The Pluripotency : Lessons from Embryonic Stem Cell Properties

1. The Pluripotency: Lessons from Embryonic Stem Cell Properties M. SaifurRohman, M.D., Ph.D. Cardiologist

2. Outline • Regenerative medicine • Historical Perspective • Stem Cells • Embryonic Stem Cells (ESC) • Pluripotency induction (iPS): A future challenge • Adult Stem Cells (ASC): A comparison • The use stem cell in medicine

3. Regenerative Medicine • Regenerative medicine aims to repair diseased or damaged tissues by replacing the affected cells with healthy, functional cells of the same type. • The prospects of this discipline have been boosted by the promise of ES cells, which are pluripotent (they can differentiate into any cell type) & which can be maintained in culture to “self-renewal” indefinitely. Blelloch, Nature 2008.-

4. Historical Perspective • Major Changes in regenerative medicine (replacement of damaged or diseased cell tissues with new cells and tissues) due to advances in stem cell technologies • First successful bone marrow transplant done in1956 on leukemic patient. Bone marrow contains adult derived hematopoietic stem cells, able to regenerate tissues similar to the specialized tissues in which they are found. • Embryonic stem cells believed to have greater potential. This line of stem cell research has been the most controversial.

5. Historical Perspective

6. Isolation & Culture of Embryonic Stem Cells(Human-1998; Mouse-1981) Method patentedU.S. patent held by Univ. Wisconsin Advantages:1) Proliferate indefinitely2) Form any tissue J. Thomson 1998

7. Why self-renew and differentiate? 1 stem cell 4 specialized cells 1 stem cell Differentiation - replaces dead or damaged cells throughout your life Self renewal - maintains the stem cell pool

8. Stem Cells: Definition • Stem cells: Primitive cells that have the capacity for extensive self-renewal, clonogenic, and the ability to differentiate into multiple cell types • Embryonic stem cells: Totipotent (pluripotent) cells derived from the inner cell mass of the blastocyst; they give rise to cells of all three germ layers • Adult stem cells: present in all renewing tissues; these cells divide for self-renewal and differentiate into multiple progenitor cell types.

9. Stem Cells: Definition • Progenitor cells: multipotential intermediate stem cells that serve as the direct precursors for tissue-specific mature cells. • Endothelial progenitor cells: cells that are present in blood and bone marrow; they are involved in angiogenesis and postnatal neovasculogenesis.

10. Stem Cells: Definition • Mesenchymal stem cells: also referred to as marrow stromal cells; these cells differentiate in vitro along multiple pathways that include cardiac myogenesis. • Hemangioblasts: primitive embryonic cells that give rise to both hemopoeitic stem cells and endothelial progenitor cells; they may also exist in adult bone marrow.

12. Mammalian development Zygote Developmental potential Oocyte Sperm Totipotent Trophoblast (extraembryonic) Inner cell mass Embryonic Stem cells (in vitro) Blastocyst Pluripotent Epiblast Primitive Multipotent Primitive streak Ectoderm (central and peripheral nervous system, epidermis, etc.) Endoderm (lung, liver, pancreas, etc.) Mesoderm (blood, heart, bone, skeletal muscle, etc.) 12 12 12 Emerging Technol Platform for SCs, 2010.-

13. Stem Cells • Embryonic Stem Cell • Adult Stem Cells

14. Human development : Stem cell perspective totipotent Loose definition Strict definition pluripotent

15. Generates every cell in the body including the placenta and extra-embryonic tissues Can form the entirehuman being Cannot form the entire human being Can generate every cell in the body except placenta and extra-embryonic tissues Become specific cell types; may or may not have plasticity

16. Characteristic of Stem Cells • Undifferentiated cells with the capacity for unlimited or prolonged self renewal that can give rise to differentiated cells • Metaplasia- the formation of one differentiated cell type from another • Slow cycling in cell division • Contact-insensitive; deficient in gap junction intercell. communication • Specific gene expression

17. Embryonic Stem Cells (ESC) Researchers extract stem cells from a 5-7 days old blastocyst. Stem cells can divide in culture to form more of their own kind, thereby creating a stem cell line. The research aims to induce these cells to generate healthy tissue needed by patients. SCAN – Stem Cell Action Network

18. Properties of Human ESC in Culture • Pluripotent- able-to form any of 200 different types of cells of the body • Self renewing in vitro- can propagate or proliferate indefinitely in the undifferentiated state • Express the enzyme telomerase (required to maintain the end of chromosomes) and Oct4 ( a master regulator of ESC pluripotency) • Maintain normal chromosome structure and complement even after long periods in culture ( unlike many other tissue cell lines)

19. Characteristics of Human ESC • Normal Karyotypes • Express high telomerase activity • Express cell surface markers of primate ESC • Maintained undifferentiated proliferation for 4-5 months and development potential to form thropoblast and, endoderm, mesoderm, ectoderm • Developed teratomas in immune deficient mice

20. ESC: What they can do differentiation embryonic stem cells PLURIPOTENT all possible types of specialized cells

21. ESC: Challenges skin grow under conditions A grow under conditions B neurons grow under conditions C embryonic stem cells blood grow under conditions D ? liver

22. Two Sources of Embryonic Stem Cells 1. Excess fertilized eggs from IVF (in-vitro fertilization) clinics 2. Therapeutic cloning (somatic cell nuclear transfer) SCAN – Stem Cell Action Network

23. Tens of thousands of frozen embryos are routinely destroyed when couples finish their treatment. • These surplus embryos can be used to produce stem cells. • Regenerative medical research aims to develop these cells into new, healthy tissue to heal severe illnesses. SCAN – Stem Cell Action Network

24. Somatic Cell Nuclear Transfer • The nucleus of a donated egg is removed and replaced with the nucleus of a mature, "somatic cell" (a skin cell, for example). • No sperm is involved in this process, and no embryo is created to be implanted in a woman’s womb. • The resulting stem cells can potentially develop into specialized cells that are useful for treating severe illnesses. SCAN – Stem Cell Action Network

26. Pluripotency signature • Pluripotent – distinct cellular marker and functions • Factors that are expressed in somatic cells or tissue specific genes must be shut down • Expression of genes for pluripotency associated factors : octamer binding transcription (Oct4) and Nanog must be initiated

28. Review : Epigenetic ?

29. Human ESC remain embryo because of epigenetic factors Molecule central that balancing act – H3K4me3 & H3K27me3 Genes that modified only by H3K4me3  contain DNA recipe to proliferate Genes that do not carry both  completely silenced in ES Science daily, Oct,8,2007

30. Embryonic stem cells regulations Specific mechanism required to disrupt  differentitation ( GCNF – Germ Cell Nuclear factor) Yamanaka et al, Jaenish et al; Thomson et al, 1998 • Maintained by 3 TF genes : • Oct4 • Sox2 • Nanog Feed-forward & feedback maintain pluripotent gene expression Oct4 – important TF - regulate a pluripotent gene expression in early embryonic development During differentiation – the expression is down regulated

32. Oct-4 (octamer-binding transcription factor 4) • Oct-4 (octamer-binding transcription factor 4) also known as POU5F1 (POU domain, class 5, transcription factor, is a protein that in humans is encoded by the POU5F1 gene • This protein is critically involved in the self-renewal of undifferentiated embryonic stem cells. As such, it is frequently used as a marker for undifferentiated cells. • Oct-4 expression must be closely regulated; too much or too little will actually cause differentiation of the cells.

33. Oct4 Upon differentiation  Oct4 is non expressed GCNF is high expressed Methylation of Oct4 gene and histone modifications  silencing Oct4 gene during differentiation Loss of DNA methylation & chromatin remodelling  no effect of repression

34. Oct4 • DNA methylation machinary : DNA Methyltranferase & Methyl DNA binding domain protein (MBDs- MBD1 & MBD2)  MBD2 binds to CpG dinucleotide (CpG" is shorthand for "—C—phosphate—G—", that is, cytosine and guanine separated by a phosphate) MBD3 bind to unmethylated CpG dinucleotide

35. Oct4 and Nanog Cells

36. ESC manage their pluripotent status by PcG mediated repressive histone lock Lysine methylation  recrut spec.Binding prot HP1 to H3 lysine 9 Histone code PRC1 (Protein regulator of cytokinesis 1) t o methylated Histon H3 lysine 27 Acetylation  nucleosome looser, > accesible to transcription factors Polycomb-group proteins Spivakov & Fischer, Nature, April 2007

37. Embryoid body ) ball like embryo)- embryoid bodies Chimeric mouse Promoter demethylationof oct3/4, Nanog and Rex1, Histon demethylation Pluripotency properties

38. Neural differentiation : AADC, DAT, ChAT, LMX1b, MAP; b-tubulin, tyrosine hydrolase Cardiac differentiation : TnTc, MEF2C, MYL2a, MyhcB , NKX2.5 Teratoma formation – teratoma is landmark for pluripotent From pluripotentto differentiated cells

39. ESC in the lab, an example : mPer2 conditional knock out generation SalI SacI-bl Intron2 DT-A Exon2 Pgk-neo SalI KpnI-bl lox lox FLP FLP Saifur Rohman, et. Upublished data,

40. Stem cell selection and implantation

41. Potential embryonic stem cell problems: • Difficult to establish and maintain • Difficult in obtaining pure cultures in the dish • Potential for tumor formation and tissue destruction • Questions regarding functional differentiation • *Hansson M et al., Diabetes 53, 2603-2609, 2004 • *Sipione S et al., Diabetologia 47, 499-508, 2004 • *Rajagopal J et al.; Science 299, 363; 2003 • *Zhang YM et al.; Circulation 106, 1294-1299; 2002

42. The Ethical Debate In favor of ESCR: • Embryonic stem cell research (ESCR) fulfills the ethical obligation to alleviate human suffering. • Since excess IVF embryos will be discarded anyway, isn’t it better that they be used in valuable research? • SCNT (Therapeutic Cloning) produces cells in a petri dish, not a pregnancy. Against ESCR: • In ESCR, stem cells are taken from a human blastocyst, which is then destroyed. This amounts to “murder.” • There is a risk of commercial exploitation of the human participants in ESCR. • Slippery slope argument: ESCR will lead to reproductive cloning. SCAN – Stem Cell Action Network

44. Cellular Plasticity • The discovery of mammalian cellular plasticity raises the possibility of reprogramming restricted cell fate, & may provide an alternative to many of the obstacles associated with using embryonic & adult stem cells in clinical applications.- • With a safe & efficient dedifferentiation process, healthy, abundant & easily accessible adult cells from a given individual could be used to generate different functional cell types to repair damaged tissues & organ.- Lyssiatis et al, Emmerging Techno Platform for SCs, 2009.- 44

45. Induced pluripotent stem cells (iPS) Pluripotent stem cell artificially derived from a non pluripotent cell by inducing a “forced “ expression of certain genes Yamanaka et al, 2006, 2007, Thomson et al, 2007, 2008

46. Induced Pluripotent Stem (iPS) Cells • Yamanaka : cell lines with some of the properties of ES cells by introducing just four transcription factors associated with pluripotency – Oct3/4, Sox2, c-Myc & Klf4 – into mouse skin fibroblast then selecting cells that expressed a marker of pluripotency, Fbx15, in response to these factors, these cells were called iPS cells.- Rossant, Nature 2007.-

47. ‘genetic reprogramming’ = add certain genes to the cell adult cell induced pluripotent stem (iPS) cell behaves like an embryonic stem cell Induced pluripotent stem cells (iPS cells) differentiation culture iPS cells in the lab all possible types of specialized cells Advantage: no need for embryos!

48. Induced pluripotent stem cells (iPS cells) genetic reprogramming pluripotent stem cell (iPS) adult cell (skin) differentiation

49. Properties of iPS identic to natural pluripotent stem cells • Expression of certain stem cell genes and protein • Chromatin methylation patterns • Doubling time • Embryoid body formation • Teratoma formation • Viable chimera formation • Potency differentiability

50. Morphology : similar to ESC, sharp edged, flat, tightly pcked Doubling time – same Stem cell markers: SSEA-3, SSEA-4, TRA-1-60, TRA – 1 -81, TREA -2-49/6E, Nanog Identity of iPS • Telomerase activity: • Stem cell genes: oct3/4, Nanog, GDF4, REXi, FGF4, ESG1, DPPA2, DPPA4 and hTERT