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A more efficient method to generate integration-free human iPS cells

Nature Methods volume 8, pages 409–412 (2011)Cite this article

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Abstract

We report a simple method, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors. We generated human iPSCs from multiple donors, including two putative human leukocyte antigen (HLA)-homozygous donors who match ∼20% of the Japanese population at major HLA loci; most iPSCs are integrated transgene-free. This method may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.

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Figure 1: Establishment of human iPSCs.
Figure 2: Characterization of pla-iPSC clones.
Figure 3: Estimated coverage of the Japanese population by HLA homozygous donors.

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Acknowledgements

We thank K. Takahashi, T. Aoi and Y. Yoshida for scientific discussion; M. Narita, T. Ichisaka, M. Ohuchi, M. Nishikawa and N. Takizawa for technical assistance; R. Kato, E. Nishikawa, S. Takeshima, Y. Ohtsu and H. Hasaba for administrative assistance; and H. Niwa (RIKEN) and J. Miyazaki (Osaka University) 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 National Institute of Biomedical Innovation, a grant from the Leading Project of Ministry of Education, Culture, Sports, Science and Technology (MEXT), a grant from Funding Program for World-Leading Innovative Research and Development on Science and Technology (FIRST Program) of Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research of Japan Society for the Promotion of Science and MEXT (to S.Y.) and Senri Life Science Foundation (to K.O.). 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, Kyoto University, Kyoto, Japan

    Keisuke Okita, Yasuko Matsumura, Yoshiko Sato, Aki Okada, Asuka Morizane, Hyenjong Hong, Masato Nakagawa, Koji Tanabe, Masayo Takahashi, Jun Takahashi & Shinya Yamanaka

  2. Department of Biological Repair, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

    Asuka Morizane & Jun Takahashi

  3. Laboratory for Retinal Regeneration, Center for Developmental Biology, RIKEN, Kobe, Japan

    Satoshi Okamoto & Masayo Takahashi

  4. Department of Tissue and Organ Development, Gifu University Graduate School of Medicine, Gifu, Japan

    Ken-ichi Tezuka & Takahiro Kunisada

  5. Department of Oral and Maxillofacial Science, Gifu University Graduate School of Medicine, Gifu, Japan

    Toshiyuki Shibata

  6. Human Leukocyte Antigen (HLA) Laboratory, Kyoto, Japan

    Hiroh Saji

  7. Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan

    Shinya Yamanaka

  8. Yamanaka Induced Pluripotent Stem Cell Project, Japan Science and Technology Agency, Kawaguchi, Japan

    Shinya Yamanaka

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

    Shinya Yamanaka

Authors
  1. Keisuke Okita
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  2. Yasuko Matsumura
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  3. Yoshiko Sato
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  4. Aki Okada
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  5. Asuka Morizane
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  6. Satoshi Okamoto
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  7. Hyenjong Hong
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  8. Masato Nakagawa
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  9. Koji Tanabe
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  10. Ken-ichi Tezuka
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  11. Toshiyuki Shibata
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  12. Takahiro Kunisada
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  13. Masayo Takahashi
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  14. Jun Takahashi
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  15. Hiroh Saji
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  16. Shinya Yamanaka
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Contributions

K.O. and S.Y. conceived the project and wrote the manuscript. K.O. constructed the vectors with H.H., M.N. and K. Tanabe, and conducted most of the experiments with Y.M., Y. S. and A.O. A.M. and J.T. carried out the differentiation experiment into dopaminergic neurons. S.O. and M.T. performed differentiation into retinal pigment epithelial cells. K. Tezuka., T.S. and T.K. established dental pulp cell lines. H.S. performed HLA haplotyping in Japanese population and supervised HLA analysis.

Corresponding authors

Correspondence to Keisuke Okita or Shinya Yamanaka.

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Competing interests

K.O., M.N. and S.Y. are filing a patent application to Japan, US and EU based on the results reported in this paper (PCT/JP2010/063733). S.Y. is a member of Scientific Advisory Board for iPS Academia Japan Inc. and iPierian Inc., which manage the patents.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Tables 1–7 and 9, 10 (PDF 1911 kb)

Supplementary Table 8

Haplotype frequency for HLA-A, HLA-B and HLA-DRB1 loci in Japanese population. (XLS 395 kb)

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Okita, K., Matsumura, Y., Sato, Y. et al. A more efficient method to generate integration-free human iPS cells. Nat Methods 8, 409–412 (2011). https://doi.org/10.1038/nmeth.1591

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