1. Induced Pluripotent Stem Cells (iPS)
The story of four little genes and a HUGE cellular change

2. 1: Stem Cells Before iPS
Embryonic Stem Cells-good source of pluripotent cells, but unethical
Somatic cell nuclear transfer-still requires oocytes

4. Stem cells

6. 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
Not fully reprogramed

7. What is a stem cell?
“A cell from which other types of cells can develop.”
“An undifferentiated cell that divides to give rise to specialized cells.”
“An undifferentiated active somatic cell that undergoes division and gives rise to other stem cells or to cells that differentiate to form specialized cells.”
A cell that undergoes asymmetric cell division, giving rise to one stem cell and one partially differentiated cell

8. Types of stem 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

9. In case you missed it… Fertilized egg up to 8-cell stage
Embryonic stem cells
Adult stem cells
(MULTIPOTENT)‏

12. 2: Mouse iPS
Techniques and theory
Optimization

13. Cell 126, 663–676, August 25, 2006.
Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University.

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

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

16. 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

17. Drug Selection Only turn on a drug resistance gene at 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

18. Drug Selection

19. So They Picked 24 Genes

20. Four Magic Genes Sox2- Self Renewal Oct4- Differentiation switch
Klf4- p53 pathway, Oncogene
c-Myc- Global Histone Acetylation, Oncogene 24

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

22. No Sox2

23. Are they really stem cells?

24. Somewhere stuck in between

25. Teratoma formation

26. Pluripotent/Multipotent?

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

28. The Next Step: 11 months later
Whitehead Institute for Biomedical Research and Department of Biology,Massachusetts Institute of Technology,

29. Better iPS cells Still working with mouse model
Used different drug selection marker
Same 4 genes
Much more closely resemble ES cells
Oct4

30. Genes expressed in iPS group with ES cells, not MEFs

31. Bisulfite Pyrosequencing
Sequencing-based analysis of DNA methylation that quantitatively measures multiple, consecutive CpG sites individually with high accuracy and reproducibility.
use of bisulfite treatment of DNA to determine its pattern of methylation.
Yields high-resolution information about the methylation status of a segment of DNA
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. 4. 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 Lin28 encodes a cytoplasmic mRNA-binding protein.
Used Oct3/4, Sox2, Nanog and Lin28
Lin28 encodes a cytoplasmic mRNA-binding protein.
Science 21 December 2007: Vol no. 5858, pp

43. Around the same time (Dec 2007)

45. 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!

46. A Cure!

47. 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

48. 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