[An experimental study on rabbit bone marrow mesenchymal stem cells double-labeled by PKH26 and 5-bromo-2'-deoxyuridine in vitro and application in cardiac patch].

Objective: To study the biological characteristic of rabbit bone marrow mesenchymal stem cells (BMSCs) double-labeled by PKH26 and BrdU in vitro, and to construct tissue engineered cardiac patch in vitro.

Methods: The BMSCs were harvested from 6-month-old New Zealand rabbits and labeled with PKH26 and BrdU. The growth and fluorescent intensity were observed by inverted phase contrast microscope, fluorescent microscope, flow cytometry, and MTT detection. The characteristics of double-labeled BMSCs differentiating into osteoblasts and adipocytes, respectively, in vitro were identified by alkaline phosphatase (ALP) staining, Alizarin red staining, Oil red O staining, immunocytochemical technique of collagen type I, and osteocalcin expression. The labeled BMSCs were seeded on the small intestinal submucosa (SIS) and co-cultured for 5-7 days to construct tissue engineered cardiac patch. The patches were tested by inverted phase contrast microscope, fluorescent microscope, scanning electron microscope, and HE staining to observe the cell proliferation.

Results: The double-labeled cells grew well and showed red fluorescence. There was no significant difference in the growth characteristic between the labeled and unlabeled cells. There was no significant difference in the expression of stem cell specific surface antigen between before labeling and after labeling. After osteogenic induction of labeled BMSCs, ALP staining and Alizarin red staining were positive, and the cells expressed collagen type I and osteocalcin. After adipocytes induction, lipid droplets could be observed in cytoplasm by Oil red O staining. After the co-culture in vitro for 5-7 days, the double-labeled cells grew well, showing a multi-layer cellular structure on the surface of SIS.

Conclusion: Rabbit BMSCs can be double-labeled with PKH26 and BrdU stably. The labeled cells still have the potential of self-renewal ability and multi potent differentiation ability; tissue engineered cardiac patch can be constructed by co-culturing labeled BMSCs and SIS in vitro.