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Induced Pluripotent Stem Cells (iPS) The story of four little genes and a HUGE cellular change 1: Stem Cells Before iPS • Embryonic Stem Cells-good source of pluripotent cells, but unethical • Somatic cell nuclear transfer-still requires oocytes Stem cells 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 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 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 In case you missed it… Fertilized egg up to 8-cell stage Embryonic stem cells Adult stem cells (MULTIPOTENT) 2: Mouse iPS – Techniques and theory – Optimization Cell 126, 663–676, August 25, 2006. Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University. Fibroblasts • • • • Are fully differentiated cells Can not become any other cell type Can only divide to make more fibroblasts Contact inhibition 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 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 Drug Selection So They Picked 24 Genes 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: a few colonies, rough surfaces • Without c-Myc: flatter cells, now know actually can do without c-myc-just very low efficiency No Sox2 Are they really stem cells? Somewhere stuck in between Teratoma formation Pluripotent/Multipotent? No baby mice! • Tried to inject into blastocyst to make baby mice but failed • Final and best test of pluripotency The Next Step: 11 months later Whitehead Institute for Biomedical Research and Department of Biology,Massachusetts Institute of Technology, Better iPS cells • • • • Still working with mouse model Used different drug selection marker Same 4 genes Much more closely resemble ES cells Oct4 Genes expressed in iPS group with ES cells, not MEFs 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 ES cell-like Methylation Gold Standard! 4. Human iPS • Human iPS • iPS used in treatment 4 months later 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 Gene expression profiles look like ES cells And protein expression DNA Methylation Profiles Differentiates into all types of cells in culture And in teratomas (injected into mice) One month later Used Oct3/4, Sox2, Nanog and Lin28 Lin28 encodes a cytoplasmic mRNA-binding protein. Science 21 December 2007: Vol. 318. no. 5858, pp. 1917 - 1920 Around the same time (Dec 2007) • 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! A Cure! 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