Tissue-engineered smooth muscle cell and endothelial progenitor cell bi-level cell sheets prevent progression of cardiac dysfunction, microvascular dysfunction, and interstitial fibrosis in a rodent model of type 1 diabetes-induced cardiomyopathy

Characterization of SMCs, EPCs, and EPC-SMC bi-level cell sheets. a SMC-EPC bi-level cell sheet manufacturing protocol. b–g Immunocytochemistry demonstrated αSMA and SM22α on SMCs (b, c), and CD31, CD34, vWF, and VEGF-R2 on EPCs (d–g). Images of isotype controls of mouse, rabbit, and goat IgG were provided (h–j). Scale bar = 50 μm. k Percentages of each antigen for SMCs and EPCs were high, and demonstrated that our protocol yielded SMCs and EPCs with high purity. l A round-shaped scaffold-free SMC-EPC bi-level cell sheet in a 35 mm-dish. m Immunostaining of the SMC-EPC bi-level cell sheet with anti-vWF (green) and anti-αSMA (red) antibodies. The cell nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. EPC endothelial progenitor cell, SMC smooth muscle cell, MSC mesenchymal stem cell, DAPI 4′,6-diamidino-2-phenylindole, αSMA α smooth muscle actin, sm22α smooth muscle protein 22-α, vWF von Willebrand factor, VEGF-R2 vascular endothelial growth factor-receptor 2, ms IgG mouse immunoglobulin G, rb IgG rabbit immunoglobulin G, gt IgG gout immunoglobulin