Protocol | Published:

Generation of mouse-induced pluripotent stem cells with plasmid vectors

Nature Protocols volume 5, pages 418428 (2010) | Download Citation

Abstract

Reprogramming of somatic cells into pluripotent stem cells has been reported by introducing a combination of several transcription factors (Oct3/4, Sox2, Klf4 and c-Myc). The induced pluripotent stem (iPS) cells from patient's somatic cells could be a useful source for drug discovery and cell transplantation therapies. However, to date, most iPS cells were made using viral vectors, such as retroviruses and lentiviruses. Here we describe an alternative method to generate iPS cells from mouse embryonic fibroblasts (MEFs) by continual transfection of plasmid vectors. This protocol takes around 2 months to complete, from MEF isolation to iPS cell establishment. Although the reprogramming efficiency of this protocol is still low, the established iPS cells are most likely free from plasmid integration. This virus-free technique reduces the safety concern for iPS cell generation and application, and provides a source of cells for the investigation of the mechanisms underlying reprogramming and pluripotency.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from $8.99

All prices are NET prices.

References

  1. 1.

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

  2. 2.

    et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

  3. 3.

    et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

  4. 4.

    et al. Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell 3, 587–590 (2008).

  5. 5.

    et al. Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 4, 11–15 (2009).

  6. 6.

    et al. Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 4, 16–19 (2009).

  7. 7.

    et al. Generation of induced pluripotent stem cell lines from Tibetan miniature pig. J. Biol. Chem. 284, 17634–17640 (2009).

  8. 8.

    et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1, 55–70 (2007).

  9. 9.

    , & Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317 (2007).

  10. 10.

    et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318–324 (2007).

  11. 11.

    & Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells advance online publication August 2009; doi:10.1002/stem.201.

  12. 12.

    et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol. 26, 101–106 (2008).

  13. 13.

    , , , & Induced pluripotent stem cells generated without viral integration. Science 322, 945–949 (2008).

  14. 14.

    et al. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 458, 771–775 (2009).

  15. 15.

    et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458, 766–770 (2009).

  16. 16.

    et al. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4, 381–384 (2009).

  17. 17.

    et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4, 472–476 (2009).

  18. 18.

    et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797–801 (2009).

  19. 19.

    , , , & Generation of mouse induced pluripotent stem cells without viral vectors. Science 322, 949–953 (2008).

  20. 20.

    et al. Variation in the safety of induced pluripotent stem cell lines. Nat. Biotechnol. 27, 743–745 (2009).

  21. 21.

    et al. Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science 321, 699–702 (2008).

  22. 22.

    et al. The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences. J. Gen. Virol. 82, 1027–1041 (2001).

  23. 23.

    , , & Efficient multicistronic expression of a transgene in human embryonic stem cells. Stem Cells 25, 1707–1712 (2007).

  24. 24.

    et al. A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell 3, 340–345 (2008).

  25. 25.

    et al. Suppression of induced pluripotent stem cell generation by the p53–p21 pathway. Nature 460, 1132–1135 (2009).

  26. 26.

    et al. Two supporting factors greatly improve the efficiency of human iPSC generation. Cell Stem Cell 3, 475–479 (2008).

  27. 27.

    et al. Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb. Nat. Cell Biol. 11, 197–203 (2009).

  28. 28.

    et al. Enhanced efficiency of generating induced pluripotent stem (iPS) cells from human somatic cells by a combination of six transcription factors. Cell. Res. 18, 600–603 (2008).

  29. 29.

    et al. Wnt signaling promotes reprogramming of somatic cells to pluripotency. Cell Stem Cell 3, 132–135 (2008).

  30. 30.

    et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat. Biotechnol. 26, 795–797 (2008).

  31. 31.

    , & Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991).

  32. 32.

    et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336, 688–690 (1988).

  33. 33.

    et al. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat. Biotechnol. 26, 916–924 (2008).

  34. 34.

    , , & Induction of pluripotent stem cells from fibroblast cultures. Nat. Protoc. 2, 3081–3089 (2007).

  35. 35.

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

  36. 36.

    et al. Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edn. 192–193 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2003).

Download references

Acknowledgements

We are grateful to Drs. M. Nakagawa, K. Yae, M. Koyanagi and K. Tanabe for scientific discussion, and to K. Takeda and T. Ishii for critical reading of the paper. We also thank T. Ichisaka, K. Okuda, M. Narita, A. Okada, N. Takizawa, R. Kato, R. Iyama, E. Nishikawa, Y. Shimazu and N. Maruhashi for technical and administrative supports. We also thank J. Miyazaki 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 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.). K.O. was a JSPS research fellow. H.H. is supported by a Japanese Government (MEXT) Scholarship.

Author information

Affiliations

  1. Center for iPS Cell Research and Application (CiRA), Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan.

    • Keisuke Okita
    • , Kazutoshi Takahashi
    •  & Shinya Yamanaka
  2. Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

    • Hyenjong Hong
    •  & Shinya Yamanaka
  3. Yamanaka iPS Cell Special Project, Japan Science and Technology Agency, Kawaguchi, Japan.

    • Shinya Yamanaka
  4. Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA.

    • Shinya Yamanaka

Authors

  1. Search for Keisuke Okita in:

  2. Search for Hyenjong Hong in:

  3. Search for Kazutoshi Takahashi in:

  4. Search for Shinya Yamanaka in:

Contributions

K.O. prepared most of the paper with the assistance of H.H., and K.T. and S.Y. provided advice and proofread the paper.

Corresponding author

Correspondence to Keisuke Okita.

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2009.231

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.