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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
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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 • Are fully differentiated cells • Can not become any other cell type • Can only divide to make more fibroblasts • Contact inhibition
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
Talk Outline • Fibroblasts and Stem Cells • Before iPS • Mouse iPS • Techniques and theory • Optimization • Human iPS • iPS used in treatment
Before iPS • Embryonic Stem Cells-good source of pluripotent cells, but unethical • Somatic cell nuclear transfer-still requires oocytes
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
Talk Outline • Fibroblasts and Stem Cells • Before iPS • Mouse iPS • Techniques and theory • Optimization • Human iPS • iPS used in treatment
If the goal is to get stem cells from normal cells, what would you need to add?
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
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
Four Magic Genes • Sox2- Self Renewal • Oct4- Differentiation switch • Klf4- p53 pathway, Oncogene • c-Myc- Global Histone Acetylation, Oncogene
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
No baby mice! • Tried to inject into blastocyst to make baby mice but failed • Final and best test of pluripotency
Better iPS cells • Still working with mouse model • Used different drug selection marker • Same 4 genes • Much more closely resemble ES cells
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
Talk Outline • Fibroblasts and Stem Cells • Before iPS • Mouse iPS • Techniques and theory • Optimization • Human iPS • iPS used in treatment
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
One month later Used Oct3/4, Sox2, Nanog and Lin28
Talk Outline • Fibroblasts and Stem Cells • Before iPS • Mouse iPS • Techniques and theory • Optimization • Human iPS • iPS used in treatment
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!
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
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