Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors

Kazim H. Narsinh; Fangjun Jia; Robert C. Robbins; Mark A. Kay; Michael T. Longaker; Joseph C. Wu

doi:10.1038/nprot.2010.173

Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors

Kazim H. Narsinh, Fangjun Jia, Robert C. Robbins, Mark A. Kay, Michael T. Longaker, Joseph C. Wu

Research output: Contribution to journal › Article › peer-review

170 Scopus citations

Abstract

Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ∼1/44 weeks.

Original language	English (US)
Pages (from-to)	78-88
Number of pages	11
Journal	Nature Protocols
Volume	6
Issue number	1
DOIs	https://doi.org/10.1038/nprot.2010.173
State	Published - Jan 2011
Externally published	Yes

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology

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10.1038/nprot.2010.173

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title = "Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors",

abstract = "Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ∼1/44 weeks.",

author = "Narsinh, {Kazim H.} and Fangjun Jia and Robbins, {Robert C.} and Kay, {Mark A.} and Longaker, {Michael T.} and Wu, {Joseph C.}",

note = "Funding Information: acknowleDGMents We are grateful to N. Sun for expert assistance with cell culture techniques. We thank Z.Y. Cheng for help with minicircle production techniques. We acknowledge funding support from Howard Hughes Medical Institute (K.H.N.); Mallinckrodt Foundation, National Institutes of Health (NIH) DP2OD004437, RC1AG036142, Burroughs Wellcome Foundation, and the American Heart Association 0970394N (J.C.W.); NIH R90DK07010301, NIH R21DE018727, NIH R21DE019274, NIH RC2DE020771, the Oak Foundation and the Hagey Laboratory for Pediatric Regenerative Medicine (M.T.L.); NIH RC1HL100490-02 (J.C.W. and M.T.L.); and NIH U01HL099776 (R.C.R.).",

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AU - Longaker, Michael T.

AU - Wu, Joseph C.

N1 - Funding Information: acknowleDGMents We are grateful to N. Sun for expert assistance with cell culture techniques. We thank Z.Y. Cheng for help with minicircle production techniques. We acknowledge funding support from Howard Hughes Medical Institute (K.H.N.); Mallinckrodt Foundation, National Institutes of Health (NIH) DP2OD004437, RC1AG036142, Burroughs Wellcome Foundation, and the American Heart Association 0970394N (J.C.W.); NIH R90DK07010301, NIH R21DE018727, NIH R21DE019274, NIH RC2DE020771, the Oak Foundation and the Hagey Laboratory for Pediatric Regenerative Medicine (M.T.L.); NIH RC1HL100490-02 (J.C.W. and M.T.L.); and NIH U01HL099776 (R.C.R.).

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Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors

Abstract

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