BS28 Origin of human coronary vasculature: differentiating endocardial cells from human embryonic stem cells (hESCs)

Background Understanding human coronary network development is crucial yet limited. In murine models, coronary endothelial cells (cECs) predominantly originate from the endocardium, a unique multipotent progenitor source with defined markers, offering potential therapeutic targets. We aim to establish a differentiation protocol for hESC-endocardium to study cEC specification, enhancing our understanding of cEC development and improving cardiac revascularization and coronary disease treatments. Methods We adapted a cardiomyocyte-based hESC differentiation protocol, adding vascular endothelial growth factor-A (VEGF-A) to induce an endocardial fate. hESC-Endocardium was isolated using magnetic activated cell sorting (MACS) with CD31 and CD144 antibodies. Time course RT-qPCR, flow cytometry, and immunostaining validated endocardial identity. Results Time course RT-qPCR showed cells transitioning through an early heart field progenitor state, with NPR3 peaking at day 7. Post-sorting, hESC-Endocardium exhibited enrichment for endocardial markers NPR3, NFATC1, and MEIS2. Flow cytometry and immunostaining confirmed CD31-positive cells coexpressing endocardial-specific markers like NPR3, ADGR6, and TEMEM100, thus validating their identity. Functionally, VEGF-A-exposed hESC-Endocardium formed tube networks in vitro. Conclusion We successfully established a protocol to differentiate and enrich hESC-Endocardium, with day 7 selected as the optimal sorting time. These cells expressed endocardial marker NPR3 and differentiated into endothelial cells capable of forming tube networks, resembling a primitive coronary plexus. Conflict of Interest None