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.

Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency

Abstract

Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to specifically express in mouse and human embryonic stem cells but not in primary fibroblasts. The bicistronic EOS vector marked emerging mouse and human iPS cell colonies with EGFP, and we used puromycin selection to aid the isolation of iPS cell lines that expressed endogenous pluripotency markers. These lines differentiated into cell types from all three germ layers. Reporter expression was extinguished upon differentiation and therefore monitored the residual pluripotent cells that form teratomas. Finally, we used EOS selection to establish Rett syndrome–specific mouse and human iPS cell lines with known mutations in MECP2.

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: EOS lentiviral vectors in mouse ES cells.
Figure 2: EOS lentiviral vectors in human ES cells.
Figure 3: EOS lentiviral vector in reprogrammed mouse iPS cells.
Figure 4: EOS lentiviral vector in reprogrammed human iPS cells.
Figure 5: EOS lentiviral vector in Rett syndrome–specific mouse and human iPS cell lines.

References

  1. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Park, I.H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008).

    Article  CAS  Google Scholar 

  7. Ellis, J. Silencing and variegation of gammaretrovirus and lentivirus vectors. Hum. Gene Ther. 16, 1241–1246 (2005).

    Article  CAS  Google Scholar 

  8. Maksakova, I.A. & Mager, D.L. Transcriptional regulation of early transposon elements, an active family of mouse long terminal repeat retrotransposons. J. Virol. 79, 13865–13874 (2005).

    Article  CAS  Google Scholar 

  9. Okumura-Nakanishi, S., Saito, M., Niwa, H. & Ishikawa, F. Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells. J. Biol. Chem. 280, 5307–5317 (2005).

    Article  CAS  Google Scholar 

  10. Tomioka, M. et al. Identification of Sox-2 regulatory region which is under the control of Oct-3/4-Sox-2 complex. Nucleic Acids Res. 30, 3202–3213 (2002).

    Article  CAS  Google Scholar 

  11. Peerani, R. et al. Niche-mediated control of human embryonic stem cell self-renewal and differentiation. EMBO J. 26, 4744–4755 (2007).

    Article  CAS  Google Scholar 

  12. Stewart, M.H. et al. Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment. Nat. Methods 3, 807–815 (2006).

    Article  CAS  Google Scholar 

  13. Bendall, S.C. et al. IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature 448, 1015–1021 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Wernig, M., Meissner, A., Cassady, J.P. & Jaenisch, R. c-Myc is dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell 2, 10–12 (2008).

    Article  CAS  Google Scholar 

  16. Takahashi, K., Okita, K., Nakagawa, M. & Yamanaka, S. Induction of pluripotent stem cells from fibroblast cultures. Nat. Protoc. 2, 3081–3089 (2007).

    Article  CAS  Google Scholar 

  17. Brambrink, T. et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2, 151–159 (2008).

    Article  CAS  Google Scholar 

  18. Stadtfeld, M., Maherali, N., Breault, D.T. & Hochedlinger, K. Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2, 230–240 (2008).

    Article  CAS  Google Scholar 

  19. Lowry, W.E. et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc. Natl. Acad. Sci. USA 105, 2883–2888 (2008).

    Article  CAS  Google Scholar 

  20. Bienvenu, T. & Chelly, J. Molecular genetics of Rett syndrome: when DNA methylation goes unrecognized. Nat. Rev. Genet. 7, 415–426 (2006).

    Article  CAS  Google Scholar 

  21. Shahbazian, M. et al. Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3. Neuron 35, 243–254 (2002).

    Article  CAS  Google Scholar 

  22. Park, I.H. et al. Disease-specific induced pluripotent stem cells. Cell 134, 877–886 (2008).

    Article  CAS  Google Scholar 

  23. Dimos, J.T. et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321, 1218–1221 (2008).

    Article  CAS  Google Scholar 

  24. Ebert, A.D. et al. Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457, 277–280 (2008).

    Article  Google Scholar 

  25. Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T. & Yamanaka, S. Generation of mouse induced pluripotent stem cells without viral vectors. Science 322, 949–953 (2008).

    Article  CAS  Google Scholar 

  26. Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G. & Hochedlinger, K. Induced pluripotent stem cells generated without viral integration. Science 322, 945–949 (2008).

    Article  CAS  Google Scholar 

  27. Shi, Y. et al. A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell 2, 525–528 (2008).

    Article  CAS  Google Scholar 

  28. Wernig, M. et al. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proc. Natl. Acad. Sci. USA 105, 5856–5861 (2008).

    Article  CAS  Google Scholar 

  29. Buzina, A. et al. β-Globin LCR and intron elements cooperate and direct spatial reorganization for gene therapy. PLoS Genet. 4, e1000051 (2008).

    Article  Google Scholar 

  30. Yeom, Y.I. et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894 (1996).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank H.R. Schöler (Max Planck Institute for Molecular Biomedicine) for providing Oct-4 promoter plasmid, T. Kitamura (Institute of Medical Science, University of Tokyo) for Plat-E cells, B. Alman and A. Lin for human fibroblast isolation and I.H. Park for advice on human iPS cell isolation. We gratefully acknowledge the assistance of T. Thompson at the Ontario Human iPS Cell Facility, SickKids The Centre for Applied Genomics Facility, SickKids ES Facility, SickKids Flow Facility and the Centre for Modeling Human Disease pathology core. This work was supported by grants from the Canadian Institutes of Health Research (MOP-10825 to D.L.M., MOP-77803 to J.R., MOP-81129 and IG1-94505 to J.E., and RMF-92090 to J.E. and D.L.M.), the Stem Cell Network (to J.R., M.B. and J.E.), the Ontario Ministry of Research and Innovation (to J.R. and J.E. for the Ontario Human IPS Cell Facility), and the International Rett Syndrome Foundation (to J.E.). A.H. is supported by a Restracomp Award from SickKids Hospital, A.Y.L.C. by a Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada, N.F. by an Ontario Council of Graduate Studies Master's Autism Scholars Award and the Ontario Student Opportunity Trust Funds Hayden Hantho Award and C.A.S. by the Stem Cell Network and the Juvenile Diabetes Research Foundation.

Author information

Authors and Affiliations

Authors

Contributions

A.H., D.L.M. and J.E. conceived the project; I.A.M. provided reagents; A.H., M.B., J.R. and J.E. designed experiments; A.H. developed the EOS vectors and performed the iPS cell reprogramming experiments; A.Y.L.C. and N.F. performed the Rett syndrome iPS cell experiments; K.V. and J.S.D. performed the hES cell experiments; C.A.S. and P.P. performed the teratoma experiments; A.H., J.R., M.B. and J.E. wrote the manuscript.

Corresponding author

Correspondence to James Ellis.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12, Supplementary Tables 1 and 2 (PDF 4397 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hotta, A., Cheung, A., Farra, N. et al. Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency. Nat Methods 6, 370–376 (2009). https://doi.org/10.1038/nmeth.1325

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1325

This article is cited by

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