Transcriptional profiling of purified endocardial and coronary vascular endothelial cells, isolated from murine embryonic hearts, at two developmental stages

Given that the Nes-gfp allele specifically labels coronary ECs, endogenous GFP and the endomucin marker (which was highly expressed in endocardial cells) were used together to separate endocardial and coronary vascular endothelial cell subpopulations in different developmental stages Overall design: For the E13.5 developmental stage, GFP+ hearts from E13.5 Nes-gfp (Tg/0) embryos were dissected and collected. Ventricles were minced (after removing atria and aortic region) and subjected to enzymatic digestion with liberase. Single-cell suspensions were stained, and the following primary immunophenotypic populations were simultaneously FACS sorted: (1)endocardial EC-enriched population: viable (dapi-)/Ter119- CD45-/CD31+/GFP-low/Emcn-high ; (2) coronary EC-enriched population: viable (dapi-)/Ter119- CD45-/CD31+/GFP+ . For the E17.5 developmental stage, hearts from E17.5 Nes-Gfp(Tg/0); Nes-CreER(Tg/0); R26 (tom/+) embryos (0.1 ug tamoxifen given at E15,5) were dissected and collected, based on GFP+ and Tomato+ fluorescence. Ventricles were minced (after removing atria and aortic region) and subjected to enzymatic digestion with liberase (Roche). Single-cell suspensions were stained, and the following primary immunophenotypic populations were simultaneously FACS sorted: (1) endocardial EC-enriched population: viable (dapi-)/Ter119- CD45-/CD31+/GFP+ ; (2A) coronary EC-enriched in (intramyocardial) arterial vessels and capillaries: viable (dapi-)/Ter119- CD45-/CD31+/GFP+/Emcn-low/Tomato+ ; (2B) coronary EC-enriched in (subepicardial) venous vessels: viable (dapi-)/Ter119- CD45-/CD31+/GFP+/Emcn-low/Tomato-/GFP-low .