# Transcriptomic Analysis of Adult Mouse Cardiac Stromal Cells Using Single-Cell qRT-PCR

## TL;DR

This study identifies a new method to isolate mouse cardiac stromal cells that are efficient for paracrine signaling and have distinct gene expression patterns.

## Contribution

A novel protocol for isolating cardiac stromal cells with higher yield and faster expansion is introduced and compared to existing methods.

## Key findings

- The collagenase–trypsin protocol yields more cells and shorter expansion time than traditional methods.
- Collagenase–trypsin cells and CDCs share similar gene profiles but differ from cardiac fibroblasts.
- CTs show an epicardial-derived fibroblast phenotype with higher Tcf21 and lower Tbx5 expression.

## Abstract

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 determine whether selected cardiac stromal cells have regenerative potential; however, their contribution to cardiac regeneration has been found to be minimal compared with that of cardiomyocyte proliferation. Despite this, transplantation of some cardiac stromal cell populations has shown therapeutic potential through paracrine signalling. The identity of the paracrine-active stromal cell populations remains unclear due to overlapping characteristics with other cardiac stromal cell populations, such as fibroblasts, mesenchymal cells, and pericytes. This study sought to clarify the transcriptional identity and heterogeneity of adult mouse cardiac stromal cells by developing a cardiac collagenase–trypsin protocol and comparing it to the established method for isolating cardiosphere-derived cells (CDCs). This novel protocol resulted in a higher cell yield and shorter expansion time, and the resulting cells showed superior survival under serum starvation compared to commercially acquired cardiac fibroblasts (CFs). Single-cell qRT-PCR analysis revealed that collagenase–trypsin cells (CTs) and CDCs share similar gene expression profiles, distinct from those of CFs. Notably, CTs exhibited higher expression of Tcf21 and lower expression of Tbx5, suggesting an epicardial-derived fibroblast phenotype, whereas Tbx5 was enriched in CDCs and CFs, reflecting heterogeneity within the cardiac fibroblast compartment. This study offers insights into the complex identity of cardiac stromal cells and concludes that CTs closely resemble CDCs but can be generated more rapidly, making them a robust and efficient source of paracrine-active cardiac stromal cells.