Influence of JAK/STAT Signaling and extracellular matrix on Chemical Resetting of human Pluripotent Stem cells

Two fundamental features characterise human pluripotent stem cells (hPSCs): they can differentiate into any adult cell type and self-renew indefinitely in vitro. In particular, two different states of human pluripotency are known: primed pluripotency, whose characteristics resemble those of the post-implantation epiblast, and naive pluripotency, resembling the pre-implantation pluripotent cells of the embryo, which retain a broader differentiation potential, as they also contribute to extraembryonic tissues like the placenta. In naive mouse stem cells, the JAK/STAT3 key signalling pathway is required to maintain pluripotency through gene expression and metabolic regulation, whereas its role in hPSCs remains poorly characterised. This work aims to elucidate the role of the JAK/STAT3 pathway in the maintenance and induction of human naive pluripotency. The latter is achieved in vitro by a chemical resetting process using a histone deacetylase inhibitor together with LIF, a JAK/STAT3 activator, which reverts primed hPSCs to the naive state. The role of the pathway was assessed by morphological and proliferation analyses, as well as by measuring gene expression and protein levels, both in the presence of the activators LIF and IL6 or upon blockade of the pathway using either a chemical inhibitor or a CRISPR interference genetic approach. From my findings, the JAK/STAT3 pathway seems to be involved in both the maintenance and acquisition of human naive pluripotency, impacting cell fate and proliferation. On the contrary, the overstimulation of the pathway mainly exerts its action on proliferation during the acquisition of naive pluripotency. Finally, naive hPSCs are commonly cultured and reset on a feeder layer of inactivated fibroblasts, whose presence might confound JAK/STAT3 pathway investigation. Therefore, I set up a feeder-free protocol for chemical resetting, which produced bona fide naive hPSCs and will be exploited in future studies.