1. Stem Cells from Skin Cells?!? The story of four little genes and a HUGE cellular change

2. Talk Outline Fibroblasts and Stem Cells Fibroblasts and Stem Cells Before iPS Mouse iPS – Techniques and theory – Optimization Human iPS iPS used in treatment

3. Fibroblasts

4. Fibroblasts Are fully differentiated cells Can not become any other cell type Can only divide to make more fibroblasts Contact inhibition

5. Stem cells

6. Stem Cells Can both make more of itself and create other, differentiated cells Totipotent Stem Cells can create everything needed to make a baby Pluripotent Stem cells can make only the cells of the baby Only Adult Stem Cells (multipotent) in your body Unipotent Cells can only make more of itself

7. Stem Cells

8. Talk Outline Fibroblasts and Stem Cells Before iPS Before iPS Mouse iPS – Techniques and theory – Optimization Human iPS iPS used in treatment

9. Before iPS Embryonic Stem Cells-good source of pluripotent cells, but unethical Somatic cell nuclear transfer-still requires oocytes

10. SCNT The basic concept is that the oocyte reprograms the DNA to be “embryonic stem cell-like” Very low efficiency No human stem cell lines have been made from SCNT Hwan Woo-Suk’s fake data Not fully reprogramed

11. Talk Outline Fibroblasts and Stem Cells Before iPS Mouse iPS Mouse iPS – Techniques and theory – Optimization Human iPS iPS used in treatment

13. If the goal is to get stem cells from normal cells, what would you need to add?

14. Retroviruses Randomly inserts DNA into genome of cells Can make special retroviruses with whatever gene you want Can’t really control how many copies of genes

15. Different lines expressed different amounts of Klf4

16. Drug Selection Only turn on a drug resistance gene when stem cell state Do this by using a gene that is only expressed in stem cells Add drug resistance to promoter region of that gene Takes around 16 days for resistance gene to be expressed- some secondary change

17. Drug Selection

18. So They Picked 24 Genes

19. Four Magic Genes Sox2- Self Renewal Oct4- Differentiation switch Klf4- p53 pathway, Oncogene c-Myc- Global Histone Acetylation, Oncogene

20. Do you really need all 4? Without Oct 3/4 or Klf: no colonies Without Sox2: rough morphology Without c-Myc: flatter cells, now know actually can do without c-myc-just very low efficiency

21. No Sox2

22. Are they really stem cells?

23. Somewhere stuck in between

24. Teratoma formation

25. Pluripotent/Multipotent?

26. No baby mice! Tried to inject into blastocyst to make baby mice but failed Final and best test of pluripotency

27. The Next Step: 11 months later

28. Better iPS cells Still working with mouse model Used different drug selection marker Same 4 genes Much more closely resemble ES cells

29. Genes expressed in iPS group with ES cells not MEFs

30. Still Integration differences

31. Bisulfite Pyrosequencing Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected Introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues

32. ES cell-like Methylation

33. Gold Standard!

34. Talk Outline Fibroblasts and Stem Cells Before iPS Mouse iPS – Techniques and theory – Optimization Human iPS Human iPS iPS used in treatment

35. 4 months later

36. Technique Basically same technique as mouse Added the mouse retrovirus receptor to the human cells to increase transfection efficiency Used facial skin cells from a 36 year old female Takes 25 days for colonies to form

37. Gene expression profiles look like ES cells

38. And protein expression

39. DNA Methylation Profiles

40. Differentiates into all types of cells in culture

41. And in teratomas (injected into mice)

42. One month later Used Oct3/4, Sox2, Nanog and Lin28

43. Talk Outline Fibroblasts and Stem Cells Before iPS Mouse iPS – Techniques and theory – Optimization Human iPS iPS used in treatment iPS used in treatment

44. Around the same time (Dec 2007)

46. Wow! Used the animal’s own cells- no immune rejection! Transfected with all four genes, but c-myc taken out after time- prevent tumors! Sickle Cell Anemia has known genetic basis-so target that gene and change it back to normal! Inject it back into the animal after radiation to reconstitute the whole blood system!

47. A Cure!

48. The Possibilities are Endless Any disease with a single genetic mutation could be easily cured! Tissue regeneration after accidents or diseases “Nanobots” Companies have already started testing iPS for therapy

49. But there are still obstacles No way FDA will approve a therapy with an oncogene Use of retroviruses can lead to mutations and cancers So many changes in the DNA can be harmful Probably hard to target to some areas