Tuning of ß-catenin activity is required to stabilize self-renewal of rat embryonic stem cells.

Stabilization of ß-catenin, through inhibition of glycogen synthase kinase 3 (GSK3) activity, in conjunction with inhibition of mitogen-activated protein kinase kinase 1/2 (MEK) promotes self-renewal of naïve-type mouse embryonic stem cells (ESC). In developmentally more advanced, primed-type, epiblast stem cells, however, ß-catenin activity induces differentiation. We investigated the response of rat ESCs to ß-catenin signaling and found that when maintained on feeder-support cells in the presence of a MEK inhibitor alone (1i culture), the derivation efficiency, growth, karyotypic stability, transcriptional profile, and differentiation potential of rat ESC cultures was similar to that of cell lines established using both MEK and GSK3 inhibitors (2i culture). Equivalent mouse ESCs, by comparison, differentiated in identical 1i conditions, consistent with insufficient ß-catenin activity. This interspecies difference in reliance on GSK3 inhibition corresponded with higher overall levels of ß-catenin activity in rat ESCs. Indeed, rat ESCs displayed widespread expression of the mesendoderm-associated ß-catenin targets, Brachyury and Cdx2 in 2i medium, and overt differentiation upon further increases in ß-catenin activity. In contrast, mouse ESCs were resistant to differentiation at similarly elevated doses of GSK3 inhibitor. Interestingly, without feeder support, moderate levels of GSK3 inhibition were necessary to support effective growth of rat ESC, confirming the conserved role for ß-catenin in ESC self-renewal. This work identifies ß-catenin signaling as a molecular rheostat in rat ESC, regulating self-renewal in a dose-dependent manner, and highlights the potential importance of controlling flux in this signaling pathway to achieve effective stabilization of naïve pluripotency.