A single-cell comparison of adult and foetal human epicardium defines the age-associated changes in epicardial activity

Re-activating quiescent adult epicardium represents a potential therapeutic approach for human cardiac regeneration. However, the exact molecular differences between inactive adult and active foetal epicardium are not known. Here, we combined foetal and adult human hearts for the first time using single-cell and single-nuclei RNA sequencing, and compared epicardial cells from both stages. We found a migratory fibroblast-like epicardial population only in the foetal heart and foetal epicardium expressed angiogenic gene programs, while the adult epicardium was solely mesothelial and immune-responsive. Furthermore, we predicted that adult hearts may still receive foetal epicardial paracrine communication, including WNT-signalling with endocardium, reinforcing the validity of regenerative strategies that administer or reactivate epicardial cells in situ. Finally, we explained graft efficacy of our human embryonic stem-cell derived epicardium model, by noting its similarity to human foetal epicardium. Overall, our study defines epicardial programs of regenerative angiogenesis absent in adult hearts, contextualises animal studies, and defines epicardial states required for effective human heart regeneration. Seven (F1-F7) foetuses were obtained following elective termination of pregnancy with full consent and were stored overnight in Hibernate-A Medium (Gibco) at 4 °C. The next day following collection, the apex and base of each heart was dissected, and dissociated. Cells were submitted for 10x library preparation for 3’ single cell sequencing on a NovaSeq 6000 (Illumina) using V3 chemistry at the Cancer Research UK (CRUK) Cambridge Institute. One sample F5 was collected as a pilot sample independently and prepared separately from the other foetal samples and only the apex attached to a careful epicardial peeling was taken and dissociated. Sample F5 was sequenced independently at the Sanger Institute using HiSeq 4000 (Illumina).