Capture of germ-line competent stem cells between rosette-stage and formative pluripotent states

The establishment of stem cells with varying pluripotency potentials is crucial for understanding the developmental progression of pre- and post-implantation embryos. Despite the advancement through the proposal of the formative pluripotent state, the model of pluripotent development remains incomplete. Here, we derived a novel type of pluripotent stem cells from mouse embryonic stem cells and sustained them in vitro through the application of MEK inhibitor PD0325901, Tankyrase inhibitor IWR1, and adenylate cyclase activator Forskolin. These cells exhibit distinct transcriptome and epigenetic profiles, resembling the epiblasts of E5.0-E5.5, and reside between rosette-like stem cells and formative stem cells. We refer to them as rfISCs (Intermediate stem cells between RSCs and FSCs). rfISCs can differentiate into germ cells in vitro and form blastocyst chimeras in vivo. Additionally, the formation of rfISCs is governed by distinct signaling pathways and transcription factors. Specifically, IWR1-mediated inhibition of Wnt signaling enhances Tcf7l1-mediated Otx2 expression, essential for rfISCs generation yet prone to inducing neural differentiation, whereas Forskolin induces Id1 transcription, suppressing neural lineage gene expression via the PKA pathway. Hence, co-overexpression of Id1 and Otx2 is sufficient to sustain rfISCs stability. The emergence of rfISCs aids in comprehending early embryo development.