Across mammals, the hypoblast derived Anterior-Visceral Endoderm (AVE) plays a conserved role in anterior-posterior body axis formation by defining the site of gastrulation initiation. In humans, research on 7 to 14 days post-fertilization human embryos remains challenging due to legal and ethical constraints, making the study of symmetry breaking mechanisms difficult. Moverover the study of in vitro prolonged cultured embryos remains observative making it hard to investigate the molecular mechanism of AVE differentiation. Stem cell models can be used to overcome this limitation, protocols to differentiate naive pluripotent stem cells to hypoblast-like cells have been published (Dattani et al. 2024). However, the current protocol simultaneously induces stem-cells derived Extraembryonic Mesoderm Cells (EXMCs), resulting in inefficient hypoblast induction. Our goal is to reveal the molecular mechanisms of human AVE differentiation, and its symmetry breaking induction by developing a new stem cells based approach

To have a higher hypoblast-like cell induction, we performed a screening of media components with quantification of the contribution of Hypoblast-like (GATA4), Trophectoderm-like (GATA3) and EXMC-like (VIM) cells using FACS. Next, we selected new marker genes to track cell fate progression of hypoblast to AVE by using human embryo scRNA-seq. To validate the potential markers, we performed IF staining on extended in vitro cultured human embryos. Using defined markers, we performed a screening of culture conditions to induce the differentiation of hypoblast-like cells to AVE along with FACS

First, we were able to successfully enhance GATA4+ differentiation up to 40% by adding FBS in the first 24H of differentiation. Next, scRNA-seq results suggested multiple markers known in mice. Among them, we selected multiple potential markers to track pre- to post implantation AVE transition and were able to confirm their expression at the protein level. We found that only 24H Activin A treatment induces 2D differentiation toward early-AVE based on the expression of AVE markers detected by FACS. Interestingly this AVE differentiation is blocked by adding MEK inhibitor, suggesting the role of FGF signaling in AVE differentiation

In this study, we identified NODAL and MEK as crucial pathways for the differentiation of early-AVE. This finding opened up the possibility to develop a new model for AVE patterning and investigate the spatiotemporal induction of human AVE