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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, K lf4, O ct4 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 GSK3β 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 .

The levels of reprogramming factors influence the induction and maintenance of pluripotency: the case of CD1 mouse strain cells