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.

  • Protocol
  • Published:

Faster generation of hiPSCs by coupling high-titer lentivirus and column-based positive selection

Nature Protocols volume 6, pages 701–714 (2011)Cite this article

Subjects

Abstract

The protocols described here address methods used in two crucial stages in the retroviral reprogramming of somatic cells to produce human induced pluripotent stem cell (hiPSC) lines. The first is an optimized method for producing lentivirus at an efficiency 600-fold greater than previously published, and it includes conjugation of the lentivirus to streptavidin superparamagnetic particles; this process takes 8 d. The second method enables the isolation of true hiPSCs immediately after somatic cell reprogramming and involves column-based positive selection of cells expressing the pluripotency marker TRA-1-81. This process takes 2 h and, as it is directly compatible with feeder-free culture, the time burden of manually identifying and mechanically propagating hiPSC colonies is reduced drastically. Taken together, these methods accelerate the production of hiPSCs and enable lines to be isolated, expanded to ∼107 cells and cryopreserved within 6–8 weeks.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1
Figure 2: Transduction of fibroblasts and anticipated results from hiPSC derivation using column enrichment.
Figure 3: Characterization of hiPSCs.

Similar content being viewed by others

References

  1. Park, I.H., Lerou, P.H., Zhao, R., Huo, H. & Daley, G.Q. Generation of human-induced pluripotent stem cells. Nat. Protoc. 3, 1180–1186 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Dick, E.P. et al. Two new protocols to enhance the production and isolation of human induced pluripotent stem cell lines. Stem Cell Res. 6, 158–167 (2011).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhou, W. & Freed, C.R. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells 27, 2667–2674 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. 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  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  11. Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618–630 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 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  PubMed  Google Scholar 

  13. Stadtfeld, M. & Hochedlinger, K. Induced pluripotency: history, mechanisms, and applications. Genes Dev 24, 2239–2263 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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  PubMed  PubMed Central  Google Scholar 

  15. 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 

  16. Chan, E.M. et al. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat. Biotechnol. 27, 1033–1037 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Merten, O.W. State-of-the-art of the production of retroviral vectors. J. Gene Med. 6 (Suppl 1): S105–S124 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Carmo, M. et al. Thermosensitivity of the reverse transcription process as an inactivation mechanism of lentiviral vectors. Hum. Gene Ther. 20, 1168–1176 (2009).

    Article  CAS  PubMed  Google Scholar 

  19. Carmo, M., Panet, A., Carrondo, M.J., Alves, P.M. & Cruz, P.E. From retroviral vector production to gene transfer: spontaneous inactivation is caused by loss of reverse transcription capacity. J. Gene Med. 10, 383–391 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Chilton, J.M. & Le Doux, J.M. Quantitative analysis of retroviral and lentiviral gene transfer to murine embryonic stem cells. J. Biotechnol. 138, 42–51 (2008).

    Article  CAS  PubMed  Google Scholar 

  21. Higashikawa, F. & Chang, L. Kinetic analyses of stability of simple and complex retroviral vectors. Virology 280, 124–131 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Zufferey, R. et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72, 9873–9880 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Nesbeth, D. et al. Metabolic biotinylation of lentiviral pseudotypes for scalable paramagnetic microparticle-dependent manipulation. Mol. Ther. 13, 814–822 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Chan, L. et al. Conjugation of lentivirus to paramagnetic particles via nonviral proteins allows efficient concentration and infection of primary acute myeloid leukemia cells. J. Virol. 79, 13190–13194 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Logan, A.C. et al. Factors influencing the titer and infectivity of lentiviral vectors. Hum. Gene Ther. 15, 976–988 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Miltenyi, S., Muller, W., Weichel, W. & Radbruch, A. High gradient magnetic cell separation with MACS. Cytometry 11, 231–238 (1990).

    Article  CAS  PubMed  Google Scholar 

  27. Grutzkau, A. & Radbruch, A. Small but mighty: how the MACS-technology based on nanosized superparamagnetic particles has helped to analyze the immune system within the last 20 years. Cytometry A 77, 643–647 (2010).

    Article  PubMed  Google Scholar 

  28. Braam, S.R. et al. Improved genetic manipulation of human embryonic stem cells. Nat. Methods 5, 389–392 (2008).

    Article  CAS  PubMed  Google Scholar 

  29. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. Baup, D. et al. Variegation and silencing in a lentiviral-based murine transgenic model. Transgenic Res. 19, 399–414 (2010).

    Article  CAS  PubMed  Google Scholar 

  31. Smith, J.R. & Whitney, R.G. Intraclonal variation in proliferative potential of human diploid fibroblasts: stochastic mechanism for cellular aging. Science 207, 82–84 (1980).

    Article  CAS  PubMed  Google Scholar 

  32. Stojkovic, P. et al. An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 23, 306–314 (2005).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  34. Burridge, P.W. et al. Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability. Stem Cells 25, 929–938 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. 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  PubMed  Google Scholar 

  36. Priddle, H. et al. Bioluminescence imaging of human embryonic stem cells transplanted in vivo in murine and chick models. Cloning Stem Cells 11, 259–267 (2009).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Medical Research Council, the Biotechnology and Biological Sciences Research Council and the British Heart Foundation. We would like to thank our collaborators at the University of Newcastle International Centre for Life who provided fibroblast samples from the BioBank in Newcastle, which is part of the MRC centre for Neuromuscular Diseases and EuroBioBank.

Author information

Authors and Affiliations

  1. Wolfson Centre for Stem Cells, Tissue Engineering & Modelling, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, UK

    Emily Dick, Elena Matsa, Lorraine E Young & Chris Denning

  2. Department of Haematological Medicine, King's College, Guy's, King's and St. Thomas' School of Medicine, The Rayne Institute, London, UK

    David Darling

Authors
  1. Emily Dick
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elena Matsa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lorraine E Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David Darling
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chris Denning
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.D. designed the experiments, carried out the work, analyzed the results and prepared the manuscript. E.M. derived the HUES-Fib line used for control experiments. L.E.Y. was involved in hiPSC setup. D.D. derived the modified 293T line, BL15, and provided methods for the calcium precipitation of lentiviral supernatants. C.D. is the principal investigator on the project and coordinates research activity and funding.

Corresponding author

Correspondence to Chris Denning.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dick, E., Matsa, E., Young, L. et al. Faster generation of hiPSCs by coupling high-titer lentivirus and column-based positive selection. Nat Protoc 6, 701–714 (2011). https://doi.org/10.1038/nprot.2011.320

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.320

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

Advanced 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