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Generation of transgene-free human induced pluripotent stem cells with an excisable single polycistronic vector

Nature Protocols volume 6, pages 12511273 (2011) | Download Citation

Abstract

The generation of induced pluripotent stem cells (iPSCs) devoid of permanently integrated reprogramming factor genes is essential to reduce differentiation biases and artifactual phenotypes. We describe a protocol for the generation of human iPSCs using a single polycistronic lentiviral vector (pLM-fSV2A) coexpressing OCT4, SOX2, KLF4 and c-MYC; this is flanked by two loxP sites in its long terminal repeats (LTRs). Human iPSC lines are established with an efficiency of up to 1% and screened to select single or low vector copy lines. To deal with potential insertional mutagenesis, the vector integrations are then mapped to the human genome. Finally, the vector is excised by transient expression of Cre recombinase (coexpressed with mCherry) through an integrase-deficient lentiviral vector. Vector-excised iPSC lines maintain all characteristics of pluripotency. This protocol can be used to efficiently derive transgene-free iPSCs from many different starting cell types in approximately 12–14 weeks.

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Acknowledgements

This work was supported by the Starr Foundation (Tri-Institutional Stem Cell Initiative, Tri-SCI-018), the New York State Stem Cell Science, NYSTEM (N08T-060) and National Heart, Blood and Lung Institute, NHLBI grant HL053750. We thank present and past members of the Sadelain, Riviere, Studer and Tomishima labs (Memorial Sloan-Kettering Cancer Center, New York) for helpful discussions and technical assistance and E. Poeschla (Mayo Clinic, Rochester, Minnesota) for providing the pCMVΔR8.91N/N plasmid.

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Affiliations

  1. Center for Cell Engineering, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Eirini P Papapetrou
    •  & Michel Sadelain

Authors

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Contributions

E.P.P. developed the protocol and wrote the paper. M.S. supervised the study and edited the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Eirini P Papapetrou.

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DOI

https://doi.org/10.1038/nprot.2011.374

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