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

Abstract

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

KLF17 promotes human naïve pluripotency but is not required for its establishment

“…Overall, our overexpression studies show that KLF17 may typically have a role in regulating naïve pluripotency in vitro. However, it is clear from our data that KLF17 expression is not necessary for establishing naïve hESCs, although our current work does not exclude the possibility that established naïve hESCs are sensitive to loss of KLF17, as recently suggested (Bayerl et al, 2020). We theorise that in a wild-type situation KLF17 may act as a peripheral pluripotency factor in human naïve pluripotency, acting alongside a core pluripotency network of OCT4 and SOX2 to maintain robustness of the pluripotent state and help to limit premature differentiation.…”

Human and Pig Pluripotent Stem Cells: From Cellular Products to Organogenesis and Beyond

“…Therefore, most human PSC chimeras have been tested using naïve or intermediate human cells, which exhibit higher efficiency [ 14 ]. However, large-scale analysis has revealed that naïve human PSCs are genome-wide hypomethylated [ 8 , 56 ], and their imprinting pattern is abnormal [ 57 ]. This loss of imprinting may prove to be carcinogenic and problematic for clinical applications.…”

“…Generally, PSCs can be categorized into primed and naïve PSC states, corresponding to the post-implantation and preimplantation epiblasts. In recent years, other new types of PSCs have been reported, such as expanded or extended potential stem cells (EPSCs) [ 3 , 4 , 5 , 6 , 7 , 8 ]. Generally, these PSCs are maintained in a medium cocktail modulating the developmentally important signaling pathways to minimize downstream differentiation but can generate various cell types when re-introduced into preimplantation embryos (in the case of mouse embryonic stem cells), or by directed or random differentiation in vitro.…”

Modeling human embryo development with embryonic and extra-embryonic stem cells

“…Specifically, two types of naïve cells showed the most similarity to the human pre-implantation epiblast both transcriptionally and epigenetically: 5i/L/A(F) 27 cells and Reset (t2iLGö/PXGL) cells 28,31,33,37 . Naïve cells vary in their capacity to respond to germ layer differentiation cues: Reset (t2iLGö/PXGL) cells require "capacitation" while other cells, such as the recently reported "HENSM" cells, do not 38,39 . The distinctions between different "naïve" hESCs raise questions about the nature of human naïve pluripotency, and how well these different protocols generate cells that recapitulate the pluripotency state of the pre-implantation epiblast.…”