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Induction of pluripotent stem cells from fibroblast cultures

Nature Protocols volume 2, pages 3081–3089 (2007)Cite this article

Abstract

Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.

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Figure 1: Schematic diagram of induced pluripotent stem (iPS) cell generation.
Figure 2: Morphologies of mouse fibroblasts used for induced pluripotent stem (iPS) cell induction.
Figure 3: Retroviral transfection system for induced pluripotent stem (iPS) cell generation.
Figure 4: Induced pluripotent stem (iPS) cells from mouse fibroblasts.

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References

  1. Yamanaka, S. Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 1, 39–49 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Egli, D., Rosains, J., Birkhoff, G. & Eggan, K. Developmental reprogramming after chromosome transfer into mitotic mouse zygotes. Nature 447, 679–685 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Silva, J., Chambers, I., Pollard, S. & Smith, A. Nanog promotes transfer of pluripotency after cell fusion. Nature 441, 997–1001 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Surani, M.A., Hayashi, K. & Hajkova, P. Genetic and epigenetic regulators of pluripotency. Cell 128, 747–762 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Niwa, H. How is pluripotency determined and maintained? Development 134, 635–646 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Boiani, M. & Schöler, H.R. Regulatory networks in embryo-derived pluripotent stem cells. Nat. Rev. Mol. Cell. Biol. 6, 872–884 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Chambers, I. & Smith, A. Self-renewal of teratocarcinoma and embryonic stem cells. Oncogene 23, 7150–7160 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Tokuzawa, Y. et al. Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development. Mol. Cell. Biol. 23, 2699–2708 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Okita, K., Ichisaka, T. & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Morita, S., Kojima, T. & Kitamura, T. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7, 1063–1066 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. McMahon, A.P. & Bradley, A. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 62, 1073–1085 (1990).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to Dr. Yoshimi Tokuzawa for earlier works that were fundamental for our experiments. We also thank Tomoko Ichisaka, Megumi Narita, Yumi Ohuchi, Rie Kato and Ryoko Iyama for technical and administrative supports, and Dr. Toshio Kitamura for pMXs retroviral vectors and Plat-E cells.

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Authors and Affiliations

  1. Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-machi, Shogoin, Sakyo-ku, 606-8507, Kyoto, Japan

    Kazutoshi Takahashi, Keisuke Okita, Masato Nakagawa & Shinya Yamanaka

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  1. Kazutoshi Takahashi
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  2. Keisuke Okita
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  3. Masato Nakagawa
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  4. Shinya Yamanaka
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Correspondence to Shinya Yamanaka.

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Takahashi, K., Okita, K., Nakagawa, M. et al. Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc 2, 3081–3089 (2007). https://doi.org/10.1038/nprot.2007.418

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