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

Eirini P. Papapetrou, Michel Sadelain

Research output: Contribution to journalArticlepeer-review

61 Scopus citations

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.

Original languageEnglish
Pages (from-to)1251-1273
Number of pages23
JournalNature Protocols
Volume6
Issue number9
DOIs
StatePublished - Sep 2011
Externally publishedYes

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