1 / 49

Stem Cells from Skin Cells?!?

Stem Cells from Skin Cells?!?. The story of four little genes and a HUGE cellular change. Talk Outline. Fibroblasts and Stem Cells Before iPS Mouse iPS Techniques and theory Optimization Human iPS iPS used in treatment. Fibroblasts. Fibroblasts. Are fully differentiated cells

proulx
Télécharger la présentation

Stem Cells from Skin Cells?!?

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

  2. Talk Outline • 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 • 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 • Techniques and theory • Optimization • Human iPS • iPS used in treatment

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

  13. 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

  14. Different lines expressed different amounts of Klf4

  15. 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

  16. Drug Selection

  17. So They Picked 24 Genes

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

  19. 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

  20. No Sox2

  21. Are they really stem cells?

  22. Somewhere stuck in between

  23. Teratoma formation

  24. Pluripotent/Multipotent?

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

  26. The Next Step: 11 months later

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

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

  29. Still Integration differences

  30. 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

  31. ES cell-like Methylation

  32. Gold Standard!

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

  34. 4 months later

  35. 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

  36. Gene expression profiles look like ES cells

  37. And protein expression

  38. DNA Methylation Profiles

  39. Differentiates into all types of cells in culture

  40. And in teratomas (injected into mice)

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

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

  43. Around the same time(Dec 2007)

  44. 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!

  45. A Cure!

  46. 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

  47. 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

More Related