Chemical conversion of human conventional PSCs to TSCs following transient naive gene activation.

In human embryos, naive pluripotent cells of the inner cell mass (ICM) generate epiblast, primitive endoderm and trophectoderm (TE) lineages, whence trophoblast cells derive. In vitro, naive pluripotent stem cells (PSCs) retain this potential and efficiently generate trophoblast stem cells (TSCs), while conventional PSCs form TSCs at low efficiency. Transient histone deacetylase and MEK inhibition combined with LIF stimulation is used to chemically reset conventional to naive PSCs. Here, we report that chemical resetting induces the expression of both naive and TSC markers and of placental imprinted genes. A modified chemical resetting protocol allows for the fast and efficient conversion of conventional PSCs into TSCs, entailing shutdown of pluripotency genes and full activation of the trophoblast master regulators, without induction of amnion markers. Chemical resetting generates a plastic intermediate state, characterised by co-expression of naive and TSC markers, after which cells steer towards one of the two fates in response to the signalling environment. The efficiency and rapidity of our system will be useful to study cell fate transitions and to generate models of placental disorders.

STAT3 and HIF1α cooperatively mediate the transcriptional and physiological responses to hypoxia.

STAT3 and HIF1α are two fundamental transcription factors involved in many merging processes, like angiogenesis, metabolism, and cell differentiation. Notably, under pathological conditions, the two factors have been shown to interact genetically, but both the molecular mechanisms underlying such interactions and their relevance under physiological conditions remain unclear. In mouse embryonic stem cells (ESCs) we manage to determine the specific subset of hypoxia-induced genes that need STAT3 to be properly transcribed and, among them, fundamental genes like Vegfa, Hk1, Hk2, Pfkp and Hilpda are worth mentioning. Unexpectedly, we also demonstrated that the absence of STAT3 does not affect the expression of Hif1α mRNA nor the stabilization of HIF1α protein, but the STAT3-driven regulation of the hypoxia-dependent subset of gene could rely on the physical interaction between STAT3 and HIF1α. To further elucidate the physiological roles of this STAT3 non-canonical nuclear activity, we used a CRISPR/Cas9 zebrafish stat3 knock-out line. Notably, hypoxia-related fluorescence of the hypoxia zebrafish reporter line (HRE:mCherry) cannot be induced when Stat3 is not active and, while Stat3 Y705 phosphorylation seems to have a pivotal role in this process, S727 does not affect the Stat3-dependent hypoxia response. Hypoxia is fundamental for vascularization, angiogenesis and immune cells mobilization; all processes that, surprisingly, cannot be induced by low oxygen levels when Stat3 is genetically ablated. All in all, here we report the specific STAT3/HIF1α-dependent subset of genes in vitro and, for the first time with an in vivo model, we determined some of the physiological roles of STAT3-hypoxia crosstalk.