Transcriptional profiling of endocardial and (arterial or venous-enriched) coronary vascular endothelial cells, purified from murine embryonic hearts at E17.5.

A specific tracing system, selectively labeling tomato+ intramyocardial vessels with the Nes-CreER driver, was used together with the Nes-Gfp reporter and endomucin marker to separate, from the same hearts, the three subsets of Ecs (ventricular endocardium, intramyocardial arterial-enriched, and subepicardial venous-enriched endothelial cells), which were subject to transcriptome profiling by RNASeq. 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 .