Abstract
This chapter describes a protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts. Human fibroblasts, cultured in fibroblast medium, are infected with a cocktail of retroviral vectors expressing the transcription factors OCT4, SOX2, KLF4, and MYC. The culture conditions are then switched to conditions that support human embryonic stem cell growth and emerging iPSC colonies that morphologically resemble human embryonic stem cell (hESC) colonies and have silenced the retroviral vectors (as evidenced by downregulation of retroviral GFP expression) that are mechanically isolated and subsequently cultured in identical fashion to hESCs. Putative iPSC lines are validated to be bona fide human iPSC lines by analyzing them for the expression of pluripotency markers and by differentiation in vitro and in vivo.
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References
Park, I.H., Zhao, R., West, J.A., Yabuuchi, A., Huo, H., Ince, T.A., Lerou, P.H., Lensch, M.W., and Daley, G.Q. (2008) Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–6.
Lowry, W.E., Richter, L., Yachechko, R., Pyle, A.D., Tchieu, J., Sridharan, R., Clark, A.T., and Plath, K. (2008) Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci USA 105, 2883–8.
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 131, 861–72.
Park, I.H., Arora, N., Huo, H., Maherali, N., Ahfeldt, T., Shimamura, A., Lensch, M.W., Cowan, C., Hochedlinger, K., and Daley, G.Q. (2008) Disease-specific induced pluripotent stem cells. Cell 134, 877–86.
Acknowledgments
The authors would like to thank In-Hyun Park, Elayne Chan, Sutheera Ratanasirintrawoot, and Iris Huo for their advice and contributions.
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© 2011 Springer Science+Business Media, LLC
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Miller, J.D., Schlaeger, T.M. (2011). Generation of Induced Pluripotent Stem Cell Lines from Human Fibroblasts via Retroviral Gene Transfer. In: Schwartz, P., Wesselschmidt, R. (eds) Human Pluripotent Stem Cells. Methods in Molecular Biology, vol 767. Humana Press. https://doi.org/10.1007/978-1-61779-201-4_5
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DOI: https://doi.org/10.1007/978-1-61779-201-4_5
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Print ISBN: 978-1-61779-200-7
Online ISBN: 978-1-61779-201-4
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