An exploration of vascular cell development in mouse embryoid bodies under hypoxia through single cell RNA-seq

Background : Differentiation of mouse stem cells (mESC) to embryonic bodies (EBs) is a widely used approach to model early stages of development. In this study, we employed single-cell transcriptomics to examine the effect of hypoxia on the differentiation of different cell populations in EBs. Results : We differentiated EBs for 8 or 10 days, with two time frames of hypoxic or normoxic exposure: a short window of 16h for the 8-day samples, and a 48h window for the 10-day samples, totaling four experimental conditions. The EBs were composed mainly of broad mesodermal progenitors, a limited number of endodermal precursors, and smaller groups of cells that were more advanced in their differentiation pathways. These developed groups included hemoendothelial progenitors, endothelium, cardiomyocytes, and monocyte and erythrocyte precursors. Both O2 and time of differentiation significantly influenced the distribution of cell types across conditions. Our findings revealed that hypoxia induces a pervasive cell cycle arrest signature across all identified cell populations within the EB. Despite the cell cycle arrest, vascular smooth muscle (vSMCs), endothelial, and cardiac cell populations exhibit expansion under hypoxic conditions, while normoxia favours an increase in the monocyte population. Differential proliferation of hemoendothelial progenitors does not appear to be the primary driver of endothelial cells expansion in hypoxia, as their distribution remained similar across treatments and depleted over time. We observed delays in the terminal differentiation pathway of vSMCs precursors in hypoxic conditions, which may contribute to their expansion and migration by switching to a synthetic phenotype. Additionally, oxygen concentration influenced the developmental path of hemangioblasts towards either an endothelial, or myeloid lineage. In the EBs, this was coupled with a shift in presomitic mesoderm differentiation towards endotome or musculoskeletal precursors, which may provide an additional source of progenitors with endothelial potential during oxygen deprivation. Conclusion : Our study provides novel insights into the cellular responses to hypoxia in the context of early embryonic development, thus unveiling potential mechanisms underlying blood vessel growth.