Human Cardiac Progenitor Cells

Isolation and Culture of CPCs

CPCs were isolated from the right atrial appendage (RAA) and left ventricle (LV) through cell dissociation using collagenase II. Due to the variation in size between RAA and LV samples, cells were seeded onto an appropriately sized plate or flask based on the weight of the tissue to minimize differences in initial seeding density. The resultant CPCs were characterized by flow cytometry for the expression of mesenchymal stem cell (MSC) markers, CD90 (thymocyte differentiation antigen, Thy-1) and CD105 (endoglin), as well as the circulating hematopoietic progenitor cell marker CD34.

Majority of the cells were negative for CD34 population, although due to the ischemic nature of the tissue collected, they still expressed CD34, which was especially more in LV CPCs (18.3% ± 3.3% in RAA CPCs vs. 24.7% ± 6.0% in LV CPCs, P = 0.23, n = 6 patients, Fig. 1A, C). CD34 negative population were then used to measure the expression of MSC markers, CD90 and CD105. CD90 expression was significantly higher in RAA CPCs compared to LV CPCs (78.7% ± 5.9% in RAA CPCs vs. 54.4% ± 11.0% in LV CPCs, P = 0.03, n = 6 patients, Fig. 1B, D). CD105 expression was high in both RAA CPCs and LV CPCs, with nearly all cells expressing the cell marker (99.%0 ± 0.6% in RAA CPCs vs. 99.3% ± 0.1% in LV CPCs, P = 0.2505, n = 6 patients, Fig. 1B, D). Further, immunocytochemical analysis confirmed the expression of cardiac transcription factor Islet-1 in the undifferentiated CPCs (Fig. 1E).

CPCs grown in differentiation medium showed positive staining for cardiomyocyte-specific markers MHC (Fig. 1F) and connexin-43 (Fig. 1G). RT-PCR analysis confirmed the expression of cardiac troponin and α-sarcomeric actin in differentiated CPCs (Fig. 1H). As expected, there was no difference in the differentiation potential between RAA and LV CPCs.

Fig. 1. Comparable expression of cell surface markers between RAA CPCs and LV CPCs (McQuaig, Ryan, et al., 2020).

Deep Comparative Transcriptome Analysis of CPCs by mRNA Sequencing

To define specific CPCs functions, human CPCs from three independent donors (CPC1-3) were compared to human bone marrow MSCs (aiming to identify putative genes related to multipotency) using mRNAseq, with human dermal fibroblasts (HDFs) as a distant reference (to discard genes expressed similarly in all cell types). After a preliminary evaluation of the impact of different culture media in CPCs vs. HDFs growth, we selected to culture each cell type in its optimal media.

CPCs, MSCs and HDFs were compared (FDR < 0.05) only for coding genes from total and differentially expressed gene (DEG) data, using replicates and/or technical duplicates of all samples at the indicated passages. CPCs mRNAseq data rendered 12,242 protein-coding genes. Normalized heat map and cluster analysis confirmed CPCs, MSCs and HDFs as cell lineages significantly different from each other (Fig. 2a, b). In addition, we confirmed that the expression profiles were not significantly affected by culture passage, as only 167 out of 11,767 total genes analyzed showed significant variations with passages.

Comparative analysis found 2,096 DEG (17.8%) for the CPCs/MSCs comparison (p.adj values < 0.05; Fig. 2c), with 1,003 highly preferentially expressed in CPCs by simultaneous comparison with HDFs (Fig. 2c). No significant differences were found in association with subcellular compartments. The majority of the top 10 upregulated genes in CPCs were specific for the CPCs/MSCs comparison (Fig. 2d) and were not found upregulated in the CPCs/HDFs comparison. Among the genes upregulated, C-X-C motif chemokine ligand 6 (CXCL6) and matrix metallopeptidase 1 (MMP1) showed the maximal differences (Fig. 2d). Other genes of interest not included among the most upregulated genes, such as GATA binding protein 4 (GATA4), calcium voltage-gated channel auxiliary subunit gamma 7 (CACNG7), G protein-coupled receptor 4 (GPR4) and cadherin 5 (CDH5), were similarly upregulated in CPCs relative to MSCs and HDFs (Fig. 2d). Aggrecan (ACAN) and cartilage oligomeric matrix protein/ thrombospondin-5 (COMP) were more clear examples of down-regulated transcripts in CPCs when compared with MSCs (Fig. 2d).

Fig. 2. RNAseq analysis of CPCs compared with MSCs and HDFs (Torán, José Luis, et al., 2019).