Abstract: Maintenance of a robust, naïve pluripotent state in mouse embryonic stem cells (mESCs) is typically established in 2i conditions (LIF, MEKi and GSK3i). Although GSK3i results in WNT-activation, the pathway seems to be dispensable in pluripotency. Since GSK3 can target over 40 substrates, and research primarily focused on its role in β-catenin stabilization, other GSK3-mediated functions in pluripotent mESCs are currently overlooked, as well as the individual roles of the different GSK3 isozymes (GSK3α and β). In this study, we unveil that inhibition of GSK3 induces a quiescent cellular state in mESCs in a dose-dependent manner, characterized by the absence of Ki67 (G0), increase in P21 and P27kip1, while maintaining pluripotency. Surprisingly, the expression of WNT/β-catenin target genes, c-Myc and n-Myc, are transcriptionally downregulated upon GSK3i in both WT and β-catenin-/- cells, indicating a β-catenin-independent mechanism controlling the downregulation of these genes in pluripotent mESCs. Functionally, priming mESCs with GSK3 inhibitors provides a protective state (increased healthy cell fraction) after cell death induction. The CRISPR/Cas9-mediated generation of GSK3α-/- and GSK3β-/- mESC cell lines revealed a differential role for the isozymes in terms of proliferation, while maintaining their pluripotent state. SP1 was predicted to be the crucial regulator of the majority of differentially expressen genes derived from our RNA-sequencing dataset of GSK3 inhibited mESCs. SP1-/- cells display a reduction in cell number, decreased c-myc at protein level, but no increase in cell death (Annexin V-positive cells). SP1-/- cells were not able to further reduce cell number in response to GSK3i. Future experiments will further elucidate the role of GSK3i in WT mESCs (proteomics), in mouse embryos (CRISPR/Cas9 KO SP1) and in human blastoids. Our results propose a new molecular mechanism for the induction of pluripotent quiescence through the inhibition of GSK3 in a β-catenin-independent manner, possibly via SP1.
