Intramyocardial Sprouting Tip Cells Specify Coronary Arterialization

The elaborate patterning of coronary arteries critically supports the high metabolic activity of the beating heart. How coronary endothelial cells coordinate hierarchical vascular remodeling and achieve arteriovenous specification remains largely unknown. Understanding the molecular and cellular cues that pattern coronary arteries is crucial to develop innovative therapeutic strategies that restore functional perfusion within the ischemic heart. Single-cell transcriptomics were used to delineate heterogeneous transcriptional states of the developing and mature coronary endothelium with a focus on sprouting endothelium and arterial cell specification. We discovered that a tip-cell-to-artery specification mechanism drives arterialization of the intramyocardial plexus and endocardial tunnels throughout life. We also identified non-overlapping intramyocardial and subepicardial tip cell populations with differential gene expression profiles and regulatory pathways, suggesting that differential sprouting programs govern the formation and specification of the venous and arterial coronary plexus. Overall design: CD31+/CD45- cardiac endothelial fractions were isolated by Fluorescent-Activated Sorting (FACS) of C57Bl6J mouse hearts at different stages. After completion of sorting, 10% DMSO was added and cell suspension frozen at -80ºC. Several rounds of single-cell suspension preparation, sorting and freezing was repeated for embryonic samples in order to get a significant number of cells: 8 weeks old adult animals (n=6), P2 postnatal (n=21), E15 (n=48) and E12 (n=37). Frozen vials of sorted CD31+/CD45- cells from E12, E15, P2 and 8wo adult hearts were thawed in a water bath at 37ºC. For E12 and E15, a dead cell removal kit (Miltenyi) was applied. For P2 and adult, re-sorting by flow cytometry and selection of live cells was performed. Then, cell suspension loaded on the Chromium controller (10X Genomics) with a targeted cell recovery of 8000-10000 per reaction. 3' gene expression libraries were prepared according to the manufacturer's instructions of the v2 Chromium Single Cell Reagent Kits (10X Genomics).