H the highest proteolytic activity group together (clones cEP1 EP5) in the same cluster, while cells with clear sprouting activity group in a different cluster (Clones cEP6 and cEP7) (Fig. 6B, right). In order to determine whether the differences observed in 3D Title Loaded From File matrix degradation of EPIC clones correlate with certain proteases, we performed MMP, ADAM and TIMP mRNA profile analyses by qPCR (Fig. 6C). For theseanalyses clones representing the most extreme proteolytic activity (cEP4), the highest sprouting activity (cEP7) and intermediate properties (cEP6) were selected. Three independent qPCR experiments were run, with GAPDH as a reference gene. The results from these analyses indicated significant changes in the expression of MMP-11 and 14; ADAM-10, 15, 17 and 19; TIMP1, 2, and 3 between different clones (Fig. 6C). To confirm the proteolytic properties of EPICs, cEP4 cells were cultured on fibrin gels containing the protease R [3?] and disease [6,7]. In vitro cell migration assays are routinely used inhibitor aprotinin. Proteolysis in these gels was significantly reduced in these cultures as compared to control (no aprotinin) ones. The supernatant from EPICS and cEP4 cultures was also used for zymography assays. The results independently confirmed the proteolytic properties of EPICs (Fig. S5). In parallel, cEP1? cell spheroids were embedded in a two-layer engineered fibrin matrix with transglutaminase(TG)-bound-BMP2 or -VEGF (Fig. S4A). Exposure to these 26001275 engineered 3D fibrin gels did not result in any observable changes in the differentiation of these cells towards endothelial or cardiomyocyte lineages (data not shown). However, some clones, when exposed to matrixbound TG-BMP-2 (cEP6, cEP7) or TG-VEGF121 (cEP7), displayed variable degrees of cell sprouting into the fibrin gel with fast outward cell migration (Fig. S4A). All the other clones did not show any sprouting response under these experimental conditions, but instead degraded the surrounding matrix afterEpicardial-Derived Interstitial CellsFigure 3. EPIC differentiation marker expression. A . EPIC express a-SMA (red) and SM22 (green). D Treatment with TGFb1,2 does not altere the number of cells expressing these two markers, but affects the phenotype of the cells which spread and elongate in culture. EPICs also express fibroblast protein markers like FSP-1 (G,H) and Collagen I (I; I9 shows the negative, non-inmune control for collagen I immunohistochemistry). J. sqPCR profiling. EPIC (left column), E9.5 proepicardium (middle columns) and E11.5 epicardium (right column). Scale bars: A,B,C,D,E,F,H,I9 = 65 mm; G = 100 mm; I = 10 mm. doi:10.1371/journal.pone.0053694.g48 hours in culture. Since most cell spheroids did not show a response to VEGF, we used as control HUVEC cells to demonstrate that TG-VEGF121 incorporated into fibrin gels is fully functional. Indeed, HUVEC cells seeded in matrix with TGVEGF121 readily migrated from the original spheroids. To evaluate TG-BMP-2 signalling activity, both BMPRI expression and pSMAD phosphorylation were confirmed (not shown) (Fig. S4A). Finally, the most extreme `sprouting’ phenotype (cEP7) was chosen for a detailed profiling. cEP7 cells migrate in response to TG-BMP2 and TG-VEGF121, as well as to bFGF embedded within fibrin (Fig. S4B). Wnt3a and Wnt5a treatments reduced the proteolytic activity of EPIC as compared to control cultures. The sprouting area for cEP7 in response to different growth factors was plotted as fold differences with respect to untreated control sets (Fig. S4B).DiscussionIn this study we have an.H the highest proteolytic activity group together (clones cEP1 EP5) in the same cluster, while cells with clear sprouting activity group in a different cluster (Clones cEP6 and cEP7) (Fig. 6B, right). In order to determine whether the differences observed in 3D matrix degradation of EPIC clones correlate with certain proteases, we performed MMP, ADAM and TIMP mRNA profile analyses by qPCR (Fig. 6C). For theseanalyses clones representing the most extreme proteolytic activity (cEP4), the highest sprouting activity (cEP7) and intermediate properties (cEP6) were selected. Three independent qPCR experiments were run, with GAPDH as a reference gene. The results from these analyses indicated significant changes in the expression of MMP-11 and 14; ADAM-10, 15, 17 and 19; TIMP1, 2, and 3 between different clones (Fig. 6C). To confirm the proteolytic properties of EPICs, cEP4 cells were cultured on fibrin gels containing the protease inhibitor aprotinin. Proteolysis in these gels was significantly reduced in these cultures as compared to control (no aprotinin) ones. The supernatant from EPICS and cEP4 cultures was also used for zymography assays. The results independently confirmed the proteolytic properties of EPICs (Fig. S5). In parallel, cEP1? cell spheroids were embedded in a two-layer engineered fibrin matrix with transglutaminase(TG)-bound-BMP2 or -VEGF (Fig. S4A). Exposure to these 26001275 engineered 3D fibrin gels did not result in any observable changes in the differentiation of these cells towards endothelial or cardiomyocyte lineages (data not shown). However, some clones, when exposed to matrixbound TG-BMP-2 (cEP6, cEP7) or TG-VEGF121 (cEP7), displayed variable degrees of cell sprouting into the fibrin gel with fast outward cell migration (Fig. S4A). All the other clones did not show any sprouting response under these experimental conditions, but instead degraded the surrounding matrix afterEpicardial-Derived Interstitial CellsFigure 3. EPIC differentiation marker expression. A . EPIC express a-SMA (red) and SM22 (green). D Treatment with TGFb1,2 does not altere the number of cells expressing these two markers, but affects the phenotype of the cells which spread and elongate in culture. EPICs also express fibroblast protein markers like FSP-1 (G,H) and Collagen I (I; I9 shows the negative, non-inmune control for collagen I immunohistochemistry). J. sqPCR profiling. EPIC (left column), E9.5 proepicardium (middle columns) and E11.5 epicardium (right column). Scale bars: A,B,C,D,E,F,H,I9 = 65 mm; G = 100 mm; I = 10 mm. doi:10.1371/journal.pone.0053694.g48 hours in culture. Since most cell spheroids did not show a response to VEGF, we used as control HUVEC cells to demonstrate that TG-VEGF121 incorporated into fibrin gels is fully functional. Indeed, HUVEC cells seeded in matrix with TGVEGF121 readily migrated from the original spheroids. To evaluate TG-BMP-2 signalling activity, both BMPRI expression and pSMAD phosphorylation were confirmed (not shown) (Fig. S4A). Finally, the most extreme `sprouting’ phenotype (cEP7) was chosen for a detailed profiling. cEP7 cells migrate in response to TG-BMP2 and TG-VEGF121, as well as to bFGF embedded within fibrin (Fig. S4B). Wnt3a and Wnt5a treatments reduced the proteolytic activity of EPIC as compared to control cultures. The sprouting area for cEP7 in response to different growth factors was plotted as fold differences with respect to untreated control sets (Fig. S4B).DiscussionIn this study we have an.
