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 ∼4 weeks.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).
Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).
Soldner, F. et al. Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964–977 (2009).
Dimos, J.T. et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321, 1218–1221 (2008).
Lee, G. et al. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461, 402–406 (2009).
Hacein-Bey-Abina, S. et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419 (2003).
Stadtfeld, M. et al. Induced pluripotent stem cells generated without viral integration. Science 322, 945–949 (2008).
Okita, K. et al. Generation of mouse induced pluripotent stem cells without viral vectors. Science 322, 949–953 (2008).
Kaji, K. et al. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 458, 771–775 (2009).
Woltjen, K. et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458, 766–770 (2009).
Yu, J. et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797–801 (2009).
Zhou, H. et al. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4, 381–384 (2009).
Jia, F. et al. A nonviral minicircle vector for deriving human iPS cells. Nat. Meth. 7, 197–199 (2010).
Miura, K. et al. Variation in the safety of induced pluripotent stem cell lines. Nat. Biotech. 27, 743–745 (2009).
Huangfu, D. et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat. Biotech. 26, 795–797 (2008).
Marson, A. et al. Wnt signaling promotes reprogramming of somatic cells to pluripotency. Cell Stem Cell 3, 132–135 (2008).
Sun, N. et al. Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells. Proc. Natl. Acad. Sci. USA 106, 15720–15725 (2009).
Aoki, T. et al. Generation of induced pluripotent stem cells from human adipose-derived stem cells without c-MYC. Tissue Eng. Part A 6, 6 (2010).
Guilak, F. et al. Clonal analysis of the differentiation potential of human adipose-derived adult stem cells. J. Cell Physiol. 206, 229–237 (2006).
Bunnell, B.A. et al. Adipose-derived stem cells: isolation, expansion and differentiation. Methods 45, 115–120 (2008).
Chen, Z.-Y. et al. Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol. Ther. 8, 495–500 (2003).
Chen, Z.Y., He, C.Y. & Kay, M.A. Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo. Hum. Gene Ther. 16, 126–131 (2005).
Galbraith, D.W., Anderson, M.T. & Herzenberg, L.A. Flow cytometric analysis and FACS sorting of cells based on GFP accumulation. Methods Cell Biol. 58, 315–341 (1999).
Pruitt, S.C., Mielnicki, L.M. & Stewart, C.C. Analysis of fluorescent protein expressing cells by flow cytometry. Flow Cytometry Protocols 263, 239–258 (2004).
Ohnuki, M., Takahashi, K. & Yamanaka, S. Generation and Characterization of Human Induced Pluripotent Stem Cells. Current Protocols in Stem Cell Biology (John Wiley & Sons, Inc., 2007).
Strutt, B., Khalil, W. & Killinger, D. Growth and differentiation of human adipose stromal cells in culture. Human Cell Culture Protocols 2, 41–56 (1996).
Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotech. 19, 971–974 (2001).
Wagner, K. & Welch, D. Feeder-free adaptation, culture and passaging of human IPS cells using complete KnockOut Serum Replacement feeder-free medium. J. Vis. Exp. 15 (2010). Published online, doi:10.3791/2236.
Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25, 402–408 (2001).
Acknowledgements
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.).
Author information
Authors and Affiliations
Contributions
K.H.N., F.J. and J.C.W. prepared most of the paper. R.C.R., M.A.K. and M.T.L. provided advice and proofread the paper.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Narsinh, K., Jia, F., Robbins, R. et al. Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nat Protoc 6, 78–88 (2011). https://doi.org/10.1038/nprot.2010.173
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2010.173
This article is cited by
-
Systematic comparison of nonviral gene delivery strategies for efficient co-expression of two transgenes in human mesenchymal stem cells
Journal of Biological Engineering (2023)
-
Induced Pluripotent Stem Cells (iPSCs) Provide a Potentially Unlimited T Cell Source for CAR-T Cell Development and Off-the-Shelf Products
Pharmaceutical Research (2021)
-
Hydrojet-based delivery of footprint-free iPSC-derived cardiomyocytes into porcine myocardium
Scientific Reports (2020)
-
Nucleic acid delivery to mesenchymal stem cells: a review of nonviral methods and applications
Journal of Biological Engineering (2019)
-
The march of pluripotent stem cells in cardiovascular regenerative medicine
Stem Cell Research & Therapy (2018)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.