PRC2 shields naïve human pluripotent cells from trophectoderm and mesoderm differentiation [scRNA-seq]

Single-cell RNA-seq reveals that Polycomb repressive complex 2 (PRC2) maintains naïve pluripotency and restricts an intrinsic capacity of pre-implantation pluripotent stem cells to give rise to trophectoderm and mesoderm lineages. Inhibition of PRC2 forces naïve hESC into an 'activated' state through which differentiation into either trophectoderm or mesoderm lineages is triggered. This trajectory is distinct from embryonic lineage specification out of the post-implantation pluripotent state, hence PRC2-mediated repression provides a highly adaptive mechanism to restrict lineage potential during early human development. Overall design: Primed and naïve cells were grown in respective media, supplemented with 10µM EZSolution™ EPZ-6438 (BioVision; 2428-5) for seven days, with daily media changes. Samples EZH2i d2, d4 and untreated control naïve as well as EZH2i d7 and untreated control primed cells were multiplexed following the manufacturer's protocol. In brief, cells were dissociated, treated with mouse feeder removal beads (naïve cells), counted and resuspended in Cell Multiplexing Oligo. After oligo incubation, cells were washed for a total of three times using DPBS-/- containing 1% BSA (Sigma Aldrich; A7284) and counted using the NucleoCounter® NC-200™ automated cell counter. At this point, cells were pooled accordingly: EZH2i d2, d4 and WT naïve cells, and EZH2i d7 and WT primed cells, respectively, and each pool was counted once more.