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Generation of mouse-induced pluripotent stem cells with plasmid vectors

Nature Protocols volume 5, pages 418–428 (2010)Cite this article

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Abstract

Reprogramming of somatic cells into pluripotent stem cells has been reported by introducing a combination of several transcription factors (Oct3/4, Sox2, Klf4 and c-Myc). The induced pluripotent stem (iPS) cells from patient's somatic cells could be a useful source for drug discovery and cell transplantation therapies. However, to date, most iPS cells were made using viral vectors, such as retroviruses and lentiviruses. Here we describe an alternative method to generate iPS cells from mouse embryonic fibroblasts (MEFs) by continual transfection of plasmid vectors. This protocol takes around 2 months to complete, from MEF isolation to iPS cell establishment. Although the reprogramming efficiency of this protocol is still low, the established iPS cells are most likely free from plasmid integration. This virus-free technique reduces the safety concern for iPS cell generation and application, and provides a source of cells for the investigation of the mechanisms underlying reprogramming and pluripotency.

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Figure 1: Schematic diagram of iPS cell generation with plasmid vectors.
Figure 2: Transfection efficiency of repeated lipofection.
Figure 3: Morphology of iPS colonies just before picking up.
Figure 4: PCR screening of genomic integration.
Figure 5: Pluripotency of plasmid-iPS cells.

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Acknowledgements

We are grateful to Drs. M. Nakagawa, K. Yae, M. Koyanagi and K. Tanabe for scientific discussion, and to K. Takeda and T. Ishii for critical reading of the paper. We also thank T. Ichisaka, K. Okuda, M. Narita, A. Okada, N. Takizawa, R. Kato, R. Iyama, E. Nishikawa, Y. Shimazu and N. Maruhashi for technical and administrative supports. We also thank J. Miyazaki for the CAG promoter. This study was supported in part by a grant from the Program for Promotion of Fundamental Studies in Health Sciences of NIBIO, a grant from the Leading Project of MEXT, a grant from Uehara Memorial Foundation and Grants-in-Aid for Scientific Research of JSPS and MEXT (to S.Y.). K.O. was a JSPS research fellow. H.H. is supported by a Japanese Government (MEXT) Scholarship.

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Authors and Affiliations

  1. Center for iPS Cell Research and Application (CiRA), Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan

    Keisuke Okita, Kazutoshi Takahashi & Shinya Yamanaka

  2. Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

    Hyenjong Hong & Shinya Yamanaka

  3. Yamanaka iPS Cell Special Project, Japan Science and Technology Agency, Kawaguchi, Japan

    Shinya Yamanaka

  4. Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA

    Shinya Yamanaka

Authors
  1. Keisuke Okita
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  2. Hyenjong Hong
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  4. Shinya Yamanaka
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Contributions

K.O. prepared most of the paper with the assistance of H.H., and K.T. and S.Y. provided advice and proofread the paper.

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Correspondence to Keisuke Okita.

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Okita, K., Hong, H., Takahashi, K. et al. Generation of mouse-induced pluripotent stem cells with plasmid vectors. Nat Protoc 5, 418–428 (2010). https://doi.org/10.1038/nprot.2009.231

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