Differentiation and Characterization of Human Pluripotent Stem Cell-Derived Cardiac Endothelial Cells for In Vitro Applications

Various protocols have been developed to generate endothelial cells for disease modeling, angiogenesis, vascular regeneration, and drug screening. These protocols often require cell sorting, as most differentiation strategies result in a heterogenous population of endothelial cells (ECs). For any given model system, one important consideration is choosing the appropriate EC subtype, as different EC populations have unique molecular signatures. Herein, we describe a protocol for cardiac EC differentiation and a protocol for endothelial cell characterization. This protocol is aimed at investigating differentiation efficiency by measuring endothelial lineage markers, CD31, VE-Cadherin, and VEGFR2 by flow cytometry. Collectively, these protocols comprise the tools required to generate cardiac ECs efficiently and reproducibly from different hPSC lines without the need for cell sorting. Our protocol adds to the panel of hPSCs for cardiac EC differentiation and addresses reproducibility concerns of hPSC-based experiments. The approaches described are also applicable for complex model generation where multiple cardiovascular cell types are involved and may assist in optimizing differentiations for different cell lineages, including cardiomyocytes, cardiac endothelial cells, and cardiac fibroblasts.