Experimental study on contact co-culture of DiI labeled rat bone marrow mesenchymal stem cells and neonatal rat cardiomyocytes on polycaprolactone film to make myocardial patch
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 a-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 a-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.
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