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Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts

Nature Biotechnology volume 26, pages 101–106 (2008)Cite this article

Abstract

Direct reprogramming of somatic cells provides an opportunity to generate patient- or disease-specific pluripotent stem cells. Such induced pluripotent stem (iPS) cells were generated from mouse fibroblasts by retroviral transduction of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc1. Mouse iPS cells are indistinguishable from embryonic stem (ES) cells in many respects and produce germline-competent chimeras2,3,4. Reactivation of the c-Myc retrovirus, however, increases tumorigenicity in the chimeras and progeny mice, hindering clinical applications3. Here we describe a modified protocol for the generation of iPS cells that does not require the Myc retrovirus. With this protocol, we obtained significantly fewer non-iPS background cells, and the iPS cells generated were consistently of high quality. Mice derived from Myc− iPS cells did not develop tumors during the study period. The protocol also enabled efficient isolation of iPS cells without drug selection. Furthermore, we generated human iPS cells from adult dermal fibroblasts without MYC.

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Figure 1: Effect of family factors and omission of Myc on the generation of iPS cells from Nanog-reporter MEFs.
Figure 2: Generation of iPS cells without the Myc retrovirus from MEFs containing the Fbxo15 reporter and the constitutively active GFP transgene.
Figure 3: The efficient isolation of iPS cells without drug selection.
Figure 4: Generation of human iPS cells without the MYC retrovirus.

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Acknowledgements

We thank K. Yae and M. Maekawa for their valuable scientific discussions; M. Narita for technical assistance; and R. Kato and R. Iyama for administrative assistance. We also thank K. Tomoda for the RNA of hES cells, T. Kitamura for the Plat-E cells and pMXs retroviral vectors, and R. Farese for the RF8 ES cells. 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.). T. A. and K.O. are JSPS research fellows.

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Author notes

  1. Masato Nakagawa and Michiyo Koyanagi: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan

    Masato Nakagawa, Michiyo Koyanagi, Koji Tanabe, Kazutoshi Takahashi, Tomoko Ichisaka, Takashi Aoi, Keisuke Okita, Yuji Mochiduki, Nanako Takizawa & Shinya Yamanaka

  2. CREST, Japan Science and Technology Agency, Kawaguchi, 332-0012, Japan

    Tomoko Ichisaka & Shinya Yamanaka

  3. Gladstone Institute of Cardiovascular Disease, San Francisco, 94158, CA, USA

    Shinya Yamanaka

  4. Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, 606-8507, Japan

    Shinya Yamanaka

Authors
  1. Masato Nakagawa
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  2. Michiyo Koyanagi
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  4. Kazutoshi Takahashi
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Contributions

M.N., M.K., K.Tanabe, K.Takahashi, T.A. and K.O. generated and characterized iPS cells. T.I. performed the chimera experiments. Y.M. prepared plasmids. N.T. characterized iPS cells. S.Y. supervised the study and wrote the manuscript.

Corresponding author

Correspondence to Shinya Yamanaka.

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Supplementary Tables 1–3 and Figures 1–3 (PDF 2890 kb)

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Nakagawa, M., Koyanagi, M., Tanabe, K. et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26, 101–106 (2008). https://doi.org/10.1038/nbt1374

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