Objective:To investigate the possible mechanism of DiI labeled bone marrow mesenchymal stem cells (BMSCs) in contact co-cultured with neonatal rat cardiomyocytes (CMs) on polycaprolactone (PCL) film to make myocardial patch.Methods:BMSCs from Sprague Dawley rats (aged 5-6 weeks) were isolated, cultured, and characterized for surface marker expression using flow cytometry. CMs from 15 neonatal rats were isolated and cultured. After cultured for 3 generations, BMSCs were labeled with DiI dye. On PCL film, DiI labeled BMSCs were co-cultured with CMs as the experimental group, and CMs were replaced with the same amount of unlabeled BMSCs in the control group. After 24 h of co-culture, the cell growth was observed under fluorescence microscope and the co-culture was observed under scanning electron microscope. Immunofluorescence staining was performed after 7 days to detect myocardial markers, including cardiac troponin T (cTnT) and α-actinin. BMSC differentiation on the PCL film was observed under a fluorescence microscope. The differentiation efficiency of BMSCs into cardiomyoid cells was analyzed by flow cytometry on days 1 and 7 of co-culture. Intercellular dye transfer was observed by staining CMs with calcein and co-culturing them with DiI-labeled BMSCs on PCL film. The cells were stained with immunofluorescence to detect the expression of connexin 43 (Cx43) and observe the relationship between gap junction and contact co-culture.Results:Flow cytometry showed strong positivity for CD90 and CD44 and negativity for CD11b/c and CD45 on BMSCs. After 24 h of co-culture, DiI labeled BMSCs glowed red on the PCL film, while unlabeled CMs did not; the number of cells on PCL film was large and cell morphology appeared normal under scanning electron microscope. On the 7th day of co-culture, some DiI labeled BMSCs expressed cTnT and α-actinin. Flow cytometry showed a higher differentiation rate of stem cells in the experimental group on day 7 compared to the control group ((20.12±0.15)% vs. (3.49±0.20)%, P<0.05). From the second day of co-culture, some BMSCs exhibited green dot fluorescence in Cx43 immunofluorescence staining; and by the third day, dye transfer test showed green fluorescence emission from some BMSCs. Conclusion:Contact co-culture of DiI labeled BMSCs and CMs on PCL film can make myocardial patch. The mechanism of contact co-culture promoting the differentiation and formation of myocardial patch may be associated with gap junctions and intercellular signal pathways mediated by gap junctions.