The document discusses induced pluripotent stem cells (iPSCs), which are derived from adult somatic cells that are reprogrammed by introducing genes associated with pluripotency. iPSCs were first generated in 2006 and resemble embryonic stem cells. They can be produced from a person's own cells and have potential applications in disease modeling, drug development, and regenerative medicine without ethical issues associated with embryonic stem cells.

1. INDUCED PLURIPOTENT STEM CELLS Presented by: VIDUR BHATIA M. Sc. (F), KUK.

2. Contents Introduction History Reprogramming of somatic cells What are iPSCs Genes responsible Production first generation Second generation Human iPSCs Complications Identity to natural pluripotent stem cells Applications Conclusions References

3. Introduction Most of the cells of a multicellular organism become more and more restricted to specific cell lineages. For the treatment of many genetic diseases Human embryonic stem cells can be used , But due to some ethics we can’t use embryo for this purpose. To avoid this problem artificially induced pluripotent stem cells came in picture, which can be created from normal somatic cells by the ectopic expression of some genes which are responsible for the pluripotency.

4. History First generated by Shinya Yamanaka et al. At Kyoto in Japan in 2006.; Second generated in mice in 2006 by same group. Alexender Meissner showed that induction of pluripotency is a slow and gradual process, 2008. Yang Chao showed that p53 siRNA and UTF1 enhances the efficiency of pluripotency, in Nov.2008.

5. Conti….. Cesor A. Sommer used a single lentivirus for all the genes required pluripotency in 2008 Yomiuri Shimbun has created the mouse kidney by the use of iPS cells 10 march,2009. James A Thomson made the use of plasmid for pluripotency induction 26 march 2009.

6. Embryonic Stem cells Totipotent Pluripotent Multipotent Unipotent

7. Reprogramming of somatic cells to ES cells Somatic cell nuclear transfer Cell fusion Treatment with the extract of the pluripotent stem cells Stable expression of defined factors ( Cowan et al.,2005) ( Wilmut et al.,1997) (Takahashi and Yamanaka, 2006.)

9. Cell Fusion Technology

10. Treatment with the extract of the pluripotent cells Permeabilised cells are exposed to cell-free extract of pluripotent cells. Limitations :- a. Limited experience with primary cells. b. Reprogrammed cells regain only some of the properties of pluripotent cell

11. Stable expression of defined factors Twenty four candidate genes play pivotal roles in the maintenance of ES cell identity base on their hypothesis. (Takahashi and Yamanaka,2006.) Myb, Kit, Gdf3, Esrrb 21 22 23 24 Ecat1 Dppa5(Esg1) Fbox15 Nanog Eras Dnmt31 Ecat8 Gdf3 Sox15 Dppa4 Dppa2 Fthl17 Sall4 Oct3/4 Sox2 Rex1 Utf1 Tcl1 Dppa3 Klf4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Symbol No.

12. Exogenous expression of Oct3/4, Sox2, Klf4 and c-Myc and factors are essentially required. But large number of factors are also required for pluripotency ….. (Takahashi and Yamanaka, August 25, 2006.)

13. iPSCs , are a type of pluripotent stem cell artificially derived from a non- pluripotent cell, typically an adult somatic cell , by inducing a "forced" expression of certain genes . first produced in 2006 from mouse cells and in 2007 from human cells WHAT ARE iPSCs ?

14. Genes responsible for pluripotency Group 1 ES cell-Specific transcription factors Essential for pluripotency in ES cell & early embryos Oct¾, Sox2, Nanog… Group 2 (Proto-oncogene's) Important for proliferation of ES cells, but not in early embryos TCL1, Stat3, c-Myc, ERas, Klf4… Group 3 Less famous Specifically expressed in ES cell But less defined function ECAT1, Esg1,Fbx15, … (Takahashi and Yamanaka, 2006.)

15. Oct3/4: Involve in the maintenance of self renewal of pluripotent cells. Repression in ES cells leads to the formation of trophoectoderm. Overexpression leads to the formation of various lineages including primitive endoderm . Sox2: Essential for embryonic development Downregulation by siRNA silencing leads to the differentiation of cell in murine ES cells .

16. Klf4 Klf4 repress p53 directly p53 protein suppress Nanog during ES cell differentiation Klf4 contributes to activation of Nanog and other ES cell-specific genes (Rowland et al ., 2005; Lin et al ., 2004) Klf4 acts as an inhibitor of c-Myc-induced apoptosis through the repression of p53 (Zindy et al ., 1998) Klf4 activates p21CIP1, thereby suppressing cell proliferation .This antiproliferation function of Klf4 inhibited by c-Myc, which suppresses the expression of p21CIP1 ( Zhang et al ., 2000; Seoane et al ., 2002 )

17. Nanog : In embryonic stem cells, Nanog, along with Oct-3/4 and Sox2, is necessary in promoting pluripotency. LIN28 : LIN28 is an mRNA binding protein expressed in embryonic stem cells and embryonic carcinoma cells associated with differentiation and proliferation. (Thomson et al.)

18. Production of iPSCs Typically derived by transfection of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts . Transfection is typically achieved through viral vectors, such as retroviruses . After 3–4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection.

19. (1)Isolate and culture donor cells. (2)Transfect stem cell-associated genes into the cells by viral vectors. (3)Harvest and culture the cells according to ES cell culture, (4)A small subset of the transfected cells become iPS cells and generate ES-like colonies.

20. First generation First generated by Shinya Yamanaka 's team at Kyoto University , Japan in 2006. four key pluripotency genes essential for the production of pluripotent stem cells were used; Oct-3/4, Sox2, c- Myc , and Klf4 . Retroviruses was used to transfect mouse fibroblasts. Cells were isolated by antibiotic selection of Fbx15 + cells.

21. Limitations:- This iPS line showed DNA methylation errors compared to original patterns in ESC lines and failed to produce viable chimeras if injected into developing embryos

22. Second generation in mice In June 2007, by the same group. These cell lines were also derived from mouse fibroblast by retroviral mediated reactivation of the same four endogenous pluripotent factors, but Instead of Fbx15, they used Nanog which is an important gene in ESCs. DNA methylation patterns and producing viable chimeras (and thereby contributing to subsequent germ-line production) indicated that Nanog is a major determinant of cellular pluripotency.

23. Limitations:- One of the four genes used (namely, c-Myc) is oncogenic , and 20% of the chimeric mice developed cancer. (In a later study, Yamanaka reported that one can create iPSCs even without c-Myc, although process takes longer and is not as efficient, but the resulting chimeras didn't develop cancer).

24. Human induced pluripotent stem cells Produced in November 2007. With the same principle used earlier in mouse models, Yamanaka had successfully transformed human fibroblasts into pluripotent stem cells using the same four pivotal genes: Oct3/4, Sox2, Klf4, and c-Myc with a retroviral system.

25. Pluripotency induction is a slow and gradual process After transfecting the cells with all of the four factors (Sox2,OCct3/4,Klf4 and c-Myc) initially only eight colonies were picked at 11th day and ten colonies on 16th day. Only one out of eight colonies from 11 day and four colonies from ten colonies from 16 day old colonies gave rise the ES like cells. (Alexender Meissner et al.;2007)

26. Complications Because of viral transfection systems, the created cells might be prone to form tumors. However Konrad Hochedlinger and his Harvard University research team successfully used an adenovirus to transport the requisite four genes into the DNA of skin and liver cells of mice. Since the adenovirus does not combine any of its own genes with the targeted host, the danger of creating tumors is eliminated.

27. Identity The generated iPSCs were remarkably similar to naturally-isolated pluripotent stem cells Cellular biological properties: Morphology , Growth properties , Stem Cell Markers , Stem Cell Genes , Telomerase Activity: Pluripotency of iPSCs : Neural Differentiation, Cardiac Differentiation , Teratoma Formation , Embryoid Body,

28. Conti….. Epigenetic reprogramming : Promoter Demethylation , Histone demethylation etc.

29. APPLICATIONS

30. iPS Cells - the Wave of Future iPSC regarded as holy grail stem cell research Studying disease models in vitro Drug screening Toxicological testing of new drugs Generating patient specific & disease specific pleuripotent stem cells Allow unprecedented access to all stages of human biology Studying development & function of human tissue Regenerative medicine

31. iPS CELLS TO CURE SC ANEAMIA

32. Cell replacement therapy seems particularly suitable for Parkinson’s disease, A common neurodegenerative disease caused by loss of midbrain dopamine neurons . Transplantation of fetal midbrain cells has been shown to restore dopamine function in animal models and in human patients . ( Parish CL, et al . (2008)) iPS can be used for treatment of Parkinson’s disease

33. Mouse kidneys created using iPS cells A team of scientists has successfully used induced pluripotent stem (iPS) cells to create kidneys inside a mouse whose parents were genetically engineered so their offspring would not be born with the organ. (Hiromitsu Nakuchi; Mar. 10, 2009))

34. Stem cells scientists at UCLA showed for the first time that human induced pluripotent stem (iPS) cells can be differentiated into electrically active motor neurons, a discovery that may aid in studying and treating neurological disorders. The motor neurons derived from the iPS cells appeared to be similar in function and efficiency to those derived from human embryonic stem cells, although further testing needs to be done to confirm that. iPSc can be used to create Electrically Active Neuron (Michael Scott on February - 25 – 2009)

35. Somatic cells can be used to generate the β -cells Pancreatic Exocrine cells can be converted into β -cells closely related to the islet β -cells. Can be used to cure diabetes. (Qiao Zhou et al .,2008)

36. Conclusion SCNT and cell fusion may use to produce the pluripotent cells but can be used only for animals, these processes can’t be shifted to human beings. iPS may be the answer of the all question of ethics and may avoid the problem of transplant rejection. In future these technique may help us in the field genetic study, research and to fight against disease.

37. Takahashi & K. Yamanaka; Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors, Cell 2006;126:663–676. Yamanaka S. & et al. ; Generation of germline- competent induced pluripotent stem cells, Nature 2007;448:313-317. Maherali N & et. al.; Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution, Cell Stem Cell 2007;1:55–70 . References

38. Stadtfeld M., Nagaya M., Utikal J., Weir G., Hochedlinger K.Induced Pluripotent Stem Cells Generated without Viral Integration. Science 2008 Sep. 25. pp.212-220. Okita K., Nakagawa M., Hyenjong H., Ichisaka T,Yamanaka S. Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors, Science, 2008 Oct 9. pp.167-178.

39. Thank you