Regenerative potential of mouse embryonic stem cell-derived PDGFRα+ cardiac lineage committed cells in infarcted myocardium

Figure 1 Implantations of platelet-derived growth factor receptor-α⁺ cardiac lineage-committed cells and αMHC⁺ cardiomyocytes in the acute myocardial infarction murine model. A: Experimental scheme of implanting either platelet-derived growth factor receptor-α (PDGFRα)⁺ cardiac lineage-committed cells (CLCs) or αMHC⁺ cardiomyocytes (CMs) into acute myocardial infarction (MI) murine model. Analyses were performed at 2 wk after implantation of approximately 1 × 10⁶ cells of PDGFRα⁺ CLCs or αMHC⁺ CMs into the left ventricular myocardium of acute MI murine model; B: Live cell image showing tdTomato⁺ cells during induction of PDGFRα⁺ CLCs from embryonic stem cells. Scale bars, 100 μm; C: Representative confocal image showing implanted tdTomato⁺ PDGFRα⁺ CLCs in the myocardial spaces (arrowheads) of the infarcted zone (dotted line and arrow), which was formed by ligation of coronary artery 1 h prior to the implantation. Scale bar, 500 μm. CLCs: Cardiac lineage-committed cells; CMs: Cardiomyocytes; PSCs: Pluripotent stem cells; ESCs: Embryonic stem cells; LIF: Leukemia inhibitory factor.

Figure 2 Implantations of platelet-derived growth factor receptor-α⁺ cardiac lineage-committed cells and αMHC⁺ cardiomyocytes equally improves contractile function and structure in the infarcted heart. A: Representative M-mode transthoracic echocardiography views of control, myocardial infarction (MI), MI+ platelet-derived growth factor receptor-α (PDGFRα)⁺ cardiac lineage-committed cells (CLCs), and MI+αMHC⁺ cardiomyocytes (CMs). Improved anterior (white arrowheads) and septal (yellow arrowheads) regional wall motion are observed in the left ventricles of MI+PDGFRα⁺ CLCs and MI+αMHC⁺ CMs; B and C: Quantifications of left ventricular internal dimension in systole (mm), ejection fraction (%) and fractional shortening (%). Each group, n = 7. ^aP < 0.05 and ^bP < 0.01 vs Con; ^cP < 0.01 vs MI; D: Gross images of hearts in control, MI, MI+PDGFRα⁺ CLCs, and MI+αMHC⁺ CMs. Scale bars, 2.5 mm; E: H and E staining of mid-sectioned hearts of control, MI, MI+PDGFRα⁺ CLCs, and MI+αMHC⁺ CMs. Scale bars, 1 mm and 50 μm in the upper and lower panels, respectively; F: Quantifications of the thickness (mm) of left ventricle in the infarcted region. Each group, n = 7. ^bP < 0.01 vs Con; ^cP < 0.01 vs MI or MI+PDGFRα⁺ CLCs. CLCs: Cardiac lineage-committed cells; CMs: Cardiomyocytes; MI: Myocardial infarction.

Figure 3 Integration and differentiation of implanted platelet-derived growth factor receptor-α⁺ cardiac lineage-committed cells in the infarcted heart. A: tdTomato-tagged platelet-derived growth factor receptor-α (PDGFRα)⁺ cardiac lineage-committed cells (CLCs) or αMHC⁺ cardiomyocytes (CMs) were implanted into the infracted myocardium and integration was confirmed by immunostaining. tdTomato⁺/α-MHC-GFP⁺ cells (white arrowheads) are implanted PDGFRα⁺ CLCs and αMHC⁺ CMs. Scale bars, 100 μm; B: Representative confocal images showing differentiation of tdTomato⁺ PDGFRα⁺ CLCs and αMHC⁺ CMs into cardiomyocytes 15 d after the implantation. αSMA-expressing cells were negative for tdTomato or α-MHC-GFP signal as shown in the inlet. Scale bars, 25 μm; C: Percentages of α-actinin⁺ CMs, CD31 endothelial cells and αSMA⁺ smooth muscle cells of the implanted PDGFRα⁺ CLCs and αMHC⁺ CMs. Each group, n = 4. Scale bars, 20 μm. CLCs: Cardiac lineage-committed cells; CMs: Cardiomyocytes; MI: Myocardial infarction; ECs: Endothelial cells; SMCs: Smooth muscle cells.

Figure 4 Proliferation and survival of implanted platelet-derived growth factor receptor-α⁺ cardiac lineage-committed cells in the infarcted heart. A: Representative confocal image of Ki-67⁺ host cardiomyocytes in the peri-infarcted region 3 d after cell injection. Dotted-lined rectangular region is magnified in right. Scale bar, 50 μm; B: Quantifications of Ki-67⁺α-actinin⁺ cardiomyocytes per 10⁴ nuclei in the peri-infarcted region 3 and 15 d after the implantation. Each group, n = 4. ^aP < 0.05 vs myocardial infarction (MI); C: Representative confocal images of revascularization within the infarcted areas 15 d after the implantation. Scale bars, 100 μm; D: Quantifications of capillary density (No. of CD31⁺ ECs/mm²) within the infarcted areas. Each group, n = 4. ^bP < 0.01 vs MI; ^cP < 0.01 vs MI+ platelet-derived growth factor receptor-α (PDGFRα)⁺ cardiac lineage-committed cells (CLCs); E: Representative confocal images of tdTomato⁺ PDGFRα⁺ CLCs and αMHC⁺ CMs 60 d after the implantation. Three independent experiments showed similar findings. Scale bars, 20 μm; F: Schematic diagram illustrating the regenerative potential of PDGFRα⁺ CLCs in the infarcted heart. CLCs: Cardiac lineage-committed cells; CMs: Cardiomyocytes; MI: Myocardial infarction.