Bistable Otx2-Id1 circuitry governs a developmental transition state in the pluripotency continuum

While current pluripotency models capture discrete embryonic stages, they inadequately resolve transitional states during peri-implantation development. Here, we establish rosette-formative intermediate stem cells (rfISCs) from mouse embryonic stem cells using the MEK Inhibitor PD0325901, the Wnt Inhibitor IWR1, and the PKA Activator Forskolin. These cells exhibit transcriptomic/epigenetic profiles mirroring those of E5.0‒5.5 epiblasts, bridging rosette-stage and formative pluripotency. rfISCs demonstrate developmental bipotency, retaining in vitro germline differentiation capacity while generating germline-competent chimeras in vivo. Mechanistically, we identified opposing signaling axes that govern rfISC identification through the regulation of lineage priming: IWR1 stabilizes Tcf7l1 to drive Otx2-mediated rfISC specification and neural priming, whereas Forskolin activates PKA to induce Id1-dependent neural suppression. This creates a bistable regulatory circuit in which Otx2/Id1 synergy maintains pluripotency plasticity under MEK inhibition. Notably, rfISCs can be directly derived from E5.25 epiblasts, confirming their physiological relevance. Our work bridges a fundamental gap in pluripotency modeling by capturing the RSC-to-FSC transition through dynamic signaling equilibria.