Elucidating the Genetic Determinants for Exit from the Naïve State of Pluripotency

Progression through states of pluripotency is required for cells in early mammalian embryos to transition away from heightened self-renewal and toward competency for lineage specification. This transition is referred to as the exit from naïve pluripotency and coincides with embryo implantation, which can be studied in vitro using mouse embryonic stem cells (ESC). To identify novel genes required for exit out of naive pluripotency, we used a CRISPR knockout mutagenesis screen targeting the mouse genome with ~90k unique sgRNAs. The screen yielded 30 high confidence candidates (FDR<5%) in both expected and unexpected aspects of cell biology. Unexpectedly, we identified a role for intracellular Ca2+ homeostasis during exit out of the naïve state of pluripotency. Mutation of a plasma membrane Ca2+ pump encoded by Atp2b1 increased intracellular Ca2+ such that it overcame effects of intracellular Ca2+ reduction, which is required for naïve exit. Persistent self-renewal of ESC was supported in both Atp2b1-/- Tcf7l1-/- double knockout ESC passaged in defined media alone (no LIF or inhibitors) and in wildtype cells passaged in media containing only calcitonin and a GSK3 inhibitor. These new findings suggest a central role for intracellular Ca2+ in safeguarding naïve pluripotency, which may impact the derivation of therapeutic cellular products from pluripotent cells.