Isolation of murine embryonic hemogenic endothelial cells

Abstract

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distincttissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and theembryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolationfor careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM.We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion andantibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots areshown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their bloodforming potential on a clonal level.