Resistance to naive and formative pluripotency conversion in RSeT human embryonic stem cells

One of the most important properties of human embryonic stem cells (hESCs) is their ability to exist in primed and naive pluripotent states. Our previous meta-analysis indicated the existence of heterogeneous pluripotent states derived from diverse naive protocols. In this study, we characterized a commercial, RSeT-based pluripotent state under various growth conditions. Notably, RSeT hESCs can circumvent the hypoxic growth conditions required by naive hESCs, although some RSeT cells (eg, H1 cells) exhibit much lower single-cell plating efficiency and display altered or significantly retarded cell growth under both normoxia and hypoxia. Importantly, RSeT hPSCs lack many transcriptomic hallmarks of naive and formative pluripotency (the phase between naive and primed states). Integrative transcriptome analysis suggests that our primed and RSeT hESCs are similar to the early stage of post-implantation embryos, in line with previously reported primary hESCs and early hESC cultures. Moreover, RSeT hESCs do not express naive surface markers such as SUSD2 and CD75 at significant levels. At the biochemical level, RSeT hESCs show differential dependence on FGF2 and co-independency on both Janus kinase (JAK) and TGFβ signaling in a cell line-specific manner. Thus, RSeT hESCs represent a previously unrecognized pluripotent state downstream of naive pluripotency. Our data suggest that human naive pluripotent potentials may be restricted in RSeT medium, which sustains FGF2 activity. Hence, this study provides new insights into pluripotent state transitions in vitro.