1) High-Resolution Dissection of the Trajectory Underlying Successful Somatic Reprogramming toward Naïve Ground-State Pluripotency
The ability to reprogram somatic cells into iPSCs with the four Yamanaka factors has provoked immense scientific interest over the past decade. Different studies sought to define inhibitors and facilitators of this process, as well as map the molecular changes accompanying cells undergoing reprogramming. However, our group decided to take a different approach to this challenge, focusing on characterizing the molecular synthesis processes underlying the successful conversion of somatic cells into iPSCs while simultaneously attempting to overlook the “noise” from populations that fail to complete the process. This gap in understanding results from the inefficiency of conventional reprogramming methods and the difficulty of prospectively isolating the rare cells that eventually correctly reprogram into iPSCs. The near-deterministic reprogramming strategies recently developed by our group (Rais et al. Nature 2013) and others open the way to the dissection of molecular events accompanying a synchronized and non-saltatory progression toward iPSCs, thereby providing an opportunity to reveal the critical molecular details needed to decipher reprogramming dynamics without the need for cell sorting or repeated passaging, which greatly bias results.
2) 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. 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.
3) SUMOylation of linker histone H1 drives chromatin condensation and restriction of embryonic cell fate identity. The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity.
