Principles of Signalling Pathway Modulation for Enhancing Human Naïve Pluripotency Induction [ChIP-seq]

Isolating human MEK/ERK signalling independent pluripotent stem cells (PSCs) with characteristics of naive pluripotency while maintaining differentiation competence and genetic integrity, remains challenging. Here we engineer reporter systems that allow screening for conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT-ßCATENIN, PKC and SRC signalling enables induction of teratoma competent naïve human PSCs, with capacity to differentiate into trophoblast stem cells (TSC) and extraembryonic endodermal (XEN) cells in vitro. Divergent signalling and transcriptional requirements for boosting naïve pluripotency were found between mouse and human. P53 depletion in human naïve PSCs endows them with competitiveness to better contribute to mouse-human cross-species chimeric embryos upon priming. Finally, MEK/ERK inhibition can be substituted with inhibition for NOTCH/RBPj, which allows inducing alternative human naïve-like PSCs with diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signalling foundations of human naïve pluripotency. ChIP-seq of the following proteins: H3K27ac, KLF17, KLF4, NANOG, OCT4, SOX2 and TFAP2C, measured in human pluropotent cells (PSCs) maintained in naïve conditions (Enhanced-NHSM), or in conventional/primed human PSCs, Measured on various cell lines.