Molecular signature of progenitor cells isolated from young and adult human hearts

Figures

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  Figure 1. Isolation and morphological comparison of c-kitpos CPCs and CDCs. (a) Schematic overview of the steps involved to isolate c-kitpos CPCs and CDCs. To isolate c-kitpos cells, a human RA appendage biopsy is minced into small pieces and collagenase digested to culture a heterogeneous mixture of single cells for 2 passages prior to MACS-based selection. To obtain CDCs, a mild collagenase-based digestion is performed on small myocardial fragments to allow outgrowth of fibroblasts and phase-bright cells from the cardiac explants. The latter are transferred onto a poly-D-Lysine coated dish to form cardiospheres. Finally, the cardiospheres are collected and grown as monolayer cells called CDCs. (b) Phase bright-field pictures of c-kitpos CPCs and CDCs derived from young and adult donors. Scale bars represent 200 µm. CPC, cardiac progenitor cell; CDC, cardiosphere-derived cell; RA, right atrial.

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  Figure 2. Differential gene expression between CDCs and c-kitpos CPCs derived from adult and young donors. (a) Heat map of Spearman correlations with the greatest differences observed between aCDCs and c-kitpos CPCs and between aCDCs and yCDCs. (b) String network analysis using the top significant genes showed clusters of transcripts that are related to cytokines and inflammation, growth factors, cell motility and extracellular matrix. (c,d) Gene ontology results using DAVID software (blue) and pathway analysis (IPA) (green) comparing aCDCs and c-kitpos aCPCs (c) and yCDCs and aCDCs (d) confirming the string network analysis. Compared to c-kitpos aCPCs, aCDCs are more enriched in inflammatory processes (c), while cell cycle pathways are more prominent in aCDCs compared to yCDCs (d). (e–g) Heatmaps of immunomodulatory genes, cell cycle markers and stem cell markers and growth factors, red = downregulated; blue = upregulated. c-kitpos yCPC (ages 5 d, 2 m, 2.5 m, 3 m, 4 m), c-kitpos aCPC (ages 62 y, 65 y, 74 y, 79 y, 82 y), yCDC (ages 4 d, 1,5 m, 3 m, 5 m, 10 y), aCDC (ages 47 y, 65 y, 70 y, 75 y, 77 y).

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  Figure 3. Phenotypic comparison of adult and young c-kitpos CPCs and CDCs. (a) Gene expression levels of CD117 (KIT), CD105, CD90, the EC marker CD31, early cardiac transcription factors GATA4 and MEF2c, SMC marker MYH11 and fibroblast marker DDR2. c-kitpos yCPC (n = 8), c-kitpos aCPC (n = 9), yCDC (n = 7) and aCDC (n = 7). Results are geometric mean with 95% CI, one-way ANOVA was performed for normally distributed data and Kruskal-Wallis test for non-normally distributed data, *p < 0.05. EC, endothelial cell; SMC, smooth muscle cell; CPC, cardiac progenitor cell; CDC, cardiosphere-derived cell. (b) Flow cytometry analysis of c-kitpos yCPCs (n = 8), c-kitpos aCPCs (n = 10), yCDCs (n = 7) and aCDCs (n = 8) for CD90, CD105, and CD117. All analyses were performed after 6 passages. Results are mean ± SEM, *p < 0.05. (c) PDT of c-kitpos yCPCs (n = 4), c-kitpos aCPCs (n = 5), yCDCs (n = 5) and aCDCs (n = 4). Results are mean ± SEM, *p < 0.05. CPC, cardiac progenitor cell; CDC, cardiosphere-derived cell; PDT, population doubling time.

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  Figure 4. Vasculogenic and angiogenic potential of adult and young c-kitpos CPCs and CDCs. (a) Analysis of network formation 6 hours after culture on Matrigel® of c-kitpos yCPCs (n = 5), c-kitpos aCPCs (n = 8), yCDCs (n = 5) and aCDCs (n = 9). (b) Quantification of number of nodes, junctions, meshes, segments and branches/mm2. Results are mean ± SEM, *p < 0.05. (c) Correlation of vasculogenic potential, expressed as number of nodes/mm2 and CD90 mRNA expression of aCDCs and yCDCs. (d) Representative images of 3D sprouting of c-kitpos yCPC (n = 2), c-kitpos aCPC (n = 2), yCDC (n = 2) and aCDC (n = 2) spheroids after 48 hours incubation. Per group, cells of 2 different donors were used and for each donor, 4 different spheroids were imaged. One representative spheroid per donor is depicted. (e) Growth factor release analysis in conditioned medium collected after 24 hours incubation of c-kitpos yCPCs (n = 9), c-kitpos aCPCs (n = 11), yCDCs (n = 7) and aCDCs (n = 10) for IGF-1, VEGF, bFGF, PlGF, HGF and SDF-1. Results are mean ± SEM, *p < 0.05 compared to aCDC and #p < 0.05 compared to yCDC. CPC, cardiac progenitor cell; CDC, cardiosphere-derived cell.

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  Figure 5. The cardiac differentiation capacity of adult and young c-kitpos CPCs and CDCs. (a) Double immunofluorescence staining of phalloidin (green) and α-sarcomeric actinin (red) revealed protein expression of a cardiac contractile protein after incubation in cardiac differentiation medium (DIFF) or regular expansion medium (CON) for 12 days. c-kitpos yCPCs (n = 5), c-kitpos aCPCs (n = 6), yCDCs (n = 5) and aCDCs (n = 5). Scale bars represent 100 µm. (b) Quantification of the immunofluorescent staining for α-sarcomeric actinin positive cells after 12 days incubation in DIFF medium. Fold change of alpha-sarcomeric actinin positive cells in DIFF treated groups compared to CON groups, results are mean ± SEM, *p < 0.05. (c) Fold change expression of GATA4, MEF2c and NKX2.5 in DIFF treated groups compared to CON groups. Results are mean ± SEM.