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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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.
Author information
Affiliations
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
Department of Biological Repair, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
- Asuka Morizane
- & Jun Takahashi
Laboratory for Retinal Regeneration, Center for Developmental Biology, RIKEN, Kobe, Japan.
- Satoshi Okamoto
- & Masayo Takahashi
Department of Tissue and Organ Development, Gifu University Graduate School of Medicine, Gifu, Japan.
- Ken-ichi Tezuka
- & Takahiro Kunisada
Department of Oral and Maxillofacial Science, Gifu University Graduate School of Medicine, Gifu, Japan.
- Toshiyuki Shibata
Human Leukocyte Antigen (HLA) Laboratory, Kyoto, Japan.
- Hiroh Saji
Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan.
- Shinya Yamanaka
Yamanaka Induced Pluripotent Stem Cell Project, Japan Science and Technology Agency, Kawaguchi, Japan.
- Shinya Yamanaka
Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA.
- 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.
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.
Corresponding authors
Correspondence to Keisuke Okita or Shinya Yamanaka.
Supplementary information
PDF files
- 1.
Supplementary Text and Figures
Supplementary Figures 1–9 and Supplementary Tables 1–7 and 9, 10
Excel files
- 1.
Supplementary Table 8
Haplotype frequency for HLA-A, HLA-B and HLA-DRB1 loci in Japanese population.