In the adult heart, epicardial stromal cells (EpiSC) are induced by myocardial infarction (MI) and are considered both as endogenous progenitor cells and source for paracrine factors involved in cardiac wound healing. EpiSC are believed to be a heterogeneous cell fraction, but details are unknown. We have taken advantage of a novel technique recently reported by us (Owenier et al. 2019, Cardiovasc Res, 116: 1047–1058) for the simultaneous isolation of EpiSC and cardiac fibroblasts (activated cardiac stromal cells, aCSC) from the infarcted mouse heart to explore the cellular composition and hierarchy of EpiSC in comparison to aCSC by single-cell RNA sequencing (scRNAseq).

At day 5 after myocardial ischemia/reperfusion, scRNAseq of EpiSC and aCSC fractions from three mouse hearts was carried out using the 10x Genomics Chromium platform. Transcriptional profiles of 13,796 EpiSC and 24,470 aCSC were captured after quality control filtering. Unbiased clustering using the Seurat R package with visualization in UMAP dimension plots identified 11 molecularly distinct EpiSC populations. Spatial distribution of key populations within the post-MI epicardium was identified by RNA in situ hybridization. RNA velocity analysis, combined with lineage tracing using Wt1-CreERT2 mice, showed that EpiSC can be grouped into three main clusters, each with its own proliferation center. EpiSC populations substantially differed in expression of epicardial progenitor markers, cardiogenic and hypoxia-responsive genes. Both EpiSC and aCSC showed a distinct and diverse expression of pro-regenerative paracrine factors.

Our results demonstrate that the post-MI epicardium constitutes a highly heterogeneous cell compartment. Major epicardial functions can now be attributed to specific cell populations, which may allow for novel treatment options to improve cardiac repair.