Exit from naive pluripotency proceeds with variable latency but without asymmetric division to generate population heterogeneity

Mouse embryonic stem (ES) cells are pluripotent cell lines established from the preimplantation epiblast. ES cells undergo transition from naive to formative pluripotency in defined conditions. Here, we used the Rex1-GFPd2 (RGd2) fluorescent reporter to track ES cells traversing the cell state boundary by long-term single-cell imaging (LTSCI). We saw no reporter re-expression, indicating irreversible differentiation. We observed only symmetric divisions, consistent with a simple population dynamics model. Genealogical analysis showed that transitions were highly correlated between sister cells. The collapse of naive identity was invariably abrupt but was preceded by a variable lag period. Across the population, however, exit times varied by more than 15 h. The delay in entering transition extended up to three generations. Thus, ES cell differentiation timing is not directly determined by cell cycle. We speculate that asynchronous departure from the naive state may safeguard pluripotency progression in the embryo founder population.