Fig 2: Creation and characterization of the smooth muscle cell (SMC)-endothelial progenitor cell (EPC) bi-level cell sheet. (A) The diagram shows the methods for engineering the cell sheet. Cell-sheet is harvested from the temperature-responsive culture dish. (B) A round-shaped scaffold-free SMC-EPC bi-level cell sheet in a 35-mm dish. (C) The bi-level cell sheet maintained CD31 positive EPC and SM22-a positive SMC in separate layers. Green indicates CD31; red, SM22-a. The cellular nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 50 µm. (D-F) Representative scanning electron microscope (SEM) images of SMC-EPC bi-level cell sheet. (D) The examination of the surface (EPC layer) of the SMC-EPC bi-level cell sheet suggested that EPCs form a smooth confluent layer with cell borders in close contact with each other, thereby forming a thin film-like layer on top of the SMC. (E) The examination of the bottom (SMC layer) of the SMC-EPC bi-level cell sheet indicated that SMC morphology varies regionally from spherical to flattened, suggesting that SMCs differentiated into an elongated and spindle-shaped dense sheet-like construct with cellular extensions forming a densely adherent layer. (F) The interface between the two layers of the SMC-EPC bi-level cell sheet (SMC-EPC border-zone) showed the development of a cross-linked structure.

Fig 3: Schematic overview of the MAC sorting strategy to obtain endothelial cells (marker: CD31) and myoblasts (marker: CD56). Both cell types are isolated from the CD45-negative cell population (depleted cells of hematopoietic origin).

Fig 4: Time course analysis of the transfection efficiency of Gel@MSN/miR-21-5p in vivo.MSNs were prelabeled with FITC (green), and miR-21-5p was prelabeled with Cy3 (red). The hydrogel (FITC-labeled Gel@MSN/miR-21-5p or Cy3-labeled Gel@MSN/miR-21-5p) was injected into the mid-myocardium of each target site in the pigs. The duration and efficiency of MSNs and miRNA delivery upon Gel@MSN/miR-21-5p injection were monitored using time course analysis at 1, 14, and 28 days after injection. (A) Histological sections of the infarct region in the Gel@MSN/miR-21-5p group were immunolabeled with the hematoxylin and eosin (H&E) macrophage marker F4/80. (B) Histological sections of the infarct region in the Gel@MSN/miR-21-5p group were immunolabeled with the endothelial marker CD31. Cell nuclei were counterstained with DAPI (blue). (C) F4/80+FITC+ and CD31+Cy3+ double-positive cells were quantified from at least eight high-resolution images acquired from at least eight different regions of each heart. (D) miR-21-5p levels were detected using real-time quantitative PCR at different time points. The transfection efficiency was determined by quantifying the miRNA level. Scale bars, 100 µm. n = 3 per group. The data are shown as means ± SD. Photo credit: Yan Li, Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.

Fig 5: Characterization of isolated SkMVECs. (A) Immunofluorescence staining of isolated SkMVECs for the endothelial cell markers CD31 (pseudo-color red) and vWF (pseudo-color red). Nuclei are DAPI-stained (blue) (B) Quantitative characterization by flow cytometry for CD31. (C) Tube formation assay of GFP-labeled isolated SkMVECs on growth factor reduced Matrigel imaged after 4 h showing endothelial networks formed in vitro (pseudo-color red).