Fig 2: BM-coated PS plates enhanced AHCF adhesion and miRcombo-mediated direct reprogramming into iCMs. Schematic representations of: (a) cell culture conditions on uncoated polystyrene (PS) and BM-coated PS dishes; (b) cell transfection (with miRcombo or negmiR) and culture on different conditions for 15 days post-transfection. Image created with Biorender.com under license. (c) Cell adhesion on uncoated and BM-coated PS dishes at different time points (0.5, 1, 2 and 4 h): cell adhesion percentage (left panel) and representative images (right panel) of adherent DAPI-stained cells. Experiments were conducted in triplicate, five fields for each sample were taken using a fluorescence microscope. Nuclei were counterstained using DAPI. Scale bar, 100 µm. (d–f) Expression of TNNT2, ACTC1 and CACNA1C cardiomyocyte genes analyzed by ddPCR in AHCFs transfected with negmiR or miRcombo and cultured on uncoated or BM-coated PS plates for 15 days. Results, expressed as DNA copies/µL, are the average of three independent experiments, each performed in triplicate. (g) Representative flow plots of cTnT+ cells in AHCFs transfected with negmiR or miRcombo and cultured on BM-coated PS plates (n = 3) for 15 days. (h) Graph showing flow cytometry percentage of cTnT+ cells in AHCFs transfected with negmiR or miRcombo and cultured on PS or BM-coated PS plates (n = 3) for 15 days. All data are expressed as the mean ± SEM. Statistical differences between the groups were determined with two-sided t-tests. * p < 0.05, ** p < 0.01 and *** p < 0.001, **** p < 0.0001.

Fig 3: Induction of cardiomyocyte markers in miRcombo-transfected AHCFs. (A,B) Gene expression of cardiomyocyte markers TNNT2 and TNNI3 in AHCFs transfected with miRcombo (red) or negmiR (blue) evaluated by ddPCR. Data are representative of three independent experiments, each performed in triplicate. (C) Representative flow plots (left panel) and percentage (right panel) of cTnT positive cells in AHCFs transfected with miRcombo (n = 3) and negmiR (n = 3) 15 days after transfection. (D–F) Gene expression of fibroblastic markers VIM, DDR2, and FSP-1 in AHCFs transfected with miRcombo (red) or negmiR (blue) by ddPCR. Data are representative of three independent experiments, each performed in triplicate. Stated P-value is versus negmiR.

Fig 4: Three-dimensional BM hydrogel enhances miRcombo-mediated cell reprogramming as assessed by immunofluorescence and calcium transient analysis. Immunofluorescence analysis: (a) cTnT expression and (b) percentage of cTnT-positive cells in miRcombo-transfected AHCFs cultured in 3D and 3D BM hydrogels for 15 days. Blue: nuclei counterstained using DAPI. Green: cTnT. Scale bar: 50 µm. (c) Percentage of cells showing Ca2+ transients, (d) slope of Ca+2 upstroke after caffeine induction (* p < 0.05) and (e) trend of Ca+2 oscillation vs. time after caffeine induction, for miRcombo-transfected cells in 3D and 3D BM hydrogels after 15 days culture time.

Fig 5: Coimmunostaining of cTNI (cardiac troponin I) and Col IV (collagen IV). A, Remote healthy myocardium, (B) lesion margin and healthy myocardium (dotted line), and (C) lesion core. A, In remote areas, baseline level of Col IV in uninjured myocardium. B, At the lesion margin, slightly increased Col IV is observed in the region with abrupt myocyte loss in comparison to adjacent region with surviving myocytes (left of the dotted line). Loss of red CTNT staining represents damage to myocytes (upper), (C) in the lesion core, significantly increased Col IV is observed where no surviving myocytes remains. Increased collagen deposition is seen in the lesion core. Color codes: cTNT: red, Col IV, cyan. Nuclei, blue. Representative images are from 10 to 20 scans per area from 6 different sections. Scale bar, 20 µm.