necessary for continued proliferation of ES cells • STAT3 and Oct-4 may interact and perhaps affect the function of a common set of target genes • Activation of ERK and SHP-2 inhibit self-renewal of ES cells • In mouse ES cells, Oct-4 expression and Gab-1 activation suppress Ras-ERK signalling pathway, and suppress induction of differentiation

27. Leukemia inhibitory factor (LIF) • Early blastocyst development and implantation • Survival for primordial germ cell • Maintenance of mouse embryonic stem (ES) cell but not human ES cell

28. Effect of LIF on self-renewal of mouse embryonic stem cells +LIF 24h +LIF 48h -LIF 24h -LIF 48h Nature 336, 684-7 (1988)

30. Regulation of self-renewal in mouse ES cells by Oct3/4, Nanog, BMP-dependent SMAD, and LIF-dependent JAK/STAT3 signaling pathways Physiol. Rev. 85: 635-678, 2005

31. Examples demonstrating the developmental potential of human ES cells in vitro • Cell Types Developed • Ectoderm, endoderm, mesoderm, and neural precursors • Cardiomyocytes • Cardiomyocytes, endodermal, hematopoietic, and neuronal cells • Neuronal, epithelial, pancreatic, urogenital, hematopoietic, muscle, bone, kidney, and heart cells • Neural epithelium, embryonic ganglia, stratified squamous epithelium, gut epithelium, cartilage, bone, smooth and striated muscle cells • Cells with properties of pancreatic -like cells • Cardiomyocytes, pigmented and nonpigmented epithelial cells, neural cells, mesenchymal cells, erythroid, macrophage, granulocyte, and megakaryocyte cells • Myeloid, erythroid, megakaryocyte colony-forming cells • Neural precursors, glial and neuronal cells: incorporation into the brain (H1, H9, H9.2 lines) • Neural precursors, glial and neuronal cells: incorporation into the brain (HES-1 line) • Neural progenitor, dopaminergic, GABAergic, glutamatergic, glycinergic neurons, astrocytes • Neural progenitor, neuronal cells • Trophoblast • Hepatocytes

32. Directed differentiation of human ES cells in vitro • Human ES cells differentiate spontaneously if removed from feeder cells and grown in suspension culture • bFGF: Epidermal epithelial cells (keratin) • Activin A:Muscle cell-like syncytium (enolase) • Retinoid acid: Neuron (neurofilament H) • Mouse BM stromal cell: Hematopoietic precursor cell (CD34)

33. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells • None of the growth factors directs differentiation exclusively to one cell type • Activin A and TGF-1 mainly induce mesodermal cells • RA, EGF, BMP-4 and bFGF activate ectodermal and mesodermal markers • NGF and HGF allow differentiation into the three germ layers • Most of the factors inhibit differentiation of specific cell types, and this inhibitory effect is more pronounced than an induction effect (PNAS 97:11307-12, 2000)

34. An ES colony + HGF + activin A + RA + bFGF + BMP-4 Schuldiner et al PNAS 97:11307-12, 2000

35. Schuldiner et al PNAS 97:11307-12, 2000

36. Schuldiner et al PNAS 97:11307-12, 2000

37. Schematic illustration for the isolation and differentiation of hES cell–derived NCS cell Nature Biotechnology 25, 1468 - 1475 (2007) Nature Biotechnology25, 1468 - 1475 (2007)

38. Adipocyte differentiation Nature Reviews Molecular Cell Biology7, 885–896 (2006)

40. Thank You

41. Trophectoderm (multipotent) Cdx2 Primitive endoderm (multipotent) Gata6 Oct4 Precusor (totopotent) Nanog Epiblast (pluripotent) Inner cell mass (pluripotent) Self-organizing transcription faactors network for ES cells self-renew Oct4: Loss of Oct4 causes differentiation of ES cells into trophectoderm. Overexpression of Oct4 results in differentiation into primitive endoderm and mesoderm. Sox2: One of the target genes of Oct4 and is required in ES cells with pluripotent sustenance. Nanog: Nanog can activate Oct4 promoter and also as transcription repressor for cell differentiation genes.

42. Maintaining pluripotency Autoinductive FGF4/Erk signaling poises ESCs for lineage entry and must be resisted to allow self-renewal. A.Oct4 and Sox2 direct expression of fgf4 and poise ES cell from lineage commitment, Elevated Erk activity provides a signal rendering pluripotent cells susceptible to lineage inductive cues. B. Self-renewal of the pluripotent ES cell state requires overcoming the fgf4/Erk signal. The actions of FGF can be 1) blocked by inhibitors; 2)reversed by constitutive Nanog expression; 3) counteracted by LIF and BMP4. Cell, 132:532,2008

43. Signaling Transduction Pathways Involved in Maintaining Mouse ESC LIF-STAT3 pathway: LIF (leukemia inhibitory factor) stimulates mESC through the gp130, which works as a heterodimer together with LIFR. Activation of gp130 leads to the activation of the JAK and STAT. Wnt pathway: Wnt/b-catenin signaling involved in the maintenance of pluripotency of ESC. Wnt signaling activation can upregulate c-Myc and STAT3 expression. BMP4 pathway: BMP4 phosphorylates Smad1/5 in mouse ES cells. Smad1/5 activation results in the expression of inhibitor of differentiation (ld) protein, which blocks the neural differentiation.

44. Induced pluripotent stem cells, iPS cells Man-made pluripotency can be achieved through induced reprogramming of somatic cells Cell stem cell 2,2,151-9,2888

45. Startrgies for the generation of pluripotent stem cells from somatic cells

46. Mouse gene combinations for iPS induction The relation of ES cell And iPS cell is unclear, they may be Similar but not identical. Nature review molecular biology 9,725,2008

47. Klf4: Serves as upstream regulator of Oct4, Sox2, Nanog, and c-Myc. C-Myc: A major downstream target for the LIF/STAT3 and the Wnt signalling pathways that support maintenance of pluripotency. Lin28: RNA binding protein. Play a central role in blocking miRNA mediated differentiation in stem cells.

48. Putative Role of the Four Factors in the Induction of iPS Cells Pluripotent stem cells are immortal and have open and active chromatin structure. Myc induces these two properties. Myc also induces apoptosis and senescence , which are suppressed by KLF4. Oct3/4 change the cell fate from tumor cells to ES cells. Forced expression of c-Myc and KLF4 alone would result in the generation of tumor cells, but not pluripotent stem cells.

49. Putative Role of the Four Factors in the Induction of iPS Cells . Oct-3/4 and Sox2 activate multiple target genes synergistically. KLF4 may also function as cofactor of Oct-3/4 and Sox2. KLF4 : Kruppel-like factors, are zinc-finger proteins.

50. Following injection into blastocysts, iPS cells contributed to mouse embryonic development Mouse E 7.5 Mouse E 13.5