TY - JOUR
T1 - Isolation and cultivation of naive-like human pluripotent stem cells based on HERVH expression
AU - Wang, Jichang
AU - Singh, Manvendra
AU - Sun, Chuanbo
AU - Besser, Daniel
AU - Prigione, Alessandro
AU - Ivics, Zoltán
AU - Hurst, Laurence D.
AU - Izsvák, Zsuzsanna
PY - 2016/2/1
Y1 - 2016/2/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84956513843&partnerID=8YFLogxK
UR - http://dx.doi.org/10.1038/nprot.2016.016
U2 - 10.1038/nprot.2016.016
DO - 10.1038/nprot.2016.016
M3 - Article
C2 - 26797457
AN - SCOPUS:84956513843
SN - 1754-2189
VL - 11
SP - 327
EP - 346
JO - Nature Protocols
JF - Nature Protocols
IS - 2
ER -