Anti-fibrotic Effects of Cardiac Progenitor Cells in a 3D-Model of Human Cardiac Fibrosis

Figure 1. Fabrication and mechanical characterization of GelMA hydrogels. (A) Schematic overview of hydrogel preparation. (B) Compressive properties of GelMA hydrogels upon varying gel percentage (w/v), 5% GelMA (n = 3) and 10% GelMA (n = 4). Statistical significance was assessed using student's t-test (two-tailed). ***p < 0.001. (C) Mass Swelling ratio (q) differs with GelMA concentration. q = wet weight/dry weight. 5% GelMA (n = 5) and 10% GelMA (n = 6). Statistical significance was assessed using student's t-test (two-tailed). ***p < 0.001.

Figure 2. Encapsulation of human fetal cardiac fibroblasts in GelMA hydrogel. (A) Trichrome blue staining demonstrates homogenous encapsulation of hfCF in the 10% GelMA hydrogel. Blue = extracellular matrix and scaffold (GelMA). Dark red = hfCF. (B) Live/Dead staining shows that cells remain viable in the 10% GelMA hydrogels until 14 days in culture. Directly after encapsulation, cells display a round morphology, whereas after 7 days cells appear more elongated and attached to the matrix (arrowheads). Live cells = green. Dead cells = red. Scale bars = 25 μm. (C) Semi-quantitative analysis of the number of live cells (n = 5). Statistical significance was assessed using one-way ANOVA.

Figure 3. Stimulating cardiac fibroblast mediated fibrosis with the pro-fibrotic mediator TGF-β1. (A) Gene expression analysis shows that hfCF have a lower α-SMA expression after 7 days of culture when encapsulated in 5% (w/v) or 10% (w/v) GelMA (n = 8). Statistical significance was assessed using one-way ANOVA with a Dunnett post-hoc test. *p < 0.05. (B) Col1a1 expression remains equal. Values are relative to the corresponding 2D cultured hfCF at day 1 (n = 8). Statistical significance was assessed using one-way ANOVA. (C) Stimulation with TGF-β1 for 7 days results in a 2.1-fold increase in α-SMA expression and (D) a 10.3-fold increase in Col1a1 expression (n = 8). Statistical significance was assessed using paired student's t-test (two-tailed). ***p < 0.001 **p < 0.01. (E,F) Immunohistochemistry shows an increased number of highly Col1a1 positive cells upon stimulation with TGF-β1 (Col1a1 = red, vimentin = green, nuclei = blue). Statistical significance was assessed using paired student's t-test (two-tailed). **p < 0.01. (G,H) Accumulation of collagen fibers in the extracellular matrix is demonstrated by picrosirius red staining and quantification (n = 4). Left panel = bright field…

Figure 4. Cardiac progenitor cells attenuate the cardiac fibroblast mediated fibrotic response. (A) Schematic overview of the experimental set-up. (B) CPC attenuate α-SMA expression in hfCF-laden GelMA in culture conditions with or without 2 ng/ml TGF-β1 stimulation (n = 13). (C) Col1a1 expression is lower in co-culture conditions (n = 12) and (D) likewise, the amount of highly Col1a1 positive cells is 2-fold lower in CPC-treated gels (n = 4). Statistical significance was assessed using repeated measures two-way ANOVA. **p < 0.01 *p < 0.05.

Figure 5. Cardiac progenitor cells act via paracrine mediators. (A) Schematic overview of the experimental set-up of transfer of co-CM to a naïve hfCF-laden hydrogel. (B) co-CM attenuates α-SMA expression in hfCF-laden GelMA in culture conditions with or without 2 ng/ml TGF-β1 (n = 5). Statistical significance was assessed using repeated measures two-way ANOVA. (C) Schematic overview of the experimental set-up of treatment of naïve hfCF-laden hydrogels with CPC conditioned medium (CM) or CPC-derived extracellular vesicles (EV). (D) CM does not influence hfCF activation. In pro-fibrotic medium the expression of α-SMA is significantly increased (n = 8). Statistical significance was assessed using repeated measures two-way ANOVA. **p < 0.01. (E) Isolated EV show similar effects as CM (n = 3). Statistical significance was assessed using repeated measures two-way ANOVA. *p < 0.05.