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


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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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



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).

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