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

Abstract

DiI-labeled BMSCs were contact co-cultured with CMs on PCL film to prepare myocardial patches. BMSCs 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. After 7 days of co-culture, cells were stained for immunofluorescence detection of myocardial markers cardiac troponin T (cTnT) and α-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. 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. Scanning electron microscopy 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 and α-actin. Flow cytometry showed that the rate of stem cell differentiation in the experimental group was significantly higher than the control group on the seven 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 successfully generated myocardial patches.

What happens to the osteoporotic bone mesenchymal stem cells? Evidence from RNA sequencing

“…Flow cytometry analysis showed that the cells were positive for CD29 and CD90, and negative for CD45. The results are consistent with existing evidences on BMSCs surface markers [20][21]: CD29 is an adhesion marker and CD90 is a marker of undifferentiated stem cells, while the lack of CD45 means the cells are not haematopoietic cells. In addition, after treating with differentiation inducers, the cells presented osteogenic and adipogenic differentiation ability.…”