Abstract
This chapter describes the methods we use to derive and characterize human induced pluripotent stem (iPS) cells. We describe in order, first our culture techniques for the starting fibroblast populations and methods for retrovirus preparation and concentration. Subsequently, a detailed iPS derivation protocol suitable for human fibroblast populations is discussed using standard retroviral vectors expressing the classic four or three reprogramming genes. Finally, we elaborate a robust technique for monitoring and identification of potential iPS cells through live staining of reprogrammed cells. We also outline steps for characterization of the resulting iPS cell lines.
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References
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282(5391):1145-1147.
Reubinoff BE, Pera MF, Fong C, Trounson A, Bongso A. (2000) Embryonic stem lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399-404.
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861-872.
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917-1920.
Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, Lerou PH, Lensch MW, Daley GQ. (2008) Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451:141-146.
Mali P, Ye Z, Hommond HH, Yu X, Lin J, Chen G, Zou J, Cheng L. (2008) Improved efficiency and pace of generating induced pluripotent stem cells from human adult and fetal fibroblasts. Stem Cells 26:1998-2005.
Lowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, Sridharan R, Clark AT, Plath K. (2008) Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci U S A 105:2883-2888.
Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ. (2008) Disease-specific induced pluripotent stem cells. Cell 134:877-886.
Dravid G, Hammond H, Cheng L. (2006) Culture of human embryonic stem cells on human and mouse feeder cells. Methods Mol Biol (Clifton, N.J.) 331:91-104.
Cheng L, Hammond H, Ye Z, Zhan X, Dravid G. (2003). Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells 21:131-142.
Ye Z, Yu X, Cheng L. (2008) Lentiviral gene transduction of mouse and human stem cells. Methods Mol Biol (Clifton, N.J.) 430:243-53.
Acknowledgments
The authors would like to thank members of the Cheng lab for helpful discussions and assistance during various aspects of this study. This work was funded in part by a grant (R01 HL073781) and a supplement from NIH.
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Mali, P., Ye, Z., Chou, BK., Yen, J., Cheng, L. (2010). An Improved Method for Generating and Identifying Human Induced Pluripotent Stem Cells. In: Ding, S. (eds) Cellular Programming and Reprogramming. Methods in Molecular Biology, vol 636. Humana Press. https://doi.org/10.1007/978-1-60761-691-7_12
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DOI: https://doi.org/10.1007/978-1-60761-691-7_12
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