The levels of reprogramming factors influence the induction and maintenance of pluripotency: the case of CD1 mouse strain cells
Por:
Covarrubias, Luis, Martinez-Sarmiento, Jose-Angel, Valencia, Concepcion, Nagy, Andras, Hernandez-Garcia, David
Publicada:
1 ene 2021
Resumen:
The amount of proteins of the regulatory pluripotency network can be
determinant for somatic cell reprogramming into induced pluripotent stem
cells (iPSCs) as well as for the maintenance of pluripotent stem cells
(PSCs). Here, we report a transposon-based reprogramming system
(PB-Booster) that allowed high expression levels of a polycistronic
transgene containing Myc, Klf4, Oct4 and Sox2 (MKOS) and showed
increased reprogramming efficiency of fresh mouse embryonic fibroblasts
(MEFs) into iPSCs under low, but not under high, MKOS expression levels.
In contrast, MEFs after 2 passages derived into a similar number of iPSC
colonies as fresh MEFs at a high MKOS dose, but this number was reduced
at a low MKOS dose. Timing of reprogramming was not affected by MKOS
expression levels but, importantly, exogenous MKOS expression in
established PSCs caused a significant cell loss. At high but not at low
MKOS expression levels, MEFs of the CD1 strain produced more initial
cell clusters than iPSCs and, although reprogrammed at a similar
efficiency as MEFs of the 129/Sv strain, iPSCs could not be maintained
in the absence of exogenous MKOS. In CD1-iPSCs, Oct4, Nanog, Rex1 and
Esrrb expression levels were reduced when compared with the levels in
PSCs derived from the 129/Sv strain. Culture of CD1-iPSCs in medium with
MEK and GSK3b inhibitors allowed their self-renewal in the absence of
exogenous MKOS, but the expression levels of Oct4, Nanog, Rex1 and Esrrb
were only partially increased. Despite the reduced levels of those
pluripotency factors, CD1-iPSC kept high capacity for contribution to
chimeric mouse embryos.Therefore, levels of regulatory pluripotency
factors influence reprogramming initiation and PSC maintenance in vitro
without affecting their differentiation potential in vivo.
Filiaciones:
Covarrubias, Luis:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Genet Desarrollo & Fisiol Mol, Cuernavaca, Morelos, Mexico
Martinez-Sarmiento, Jose-Angel:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Genet Desarrollo & Fisiol Mol, Cuernavaca, Morelos, Mexico
Valencia, Concepcion:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Genet Desarrollo & Fisiol Mol, Cuernavaca, Morelos, Mexico
Nagy, Andras:
Mt Sinai Hosp, Lunenfeld Tanenbaum Res Inst, Toronto, ON, Canada
Hernandez-Garcia, David:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Genet Desarrollo & Fisiol Mol, Cuernavaca, Morelos, Mexico
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