In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states (II, single-cell RNA-Seq data)

Naive pluripotent cells in the implanting mouse blastocyst generate a rosette structure before undergoing lumenogenesis to form the egg cylinder. Simultaneously, they acquire primed pluripotency, the ability to differentiate into the primary germ layers. The existence of discrete intermediate pluripotent states during this transition has not been demonstrated. We identify here a distinct rosette pluripotent state, defined by co-expression of naive factors with transcription factor OTX2. Downregulation of WNT signals in the blastocyst drives transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells (RSCs), a self-renewing naive-primed intermediate. RSCs gain a unique epigenome that includes erasure of constitutive heterochromatin and bivalent marking of primed pluripotency genes. Notwithstanding this primed chromatin landscape, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate where control over pluripotency progression and morphogenesis pivots from WNT to MEK signals. Overall design: Mouse embryonic stem cells were differentiated towards rosette- and primed pluripotency and sorted into 384-well plates. Afterwards, single-cell transcriptome profiles were obtained using SORT-seq (Muraro, M.J. et al. A Single-Cell Transcriptome Atlas of the Human Pancreas. Cell Syst 3, 385-394 e383 (2016)).