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Nat Protoc. 2016 Feb;11(2):327-46. doi: 10.1038/nprot.2016.016. Epub 2016 Jan 21.

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

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Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany.
Paul Ehrlich Institute, Division of Medical Biotechnology, Langen, Germany.
Department of Biology and Biochemistry, University of Bath, Bath, UK.


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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