Single cell mRNA sequencing of de novo hematopoiesis from the fetal lung

Hemogenic endothelium, which gives rise to the earliest HSCs, is thought to be isolated to very early stages of development, peaking at E10.5 in mice. However, our data suggests there may be hemogenic endothelium capable of giving rise to de-novo HSPCs in the fetal lungs of mice at E17. We've adapted a platform to recapitulate the ex-vivo development of HSPCs from E17 lung endothelium. This sequencing experiment will help us to validate whether what we are seeing in culture is indeed endothelial to hematopoietic transition (EHT) and possibly identify the subset of endothelial cells that give rise to HSPCs. E17 timed pregnant Wild Type C57BL6 mice are sacrificed at E17. The fetal lungs are isolated and digested with liberase into a single cell suspension. These cells are frozen down in 10% DMSO and replated on a thin later of diluted matrigel with HSPC media (containing mTPO, IL6, SCF, FGF, VEGF, Flt3). On D3, wells are washed with PBS and replaced with fresh media. On D4, D5 and D6, suspension cells are collected prepped for 10x. Adherent layers are digested with accutase, and sorted to enrich for live-VECAD+ cells, which are then prepped for 10x.