Figure 6

Histological Assessment. (A) Representative images of hematoxylin–eosin stained myocardial sections along the short axis for untreated control, smooth muscle cell (SMC)-endothelial progenitor cell (EPC) bi-level cell sheet, and sham surgery control groups. (B) Representative images of Masson’s Trichrome stained myocardial sections of each animal group. (C) The representative periodic acid-Schiff stained myocardial section along the short axis in the remote non-infarct myocardium for untreated control, SMC-EPC bi-level cell sheet, and sham surgery control groups. Scale bar = 50 μm. (D) Representative vWF and alpha-smooth muscle actin (α-SMA) staining of the border zone myocardium for untreated control, SMC-EPC bi-level cell sheet, and sham surgery control groups. Red indicates von Willebrand factor (vWF); green, α-SMA; blue, nuclei. (E) Representative images of Prussian blue-stained myocardial sections along the short axis 8 weeks after iron-labeled SMC-EPC bi-level cell-sheet transplantation. Iron-positive cells were detected in the attached cell sheet (black arrow) and host myocardium (arrowhead). (F) Immediately after cell sheet transplantation, immunoconfocal microscopy demonstrated human leukocyte antigen (HLA)-positive cell sheet constructs attached to the epicardial membrane of the host myocardium. Green indicates HLA; blue, nuclei. Scale bar = 50 μm. (G) One week after cell sheet transplantation, immunoconfocal microscopy showed CD31, α-SMA-positive, and Notch3-positive cells in the host myocardium, whereas CD31 and α-SMA-positive, but Notch3-negative cell sheet construct was attached to the host myocardium. Green indicates Notch3; red, α-SMA; yellow, CD31; blue, nuclei. (H) Representative Notch3, CD31, and α-SMA staining of the border zone myocardium for the SMC-EPC bi-level cell sheet transplanted group. Green indicates Notch3; red, α-SMA; yellow, CD31; blue, nuclei. (I) Fate tracking of transplanted human-derived EPCs performed with anti-HLA and anti-vascular endothelial growth factor receptor 2 (VEGFR2). Immunostaining for HLA and VEGFR2 showed that transplanted EPCs over the border zone contributed to myocardium neovascularization 8 weeks after transplantation. Green indicates HLA; red, VEGFR2; blue, nuclei. Scale bar = 50 μm. (J) Fate tracking of transplanted human male-derived SMCs performed with fluorescence in-situ hybridization to identify male SMCs in the female recipient for SRY box transcription factor 1 (SOX1) gene of male cells. Immunostaining with an antibody against SOX1 and α-SMA demonstrated that SOX1-positive SMCs originating from the transplanted bi-level cell sheet migrated into the treated myocardial tissues and contributed partly to myocardium neovascularization 8 weeks after transplantation. Green indicates SOX1; red, α-SMA; blue, nuclei. Scale bar = 200 μm.