Human Cardiac Mesenchymal Stromal Cells From Right and Left Ventricles Display Differences in Number, Function, and Transcriptomic Profile

Figures

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  FIGURE 1. A higher amount of cardiac mesenchymal stromal cells (C-MSC) is present in the RV when compared to the left one. (A) Immunofluorescence staining on human left ventricular (LV) and right ventricular (RV) total tissue for the mesenchymal markers CD29, CD44, and CD105. On the left, representative images are shown. The red staining is relative to the mesenchymal markers, the blue signal marks the nuclei (Hoechst 33342). On the right, the quantification of the percentage of positive cells is reported. The scale bar indicates 50 μm. n = 13 each; *p < 0.05 (paired t-test). (B) C-MSC were isolated from either LV or RV human samples. A higher amount of C-MSC was obtained from RV samples. n = 6 each; *p < 0.05, **p < 0.01 (paired t-test).

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  FIGURE 2. The isolated left ventricle (LV) and right ventricle (RV) cardiac mesenchymal stromal cells (C-MSC) are not different for surface marker expression. LV and RV C-MSC were characterized for surface marker expression, showing no differences. Mesenchymal markers CD29, CD44, and CD105, endothelial markers CD31 and CD34, hematopoietic markers CD14 and CD45, the fibroblast marker CD90, and the alloreactivity marker HLA-DR have been used. n = 6 each (paired t-test).

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  FIGURE 3. Higher apoptosis in growth conditions characterizes left ventricle (LV) cardiac mesenchymal stromal cells (C-MSC) with respect to right ventricle (RV) cells. RV C-MSC show greater motility when compared to LV cells. (A) The growth curves of LV and RV C-MSC in the culture medium are reported. Cells were cultured for 4 days, and the number of cells was counted every 24 h. No differences in growth curve slopes have been obtained. n = 6 each (linear regression slope comparison). (B) The percentage of apoptotic and necrotic cells was evaluated in the culture medium. In the left panel, representative flow cytometry histograms comparing LV and RV C-MSC patterns are shown. As shown in the right graphs, a higher percentage of apoptotic (measured in #1 interval) LV C-MSC than RV cells has been found. No differences between the necrotic (measured in #2 interval) cell amount were recorded. n = 6 each; *p < 0.05 (paired t-test). (C) The apoptosis rate of LV and RV C-MSC for different time-points is reported. Cells were cultured for 5 days in growth medium. The Annexin V count normalized on the percentage of confluence is shown. For all the time-points, a trend of higher apoptosis was measured…

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  FIGURE 4. No differences in adipogenic differentiation between left ventricle (LV) and right ventricle (RV) cardiac mesenchymal stromal cells (C-MSC) have been observed. (A) The upper panels show Oil Red O staining representative images of LV and RV C-MSC cultured for 72 h and 1 week in the adipogenic medium (AM). The scale bar indicates 50 μm. The quantification of cell lipid accumulation is provided in the bottom panel. n = 6 each (paired t-test). (B) qRT-PCR analysis of PPARγ, FABP4, and PLIN1 expression in LV and RV C-MSC cultured for 72 h and 1 week in AM, normalized on the housekeeping gene GAPDH. 2–ΔΔ Ct ratio is shown: 2–ΔΔ Cts of each group are normalized on 2–ΔΔ Ct values of LV C-MSC cultured in AM for 72 h. n = 6 each (paired t-test). (C) The protein extracts of LV and RV C-MSC cultured for 72 h and 1 week have been analyzed by Western blot. The densitometric analysis of PPARγ, FABP4, and PLIN1 normalized on the housekeeping protein GAPDH is shown. n = 6 each (paired t-test).

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  FIGURE 5. Right ventricle (RV) cardiac mesenchymal stromal cells (C-MSC) produce more collagen than left ventricle (LV) cells. (A) Collagen production quantification with Sircol assay in supernatants (left graph) and cells (right graph) of LV and RV C-MSC cultured for 5 days in the basal medium with 2% serum. As shown in the graphs, RV cells produce more collagen than LV cells. *p < 0.05; n = 6 each (paired t-test). (B) Western blot analysis of COL1A1, the more expressed collagen type, confirmed Sircol result. A trend of higher expression of COL1A1 was evaluated in RV C-MSC. n = 6 each (paired t-test).

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  FIGURE 6. Differential gene expression between left ventricle (LV) and right ventricle (RV) cardiac mesenchymal stromal cells (C-MSC). (A) Scatterplot of the log2 fold change (FC) vs. the significance (x- and y-axes, respectively) for the paired comparison of LV vs. RV C-MSC. Red and blue dots represent genes overexpressed in LV and RV C-MSC, respectively. Light red and light blue dots are genes significant at the nominal P-value < 0.01, whereas red and blue dots represent significant differentially expressed (DE) genes that withstood adjustment for multiple testing (adjusted P-value < 0.05). Ten of the top DE genes with the highest combined rank score (the product of the log2 FC × likelihood ratio) are shown. (B) Pie chart of the percentage of coding (pink) and non-coding (light blue) differentially expressed genes; more than 80% of DE genes are protein coding.

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  FIGURE 7. Enrichment map for left ventricle (LV) and right ventricle (RV) cardiac mesenchymal stromal cells (C-MSC). The enrichment network shows the pathway gene sets (nodes) that are significantly associated either with LV or RV ventricles (false discovery rate < 0.05). Node color refers to the association with the phenotype (LV = red, RV = blue); node gradient color is proportional to the gene-set normalized enrichment score (NES), from lower (light) to higher (dark); node size is proportional to the gene-set size. Edges connect related pathways. Edge thickness is proportional to the similarity between two pathways, for a cutoff = 0.25 of the combined Jaccard plus overlap coefficient.