Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Generation of germline-competent induced pluripotent stem cells

Abstract

We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression. These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras. Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells. The four transgenes (Oct3/4, Sox2, c-myc and Klf4) were strongly silenced in Nanog iPS cells. We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation. Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene. Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Nanog-GFP-IRES-Puro r transgenic mice.
Figure 2: Generation of iPS cells from MEFs of Nanog-GFP-IRES-Puro r transgenic mice.
Figure 3: Characterization of Nanog iPS cells.
Figure 4: Gene expression in Nanog iPS cells.
Figure 5: DNA methylation of ES-cell-specific genes and imprinting genes.
Figure 6: Germline chimaeras from Nanog iPS cells.

References

  1. Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998)

    Article  ADS  CAS  Google Scholar 

  2. Hochedlinger, K. & Jaenisch, R. Nuclear reprogramming and pluripotency. Nature 441, 1061–1067 (2006)

    Article  ADS  CAS  Google Scholar 

  3. Niwa, H., Miyazaki, J. & Smith, A. G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nature Genet. 24, 372–376 (2000)

    Article  CAS  Google Scholar 

  4. Nichols, J. et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998)

    Article  CAS  Google Scholar 

  5. Avilion, A. A. et al. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 17, 126–140 (2003)

    Article  CAS  Google Scholar 

  6. Cartwright, P. et al. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development 132, 885–896 (2005)

    Article  CAS  Google Scholar 

  7. Li, Y. et al. Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4. Blood 105, 635–637 (2005)

    Article  CAS  Google Scholar 

  8. 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  Google Scholar 

  9. 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  Google Scholar 

  10. Kuroda, T. et al. Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol. Cell. Biol. 25, 2475–2485 (2005)

    Article  CAS  Google Scholar 

  11. Rodda, D. J. et al. Transcriptional regulation of Nanog by OCT4 and SOX2. J. Biol. Chem. 280, 24731–24737 (2005)

    Article  CAS  Google Scholar 

  12. Mitsui, K. et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642 (2003)

    Article  CAS  Google Scholar 

  13. Chambers, I. et al. Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655 (2003)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  15. Copeland, N. G., Jenkins, N. A. & Court, D. L. Recombineering: a powerful new tool for mouse functional genomics. Nature Rev. Genet. 2, 769–779 (2001)

    Article  CAS  Google Scholar 

  16. Testa, G. et al. Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nature Biotechnol. 21, 443–447 (2003)

    Article  CAS  Google Scholar 

  17. Cherry, S. R., Biniszkiewicz, D., van Parijs, L., Baltimore, D. & Jaenisch, R. Retroviral expression in embryonic stem cells and hematopoietic stem cells. Mol. Cell. Biol. 20, 7419–7426 (2000)

    Article  CAS  Google Scholar 

  18. Chen, T., Ueda, Y., Xie, S. & Li, E. A novel Dnmt3a isoform produced from an alternative promoter localizes to euchromatin and its expression correlates with active de novo methylation. J. Biol. Chem. 277, 38746–38754 (2002)

    Article  CAS  Google Scholar 

  19. Davis, T. L., Yang, G. J., McCarrey, J. R. & Bartolomei, M. S. The H19 methylation imprint is erased and re-established differentially on the parental alleles during male germ cell development. Hum. Mol. Genet. 9, 2885–2894 (2000)

    Article  CAS  Google Scholar 

  20. Sato, S., Yoshimizu, T., Sato, E. & Matsui, Y. Erasure of methylation imprinting of Igf2r during mouse primordial germ-cell development. Mol. Reprod. Dev. 65, 41–50 (2003)

    Article  CAS  Google Scholar 

  21. Brandeis, M. et al. The ontogeny of allele-specific methylation associated with imprinted genes in the mouse. EMBO J. 12, 3669–3677 (1993)

    Article  CAS  Google Scholar 

  22. Labosky, P. A., Barlow, D. P. & Hogan, B. L. Mouse embryonic germ (EG) cell lines: transmission through the germline and differences in the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines. Development 120, 3197–3204 (1994)

    CAS  PubMed  Google Scholar 

  23. Kato, Y. et al. Developmental potential of mouse primordial germ cells. Development 126, 1823–1832 (1999)

    CAS  PubMed  Google Scholar 

  24. Lee, J. et al. Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells. Development 129, 1807–1817 (2002)

    Article  CAS  Google Scholar 

  25. Geijsen, N. et al. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427, 148–154 (2004)

    Article  ADS  CAS  Google Scholar 

  26. Meiner, V. L. et al. Disruption of the acyl-CoA:cholesterol acyltransferase gene in mice: evidence suggesting multiple cholesterol esterification enzymes in mammals. Proc. Natl Acad. Sci. USA 93, 14041–14046 (1996)

    Article  ADS  CAS  Google Scholar 

  27. 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  Google Scholar 

  28. 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  Google Scholar 

Download references

Acknowledgements

We thank K. Takahashi, M. Nakagawa and T. Aoi for scientific discussion; M. Maeda for histological analyses; M. Narita, J. Iida, H. Miyachi and S. Kitano for technical assistance; and R. Kato, R. Iyama and Y. Ohuchi for administrative assistance. We also thank T. Kitamura for Plat-E cells and pMXs retroviral vectors, and R. Farese for RF8 ES cells. This study was supported in part by a grant from the Uehara Memorial Foundation, the Program for Promotion of Fundamental Studies in Health Sciences of NIBIO, a grant from the Leading Project of MEXT, and Grants-in-Aid for Scientific Research of JSPS and MEXT (to S.Y.). K.O. is a JSPS research fellow. The microarray data are deposited in GEO under accession number GSE7841.

Author Contributions K.O. conducted most of the experiments in this study. T.I. performed manipulation of mouse embryos to generate Nanog-GFP transgenic mice. T.I. also maintained the mouse lines. S.Y. designed and supervised the study, and prepared the manuscript. S.Y. also performed computer analyses of DNA microarray data.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Shinya Yamanaka.

Ethics declarations

Competing interests

The microarray data are deposited in GEO under accession number GSE7841. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-13 with Legends and Supplementary Table 1, which show detailed characterization of Nanog-iPS cells. (PDF 1076 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Okita, K., Ichisaka, T. & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317 (2007). https://doi.org/10.1038/nature05934

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature05934

This article is cited by

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.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing