Preparation of myocardial patches from DiI-labeled rat bone marrow mesenchymal stem cells and neonatal rat cardiomyocytes contact co-cultured on polycaprolactone film

Abstract

To provide better treatment of myocardial infarction, DiI-labeled bone marrow mesenchymal stem cells (BMSCs) were contact co-cultured with cardiomyocytes (CMs) on polycaprolactone (PCL) film to prepare myocardial patches. BMSCs from Sprague Dawley rats were isolated, cultured, and characterized for expression of surface markers by flow cytometry. CMs were isolated from suckling rats. After BMSCs were cultured for three generations, they were labeled with DiI dye. DiI-labeled BMSCs were co-cultured with CMs on PCL film in the experimental group, while CMs were replaced with the same amount of unlabeled BMSCs in the control group. After 24 h, cell growth was observed by light microscopy and cells were fixed for scanning electron microscopy (SEM). After 7 d of co-culture, cells were stained for immunofluorescence detection of myocardial markers cardiac troponin T (cTnT) andα-actin. Differentiation of BMSCs on PCL was observed by fluorescence microscopy. The efficiency of BMSC differentiation into CMs was analyzed by flow cytometry on the first and seventh days of co-culture. CMs were stained with calcein alone and contact co-cultured with DiI-labeled BMSCs on PCL film to observe intercellular dye transfer. Finally, cells were stained for immunofluorescence detection of connexin 43 (Cx43) expression and to observe the relationship between gap junctions and contact co-culture. BMSCs were identified by flow cytometry as strongly positive for CD90 and CD44H, and negative for CD11b/c and CD45. After co-culture for 24 h, cells were observed to have attached to PCL by light microscopy. Upon appropriate excitation, DiI-labeled BMSCs exhibited red fluorescence, while unlabeled CMs did not. SEM revealed a large number of cells on the PCL membrane and their cell state appeared normal. On the seventh day, some DiI-labeled BMSCs expressed cTnT andα-actin. Flow cytometry showed that the rate of stem cell differentiation in the experimental group was significantly higher than the control group on the seventh day (20.12% > 3.49%,P< 0.05). From the second day of co-culture, immunofluorescence staining for Cx43 revealed green fluorescent puncta in some BMSCs; from the third day of co-culture, a portion of BMSCs exhibited green fluorescence in dye transfer tests. Contact co-culture of DiI-labeled BMSCs and CMs on PCL film generated primary myocardial patches. The mechanism by which contact co-culture promoted differentiation of the myocardial patch may be related to gap junctions and gap junction-mediated intercellular signaling pathways.

Keywords: DiI labeling; contact co-culture; gap junction; myocardial patch; stem cell therapy.