Promoting early neovascularization by allotransplanted adipose-derived Muse cells in an ovine model of acute myocardial infarction

Figures

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  Fig 1. Flow cytometry and gene expression analysis. Labeling of A: AT-Muse cells and B: ASCs with antibodies anti- SSEA3, CD45, CD44, CD166 and controls. C: The % of SSEA3+ cells was higher in the AT-Muse group than in the ASCs group. D: Immunofluorescence image confirming the labeling of SSEA3 (green) and DAPI (blue) in AT-Muse cells at P4 in vitro. Scale bar = 100 μm. E: AT-Muse cells were CD166+, CD44+ and CD45- confirming their immunophenotype. F-I: The AT-Muse group showed higher expression of OCT4, NANOG and S1PR2 than the ASCs group. Results are means ± SD. *p<0.05, Mann-Whitney U test.

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  Fig 2. Angiogenic potential of AT-Muse cells. A-C: VEGF, ANG, and PGF gene expression was higher in the AT-Muse group than in the ASCs group. D: Tube formation assay: the AT-Muse group showed an increased number of rings per area (mm2) compared to the ASCs group. E-F: Representative images of rings formed in the AT-Muse and ASCs groups. G-H: VEGF protein expression was higher in the AT-Muse group than in the ASCs group. Scale bar 1000 μm. Results are mean ± SD. ** p < 0.01; *p < 0.05, Mann-Whitney U test.

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  Fig 3. Differentiation of PKH26+ cells. A-F: Fluorescence microscopy analysis revealed the presence of PKH26+ cells at 7 days post-AMI in the infarct border zone of AT-Muse group. A: PKH26+ Muse cells (red), Desmin (green), and DAPI (blue) are merged. B: PKH26+ Muse cells (red), Sarcomeric actin (green), and DAPI (blue) are merged. C: PKH26+ Muse cells (red), Troponin T (green), and DAPI (blue) are merged. D: PKH26+ Muse cells (red), Connexin-43 (green), and DAPI (blue) are merged. E: PKH26+ Muse cells (red), Lectin (green), and DAPI (blue) are merged. F: PKH26+ Muse cells (red), Smooth muscle, actin (green) and DAPI (blue) are merged. One cell is enlarged in the top left box (white arrow). Bars = 50 μm.

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  Fig 4. Microvascular density at 7 days post-AMI. A-B: Arteriolar and capillary densities were significantly higher in the AT-Muse group than in the Vehicle group. C-D: Representative slices of the infarct border zone for the AT-Muse and Vehicle groups. Arterioles were stained with an anti-smooth muscle actin antibody and counterstained with hematoxylin (bars = 100 μm). E-F: Representative slices of the infarct border zone for the AT-Muse and Vehicle groups showing capillaries stained with a biotinylated lectin and counterstained with hematoxylin (bars = 100 μm). Results are means ± SD. *p<0.05, Mann-Whitney U test.

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  Fig 5. Cycling cells and protein expression of angiogenic markers at 7 days post-AMI. A: The number of Ki67+ cells in the AT-Muse group was significantly higher than in the Vehicle group. B: Representative image of Ki67+ nuclei (brown) counterstained with sarcomeric actin (violet) in the AT-Muse group (bar = 20 μm). C-D: The VEGF had higher gene and protein expression in the infarct border zones of the hearts of AT-Muse group in comparison with the Vehicle group, respectively. E: The CD34 protein expression was higher than in the Vehicle group. F: representative western blot images for VEGF and CD34. Results are means ± SD. *p<0.05, Mann-Whitney U test.

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  Fig 6. Biodistribution analysis of AT-Muse cells. A: High number of PKH26-labeled ovine AT-Muse cells were found in the infarct border cross sections. Some Muse cells were found in the lungs although no Muse cells were found in spleen, liver, kidney, retina, gonad or in the Vehicle group. Results are means ± SD; ****p<0.0001, two-way ANOVA with Tukey’s post-hoc test for multiple comparisons. B: Representative microscopic merged image of infarct border zone of heart injected with Muse, PKH26+ cells (red) and DAPI (blue). C: Representative microscopic merged image from lung tissue of AT-Muse group, PKH26+ cells (red) and DAPI (blue).