Co-culture of human cardiomyocyte and human amnion epithelial stem cells in amnion bilayer matrix for cardiomyogenesis
Our study developed a culture system to deliver cells for the regeneration of infarcted myocardial tissue. Human cardiomyocytes (hCardio) and human amnion epithelial stem cells (hAESC) were co-cultured in a biological scaffold forming a 3-D matrix, prepared as a heart patch candidate. The current investigation was aimed for assessment of hCardio-to-hAESC seeding ratio for the best conditions to provide cardiomyogenesis, i.e., 1:5 or the 1:6. This ratio corresponded to optimal cell number of 500,000 cells/cm<sup>2</sup>, thus providing a 12-cm<sup>2</sup> heart patch. This cell ratio was also published in our previous study using human adipose stem cells and hCardio. The hCardio was isolated from the heart tissue taken during surgical correction performed in patients with right ventricular hypertrophy. The cell isolates expressed cTnT (10.7%), cKit/CD117 (16%), ICAM (94%), and PECAM+/VCAM- 33%. The hAESC was isolated from an elected donor at Caesarean section. It showed expression of TRA-1-60 (82.4%), SSEA-4 (28.2%), Oct-3/4 (2.9%), Nanog (11.4%), without expression of immune antigens, e.g., HLA-DR (0%), HLA-ABC (0.2%) as well as low-level expression of mesenchymal stem cell (MSC) markers, i.e., CD73 (20.2%), CD90 (0.4%), CD105 (59.2%). These cells did not exhibit Lin phenotype (CD3, CD14, CD16, CD19, CD20, CD56). At both seeding ratios, the co-culture matrix also released TNF-α at low levels (<1 pg/ml) throughout cultivation from day 1 to day 8. Both groups had shown a consistent growth of cells over time. Confocal images showed that the cell population has expanded and migrated to the deeper plane up to 140 µm after 5 days of incubation in both 1:5 and 1:6 groups. The cells seemed to connect and form projections, starting by day 5, being more obvious among the 1:5 group. They proliferated rapidly until no solitary cells were observed after day 8 of co-culture. Expression of the cardiomyogenesis genes, e.g., cTnT and ACTN2 in the 1:6 group were expressed, similarly to normal cardiomyocytes after eight days of cultivation. MHC genes in the 1:6 group were also expressed, although less than 1-fold to normal cardiomyocytes. This study indicated that hCardio and hAESC at 1:6 ratio seeded on amnion bilayer could support cardiomyogenesis derivation from the progenitor cells.