G2M splits mouse embryonic stem cells into naïve and formative pluripotency states

ABSTRACT Embryonic stem cells (ESCs) express heterogeneous levels of pluripotency and developmental transcription factors (TFs) and their cell cycle is unsynchronised when grown in the presence of serum. Here, we asked whether the cell cycle and developmental heterogeneities of ESCs are coordinated by determining the state identities of G1- and G2M-enriched mouse ESCs (mESCs) at single cell resolution. We found that G2M cells were not all the same and demonstrate their split into the naïve and formative (intermediate) pluripotency states marked by high or low Esrrb expression, respectively. The naïve G2M sub-state resembles ‘ground’ state pluripotency of the LIF/2i cultured mESCs. The naïve and formative G2M sub-states exist in the pre- and post-implantation stages of the mouse embryo, respectively, verifying developmental distinction. Moreover, the G2M sub-states partially match between the mouse and human ESCs, suggesting higher similarity of transcriptional control between these species in G2M. Our findings propose a model whereby G2M separates mESCs into naïve and formative pluripotency states. This concept of G2M-diverted pluripotency states provides new framework for understanding the mechanisms of pluripotency maintenance and lineage specification in vitro and in vivo, and the development of more efficient and clinically relevant reprogramming strategies.