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Derivation of Induced Pluripotent Stem Cells by Lentiviral Transduction

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Human Pluripotent Stem Cells

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

Abstract

This chapter provides a method for reprogramming human dermal fibroblasts into induced pluripotent stem cells (iPSCs) using three lentiviruses containing cDNAs for OCT4 and SOX2, KLF4 and C-MYC, and NANOG and LIN28, respectively. Lentiviral vectors are based on the human immunodeficiency virus (HIV) and provide an effective means for the delivery, integration, and expression of exogenous genes in mammalian cells. Lentiviruses are attractive gene delivery vehicles as they are able to infect both proliferating and nonproliferating cells. Lentiviruses stably integrate into the genome without incurring cellular toxicity and can maintain sustained transgene expression during prolonged host cell proliferation and differentiation. In this protocol, we describe how to prepare lentiviruses, stably transduce human fibroblasts, and identify bona fide iPSC colonies based on morphological similarity to human embryonic stem cell (ESC) colonies and live-cell immunological staining using cell-surface markers of human PSCs such as Tra-1-60 and Tra-1-81.

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Acknowledgments

This work has been funded by the National Institutes of Health (T15HL074286, R21MH087925, R01HD059967). The NCI Preclinical Repository supplied FGF-2. We also recognize Richard Pepple for proof-reading this chapter.

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Authors and Affiliations

  1. Neuroscience Laboratories, Centers for Neuroscience and Translational Research, CHOC Research Institute, Children’s Hospital of Orange County Research Institute, Orange, CA, USA

    Hubert E. Nethercott

  2. Neuroscience Laboratories, Centers for Neuroscience and Translational Research, Children’s Hospital of Orange County Research Institute, Orange, CA, USA

    David J. Brick & Philip H. Schwartz

Authors
  1. Hubert E. Nethercott
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  2. David J. Brick
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  3. Philip H. Schwartz
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Corresponding author

Correspondence to Philip H. Schwartz .

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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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Nethercott, H.E., Brick, D.J., Schwartz, P.H. (2011). Derivation of Induced Pluripotent Stem Cells by Lentiviral Transduction. 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_6

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  • DOI: https://doi.org/10.1007/978-1-61779-201-4_6

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  • 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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