Retinoic acid induces NELFA‐mediated 2C‐like state of mouse embryonic stem cells associates with epigenetic modifications and metabolic processes in chemically defined media

OBJECTIVES: Mouse embryonic stem cells (ESCs) are derived from the inner cell mass of blastocyst‐stage embryos and cultured in different culture media with varied pluripotency. Sporadically, a small population of ESCs exhibit 2‐cell stage embryonic features in serum containing medium. However, whether ESCs can transit into 2‐cell embryo‐like (2C‐like) cells in the chemically defined media remains largely unknown. MATERIALS AND METHODS: We established a robust in vitro induction system, based on retinoic acid (RA) containing chemically defined media, which can efficiently increase the subpopulation of 2C‐like cells. Further test the pluripotency and 2C features of ESCs cultured in RA. 2C reporter‐positive cells were selected by FACS; the level of protein was detected via immunofluorescence staining and western blot; the level gene expressions were measured by RNA‐seq. RESULTS: Retinoic acid drives a NELFA (negative elongation factor A)‐mediated 2C‐like state in mouse ESCs, characterized with 2C‐specific transcriptional networks and the ability to contribute trophectoderm (TE) when injected into developing embryos. In addition, RA treatment triggers DNA hypomethylation, active histone modification, suppressed glycolysis metabolism and reduced protein synthesis activity of ESCs. CONCLUSIONS: We showed that RA has a broader role in 2C‐like cells state, not only is one of the upstream regulators of the 2C‐like state in chemically defined media but also illuminates genetic and epigenetic regulations that govern ESCs to 2C‐like transition.