Establishment and characterization of an immortalized epicardial cell line

Figures

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  FIGURE 1. Epicardium constitutes a low proportion of cardiac cells. A‐H, Immunohistochemistry for WT1 and RALDH2 was performed on E9.5‐E12.5 mouse heart sections. TNNT2 was used to visualize cardiomyocytes (red) and DAPI was used to visualize nuclei (blue). PE, proepicardium. IFT, inflow tract. LV, left ventricle. Scale bar, 100 µm. I, Immunohistochemistry for GFP (green), WT1 (yellow), TNNT2 (red) and DAPI (blue) was performed on E12.0 WT1GFPCre/+ mouse heart sections. Scale bar, 20 µm. J, Proportion of epicardial cells as analysed by flow cytometry‐based on their GFP expression. K, Fraction of epicardial cells of E12.5 WT1GFPCre/+ mouse heart (n = 6 per group). L, Cell immunostaining for WT1 (green) and DAPI (blue) after FACS. Scale bar, 20 µm

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  FIGURE 2. Primary epicardial cells isolation and evaluation of cell purity. A, A schematic outline of primary epicardial cells isolation workflow. B, Bright‐field images show cell morphology of primary epicardial cells. Scale bar, 100 µm. C, Immunohistochemistry for WT1 (green) and DAPI (blue) on primary epicardial cells. Scale bar, 50 µm. D, qRT‐PCR analysis of epicardial markers (Wt1 and Raldh2) compared to myocardial gene expression (Nkx2‐5 and Tnnt2) in corresponding epicardium‐depleted E11.5 hearts (n = 4 per group). Epi, primary epicardial cells. ND, not determined, ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. E, Wholemount image of the heart of E11.5 WT1+/+ ; Rosa26‐RFP f/+ and WT1GFPCre/+ ; Rosa26‐RFP f/+ mouse and fluorescence images of RFP expression of migrated primary epicardial cells. Upper scale bar, 100 µm; Lower scale bar, 25 µm

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  FIGURE 3. Evaluation of cell morphology and gene expression over passaging of primary epicardial cells. A, Bright‐field images show primary epicardial cell morphology. Scale bar, 25 µm. B, Fold changes in gene expression (n = 4 per group). EMT, epithelial to mesenchymal transition. NS, not significant, ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001

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  FIGURE 4. Evaluation of cell morphology and gene expression of EpiSV40. A, Schematic outline of the EpiSV40 preparation workflow. B, Bright‐field images show cell morphology of different passages of EpiSV40 and the primary epicardial cells P1. Scale bar, 25 µm. C, Fold changes in gene expression (n = 4 per group). EMT, epithelial to mesenchymal transition. NS, not significant, ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001. D, Immunohistochemistry for WT1 (green), SV40 T Ag (red) and DAPI (blue) was performed on EpiSV40. Scale bar, 20 µm. E, Karyotype analysis of EpiSV40 shows normal chromosome architecture. F, ddPCR analysis of copy number of β‐actin and SV40‐LT gene of EpiSV40 (n = 5 per group). G, Fold changes in gene expression of SV40‐LT (n = 4 per group). ND, not determined. H, Representative Western blot analysis of expression of SV40 T Ag

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  FIGURE 5. Proliferative capacity of primary epicardial cells and EpiSV40. A, Proliferating cells were detected via EdU (green) and DAPI (blue). Scale bar, 25 µm. B, Proliferating cells were detected via pH3 (green) and DAPI (blue). Scale bar, 25 µm. C, Proliferating cells were detected via Ki67 (green) and DAPI (blue). Scale bar, 25 µm. D, Relative ratio of proliferative rate via EdU, pH3 and Ki67 (n = 4 per group). NS, not significant, ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001

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  FIGURE 6. The migration and differentiation capacities of primary epicardial cells and EpiSV40. A, Representative images of cell migration of primary epicardial cells. Scale bar, 100 µm. B, Representative images of cell migration of EpiSV40. Scale bar, 100 µm. C, Migration of primary epicardial cells is presented as a percentage of the original scratch area (n = 6 per group). D, Migration of EpiSV40 is presented as a percentage of the original scratch area (n = 6 per group). E, Fold changes in gene expression (n = 4 per group). NS, not significant, ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001. F, Primary epicardial cells and EpiSV40 were stained with β‐catenin (green) and DAPI (blue). Scale bar, 20 µm. G, Primary epicardial cells and EpiSV40 were stained with α‐smooth muscle actin (α‐SMA) (green) and DAPI (blue). Scale bar, 20 µm