17 Different conditions have been devised to isolate MEK/ERK signalling independent human 18 naïve pluripotent stem cells (PSCs) that are distinct from conventional primed PSCs and better 19 correspond to pre-implantation developmental stages. While the naïve conditions described thus 20 far endow human PSCs with different extents of naivety features, isolating human pluripotent cells 21 that retain characteristics of ground state pluripotency while maintaining differentiation potential 22 and genetic integrity, remains a major challenge. Here we engineer reporter systems that allow 23 functional screening for conditions that can endow both the molecular and functional features 24 expected from human naive pluripotency. We establish that simultaneous inhibition of SRC-NFκB, 25 WNT/ßCATENIN and PKC signalling pathways is essential for enabling expansion of teratoma 26 competent fully naïve human PSCs in defined or xeno-free conditions. Divergent signalling and 27 transcriptional requirements for maintaining naïve pluripotency were found between mouse and 28 human. Finally, we establish alternative naïve conditions in which MEK/ERK inhibition is 29 substituted with inhibition for NOTCH/RBPj signalling, which allow obtaining alternative human 30 naïve PSCs with diminished risk for loss of imprinting and deleterious global DNA 31 hypomethylation. Our findings set a framework for the signalling foundations of human naïve 32 pluripotency and may advance its utilization in future translational applications.33 34 35 36 37 38 39 40 Highlights of key findings: 1 2 • Combined inhibition of SRC, WNT and PKC signaling consolidates human naïve pluripotency 3 • Stable expansion of DNA/RNA methylation-independent and TGF/ACTIVIN-independent 4 human naïve PSCs 5 • Opposing roles for ACTIVIN and WNT/ßCATENIN signaling on mouse vs. human naive 6 pluripotency 7 • 2i and MEK/ERKi independent alternative human naïve PSC conditions via inhibiting 8 NOTCH/RBPj signaling
34The Nucleosome Remodeling and Deacytelase (NuRD) complex is a co-repressive 35 complex involved in many pathological and physiological processes in the cell. Previous studies 36 have identified one of its components, Mbd3, as a potent inhibitor for reprogramming of somatic 37 cells to pluripotency. Following OSKM induction, early and partial depletion of Mbd3 protein 38 followed by applying naïve ground-state pluripotency conditions, results in a highly efficient and 39 near-deterministic generation of mouse iPS cells. Increasing evidence indicates that the NuRD 40 complex assumes multiple mutually exclusive protein complexes, and it remains unclear whether 41 the deterministic iPSC phenotype is the result of a specific NuRD sub complex. Since complete 42 ablation of Mbd3 blocks somatic cell proliferation, here we aimed to identify alternative ways to 43 block Mbd3-dependent NuRD activity by identifying additional functionally relevant components 44 of the Mbd3/NuRD complex during early stages of reprogramming. We identified Gatad2a (also 45 known as P66α), a relatively uncharacterized NuRD-specific subunit, whose complete deletion 46does not impact somatic cell proliferation, yet specifically disrupts Mbd3/NuRD repressive activity 47 on the pluripotency circuit during both stem cell differentiation and reprogramming to pluripotency.
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