Tripartite Inhibition of SRC-WNT-PKC Signalling Consolidates Human Naïve Pluripotency

Different conditions have been devised to isolate MEK/ERK signalling independent human naïve pluripotent stem cells (PSCs) that are distinct from conventional primed PSCs and better correspond to pre-implantation developmental stages. While the naïve conditions described thus far endow human PSCs with different extents of naivety features, isolating human pluripotent cells that retain characteristics of ground state pluripotency while maintaining differentiation potential and genetic integrity, remains a major challenge. Here we engineer reporter systems that allow functional screening for conditions that can endow both the molecular and functional features expected from human naive pluripotency. We establish that simultaneous inhibition of SRC-NFκB, WNT/ßCATENIN and PKC signalling pathways is essential for enabling expansion of teratoma competent fully naïve human PSCs in defined or xeno-free conditions. Divergent signalling and transcriptional requirements for maintaining naïve pluripotency were found between mouse and human. Finally, we establish alternative naïve conditions in which MEK/ERK inhibition is substituted with inhibition for NOTCH/RBPj signalling, which allow obtaining alternative human naïve PSCs with diminished risk for loss of imprinting and deleterious global DNA hypomethylation. Our findings set a framework for the signalling foundations of human naïve pluripotency and may advance its utilization in future translational applications. Highlights of key findings Combined inhibition of SRC, WNT and PKC signaling consolidates human naïve pluripotency Stable expansion of DNA/RNA methylation-independent and TGF/ACTIVIN-independent human naïve PSCs Opposing roles for ACTIVIN and WNT/ßCATENIN signaling on mouse vs. human naive pluripotency 2i and MEK/ERKi independent alternative human naïve PSC conditions via inhibiting NOTCH/RBPj signaling