Transcriptomic analysis of adult mouse cardiac stromal cells using single-cell qRT-PCR

Fate mapping studies have challenged the longstanding view of the adult mammalian heart as a post-mitotic organ, suggesting limited cardiomyocyte renewal. This has spurred efforts to identify cardiac progenitor cell (CPC) populations, but their contribution to cardiac regeneration has been found to be minimal compared to cardiomyocyte proliferation. Despite this, CPC transplantation has shown therapeutic potential through paracrine signalling. The identity of CPCs remains unclear due to overlapping characteristics with other cardiac stromal cell populations such as fibroblasts, mesenchymal cells, and pericytes. This study sought to optimise the isolation of CPCs by developing a cardiac collagenase-trypsin (CT) protocol, which was compared to the established method of isolating cardiosphere-derived cells (CDCs). The CT protocol resulted in a higher cell yield and reduced expansion time, with both CTs and CDCs showing superior survival potential under serum starvation compared to commercially acquired cardiac fibroblasts (CFs). Single-cell qRT-PCR analysis revealed that CTs and CDCs share a similar gene expression profile, distinct from CFs, characterised by the enrichment of cardiogenic transcription factors. Notably, CTs exhibited higher expression of Tcf21 and lower Tbx5 , suggesting an epicardial-derived fibroblast phenotype, whereas Tbx5 was enriched in CDCs and CFs. Additionally, CTs showed an enrichment of macrophage-associated genes Mrc1 and Csf1r , possibly due to the transdifferentiation of macrophages to or from a fibroblast phenotype in a subset of CTs. The study concludes that CTs represent a robust and efficient source of CPCs with therapeutic potential and offers insights into the complex identity of cardiac stromal cells.