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Generation of Induced Pluripotent Stem Cell Lines from Human Fibroblasts via Retroviral Gene Transfer

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Human Pluripotent Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 767))

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

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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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Authors and Affiliations

  1. Dr. Lorenz Studer Laboratory, Developmental Biology Department, Gerstner Sloan-Kettering Graduate School, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    Justine D. Miller

  2. hESC Core Facility, Hematology/Oncology and Stem Cell Program, Harvard Medical School, Children’s Hospital Boston, Boston, MA, USA

    Thorsten M. Schlaeger

Authors
  1. Justine D. Miller
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  2. Thorsten M. Schlaeger
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Corresponding author

Correspondence to Thorsten M. Schlaeger .

Editor information

Editors and Affiliations

  1. Research Institute, CHOC Human Neural Stem Cell Resource, Children's Hospital of Orange County, S. Main Street 455, Orange, 92868-3874, California, USA

    Philip H. Schwartz

  2. Beckman Research Institute, Center for Applied Technology Developmen, City of Hope, East Duarte Road 1500, Duarte, 91010, California, USA

    Robin L. Wesselschmidt

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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

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-200-7

  • Online ISBN: 978-1-61779-201-4

  • eBook Packages: Springer Protocols

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