Principles of signaling pathway modulation for enhancing human naive pluripotency induction

Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/beta-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro. Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.

Automated summary

Isolating human MEK/ERK signaling-independent pluripotent stem cells with naive pluripotency characteristics remains challenging, but synergistic inhibition of WNT/beta-CATENIN, PKC, and SRC signaling can induce teratoma-competent naive human PSCs. The study found divergent signaling and transcriptional requirements for boosting naive pluripotency between mice and humans, and P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos. Also, MEK/ERK inhibition can be substituted with NOTCH/RBPj inhibition to induce alternative naive-like hPSCs with diminished risk for deleterious global DNA hypomethylation.