Protocol | Published:

Isolation and cultivation of naive-like human pluripotent stem cells based on HERVH expression

Nature Protocols volume 11, pages 327346 (2016) | Download Citation

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Abstract

The ability to derive and stably maintain ground-state human pluripotent stem cells (hPSCs) that resemble the cells seen in vivo in the inner cell mass has the potential to be an invaluable tool for researchers developing stem cell–based therapies. To date, derivation of human naive-like pluripotent stem cell lines has been limited to a small number of lineages, and their long-term culturing remains problematic. We describe a protocol for genetic and phenotypic tagging, selecting and maintaining naive-like hPSCs. We tag hPSCs by GFP, expressed by the long terminal repeat (LTR7) of HERVH endogenous retrovirus. This simple and efficient protocol has been reproduced with multiple hPSC lines, including embryonic and induced pluripotent stem cells, and it takes 6 weeks. By using the reporter, homogeneous hPSC cultures can be derived, characterized and maintained for the long term by repeated re-sorting and re-plating steps. The HERVH-expressing cells have a similar, but nonidentical, expression pattern to other naive-like cells, suggesting that alternative pluripotent states might exist.

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Change history

  • 29 October 2018

    In the published version of this paper, the authors omitted a funding source. L.D.H. acknowledges support from the European Research Council (Advanced Grant ERC-2014-ADG 669207). The original article has not been corrected.

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Acknowledgements

Z. Izsvák is funded by European Research Council (ERC) grant ERC-2011-AdG 294742. L.D.H. is funded by Medical Research grant MR/L007215/1. We thank E.È. Nagy for her assistance in specifying experimental details.

Author information

Affiliations

  1. Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany.

    • Jichang Wang
    • , Manvendra Singh
    • , Chuanbo Sun
    • , Daniel Besser
    • , Alessandro Prigione
    •  & Zsuzsanna Izsvák
  2. Paul Ehrlich Institute, Division of Medical Biotechnology, Langen, Germany.

    • Zoltán Ivics
  3. Department of Biology and Biochemistry, University of Bath, Bath, UK.

    • Laurence D Hurst

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Contributions

Z. Izsvák, L.D.H. and J.W. wrote the manuscript. J.W. designed and performed experiments, and provided technical details to the Protocol. M.S. performed bioinformatic, cross-culture comparative studies. C. Sun assisted in cell culture and gene expression analysis. Z. Ivics provided critical advice. D.B. and A.P. provided materials.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Laurence D Hurst or Zsuzsanna Izsvák.

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DOI

https://doi.org/10.1038/nprot.2016.016

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