Novel technique for the simultaneous isolation of cardiac fibroblasts and epicardial stromal cells from the infarcted murine heart

Myocardial infarction (MI) leads to activation of cardiac fibroblasts (aCFs) and at the same time induces the formation of epicardium-derived cells at the heart surface. To discriminate between the two cell populations, we elaborated a fast and efficient protocol for the simultaneous isolation and characterization of aCFs and epicardial stromal cells (EpiSCs) from the infarcted mouse heart. For the isolation of aCFs and EpiSCs, infarcted hearts (50 min ischaemia/reperfusion) were digested by perfusion with a collagenase-containing medium for only 8 min, while EpiSCs were enzymatically removed from the outside by applying mild shear forces via a motor driven device. Cardiac fibroblasts (CFs) isolated from unstressed hearts served as control. Microarray analysis of CFs, aCFs, and EpiSCs on day 5 post-MI revealed a unique gene expression pattern in the EpiSC fraction, which was enriched for epithelial markers and epithelial to mesenchymal transition-related genes. Compared to aCFs, 336 significantly altered gene entities were identified in the EpiSC fraction. Microarray data identified Ddah1 and Cemip to be highly up-regulated in aCFs compared to CFs. In this study, several differentially expressed markers for aCFs and EpiSCs were identified, underlining the importance of cell separation to study heterogeneity of stromal cells in the healing process after MI. 15 total samples (five biological replicates each) were analyzed. aCF and EpiSC samples were compared to each other and CF samples were compared to aCF samples. Data analyses on Affymetrix CEL files obtained from the microarray experiment were conducted with GeneSpring GX software (Vers. 12.5; Agilent Technologies). Probes within each probe set were summarized by GeneSprings’ ExonRMA16 algorithm after quantile normalization of probe-level signal intensities across all samples to reduce interarray variability. Input data preprocessing was concluded by baseline transformation to the median of all samples. After grouping of samples according to their respective experimental condition, a given probe set had to be expressed above background (i.e., fluorescence signal of that probe set was detected within the 20th and 100th percentiles of the raw signal distribution of a given array) in all five replicates in at least one of three conditions to be further analyzed in pairwise comparisons. Differential gene expression was statistically determined by Moderated t test. The Resulting P values were corrected for multiple testing by Bonferroni-correction. A P<0.05 was considered significant.