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Open Access Highly Accessed Methodology article

Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors

Karim Si-Tayeb1, Fallon K Noto1, Ana Sepac2, Filip Sedlic2, Zeljko J Bosnjak2, John W Lough1 and Stephen A Duncan1*

Author Affiliations

1 Department of Cell Biology, Neurobiology and Anatomy, The Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA

2 Department of Anesthesiology, The Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA

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BMC Developmental Biology 2010, 10:81  doi:10.1186/1471-213X-10-81

Published: 3 August 2010

Abstract

Background

The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS) cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry.

Results

Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture.

Conclusions

Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.