Adapting Induced Pluripotent Stem Cells For Clinical UseDana WymanProfessor BrutlagGenomics and MedicineIntroductionYamanaka Method4 Genes:Oct4 Sox2Klf4cMycHuman Dermal Fibroblasts Induced Pluripotent Stem Cells7 days hES medium +MEFImmunofluorescence TestES Cells (+ control)Untransduced Fibroblasts (- control) Oct 4-transduced FibroblastsBlue (DAPI): nuclear membrane of all cellsRed : Cells containing the Oct 4 protein (expressing the Oct 4 gene)Alkaline Phosphatase (AP) StainThese 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 cellApproaches to iPSC GenerationIntegrating Vectors•Retro/LentivirusesExcisable Vectors•Lox-CRE mediated excisions•TransposonsNon-Integrating Vectors•Direct Delivery of Proteins•MicroRNASafer Viral Integration•Using fewer reprogramming factors•Specifically avoiding Klf4 and c-Myc•Using a single expression “cassette” rather than multiple vectorsExcisable Vectors•piggyBac transposons•Capable of carrying 10 kilobases of DNA•Can be excised seamlessly using piggyBac transposasehttp://www.transposagenbio.com/page.php?page=our-technologyProteinhttp://www.3dchem.com/molecules.asp?ID=53Protein 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 occurMicroRNA•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•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:
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