Adapting Induced Pluripotent Stem Cells For Clinical Use Dana Wyman Professor Brutlag Genomics and Medicine Introduction Yamanaka Method 7 days hES medium MEF 4 Genes Oct4 Sox2 Klf4 cMyc Human Dermal Fibroblasts Induced Pluripotent Stem Cells Immunofluorescence Test Oct 4 transduced Fibroblasts ES Cells control Blue DAPI nuclear membrane of all cells Red Cells containing the Oct 4 protein expressing the Oct 4 gene Untransduced Fibroblasts control Alkaline Phosphatase AP Stain These AP positive iPSC colonies are expressing surface antigens characteristic of ES cells as indicated by the red stain Problems with the Yamanaka Method Carcinogenic reprogramming factors Viral integration into the genome Interferes with endogenous gene expression Can lead to tumors Large number of viral integrations per cell Approaches to iPSC Generation Integrating Vectors Retro Lentiviruses Excisable Vectors Lox CRE mediated excisions Transposons Non Integrating Vectors Direct Delivery of Proteins MicroRNA Safer Viral Integration Using fewer reprogramming factors Specifically avoiding Klf4 and c Myc Using a single expression cassette rather than multiple vectors Excisable Vectors piggyBac transposons Capable of carrying 10 kilobases of DNA Can be excised seamlessly using piggyBac transposase http www transposagenbio com page php page our technology Protein Protein Delivery Purified reprogramming proteins are attached to a cell penetrating peptide No integration into the genome No nucleic acids involved More time consuming and less efficient than viral integration Multiple rounds of protein treatment may be required for full reprogramming to occur http www 3dchem com molecules asp ID 53 MicroRNA A subset of microRNA exclusively found in ESCs is thought to regulate expression of developmentally important genes Used with other methods to increase their efficiency Can replace the reprogramming factor c Myc Sources Gangaraju V K Lin H MicroRNAs Key regulators of stem cells 2009 Nature Reviews Molecular Cell Biology 10 2 pp 116 125 doi 10 1038 nrm2621 http www nature com nrm journal v10 n2 full nrm2621 html Judson R L Babiarz J E Venere M Blelloch R 2009 Embryonic stem cell specific microRNAs promote induced pluripotency Nat Biotechnol http www nature com nbt journal v27 n5 full nbt 1535 html Kim D et al Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins Cell Stem Cell 4 472 476 2009 http www genetics uab edu Document Travis 202 20031510 pdf Seifinejad A Tabebordbar M Baharvand H Boyer LA et al 2010 Progress and promise towards safe induced pluripotent stem cells for therapy Stem Cell Rev Rep http www springerlink com content 4qr235667l331p66 fulltext html CR50 Takahashi K Tanabe K Ohnuki M Narita M Ichisaka T Tomoda K and Yamanaka S 2007 Induction of pluripotent stem cells from adult human fibroblasts by defined factors Cell 1 12 doi 10 1016 j cell 2007 11 019 Winokur S 2009 iPS Project Unpublished protocol and raw data Yusa K Rad R Takeda J Bradley A Generation of transgene free induced pluripotent mouse stem cells by the piggyBac transposon 2009 Nature Methods 6 5 pp 363 369 doi 10 1038 nmeth 1323 Zhou H Wu S Joo JY Zhu S Han DW Lin T Trauger S Bien G Yao S Zhu Y et al 2009 Generation of induced pluripotent stem cells using recombinant proteins Cell Stem Cell 4 381 384 http www ncbi nlm nih gov pubmed 19398399 dopt Abstract Stadtfield M Hochedlinger K 2010 Induced Pluripotency History Mechanisms and Applications Genes Dev 2010 24 2239 2263 http genesdev cshlp org content 24 20 2239 long ref 216 picture on first slide http btech info blogspot com
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