Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells

Abstract

Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential. Furthermore, we find that female ES cells cultured in conventional serum plus LIF medium phenocopy male ES cells cultured in 2i/L. Mechanistically, we demonstrate that the inhibition of Mek1/2 is predominantly responsible for these effects, in part through the downregulation of DNA methyltransferases and their cofactors. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as the developmental potential of ES cells. Taken together, our data suggest that, although short-term suppression of Mek1/2 in ES cells helps to maintain an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential

Naive Pluripotent Stem Cells as a Model for Studying Human Developmental Epigenomics: Opportunities and Limitations

“…The difference in DNA methylation stability between hESCs and the embryo might not be restricted to human cells, as two recent reports showed that naive mouse ESCs lose their imprints when cultured in media containing MEK1/2 inhibitor [20,21]. These studies contrast with several previous reports that demonstrated imprint stability, and the reasons for the discrepant findings are not obvious.…”

“…These studies contrast with several previous reports that demonstrated imprint stability, and the reasons for the discrepant findings are not obvious. Loss of imprints in mouse ESCs might be associated with defective developmental potential [20,21], although this needs to be examined more systematically and also in the context of naive hESC differentiation.…”

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Naïve-like State with Improved Multilineage Differentiation Potency

“…Similar epigenomic aberrations, especially at imprinted loci, have been detected in mouse ESC following prolonged exposure to the LIF-2i cocktail 14 Interestingly, some reversion culture systems have reported global improvements in specific attributes of functional pluripotency of PSC such as the enhanced capacity for in vivo trophectoderm contribution 2,15 .…”

Epigenetic foundations of pluripotent stem cells that recapitulate in vivo pluripotency

“…However, it has not been fully elucidated whether genetic and epigenetic stabilities of 2i-treated ES cells are stable. Recent studies demonstrated that derivation of ES cells directly from blastocysts in 2i/LIF and prolonged culture of ES cells in 2i/LIF result in loss of DNA methylation at imprinted loci 74,75 (Figure 4). Surprisingly, such imprint eroded ES cells compromise autonomous developmental potential by tetraploid embryo complementation and nuclear transfer 74,75 (Figure 4).…”

“…Mechanistically, the inhibition of MEK1/2 is responsible for these opposing effects. In addition, replacement of the MEK1/2 inhibitor with an Src inhibitor (a2i includes Src inhibitor; CGP77675 and CHIR99021) preserves genetic and epigenetic stability as well as autonomous developmental potential 74,75 (Figure 4). Given that many laboratories have implemented 2i culture condition as standard practice since the discovery of 2i, these findings should be taken into account for future experiments using 2i-treated ES cells.…”