The Study To Differentiate Cardiomyocytes Under Co-cultured BMSCs And Cardiomyocytes

ObjectiveBone marrow stromal stem cells (BMSCs) are to be multipotent cells, which are present in bone marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, and fat et al. Under various conditions, these cells could be differentiated the adipocytic, chondrocytic, or osteocytic lineages. By finding indicated, miazines chemical materials 5-aza can induce to differentiate bone marrow stromal stem cells into cardiomyocytes, it make BMSCs to become one of the most future cells for cellular transplantation in treatment ischemic heart disease.The purpose of this study were to growth characteris of cultured murine marrow stromal stem cells; to investigate the differentiate potential of BMSCs under co-cultured BMSCs and cardiomyocytes without any inducement; and by GFP transfect BMSCs, observe expression ability of GFP signed BMSCs. Based the experiment, we expect to understand the biological characters of BMSCs further more, and offer reliable bases before they are applied to clinic.Methods1. A population of homogeneous murine marrow stromal stem cells were isolated and cultured from bone marrow by their abilities to proliferate in culture with an attached well-spread morphology. And the shapes and structures of the cells were observed by phase contrast microscope.2. A population of homogeneous murine cardiomyocytes were isolated and cultured with a difference-speed adherence method. And the shapes and structures of the cells were observed by phase contrast microscope.3. GFP plasmid transfect the third generation BMSCs. Observe the transfection efficiency of cells by Olympus fluorescence phase contrast microscope.4. Centrifuge and collect after transfected BMSCs and co-cultured with cardiomyocytes by ratio 1:3. And the shapes and structures of the cells were observed by Olympus fluorescence phase contrast microscope.Results1. Morphologic observation of BMSCs: 24h after the initial seeding, BMSCs attached the surface of foster. There were distinct formation of clonies later, and the shape, the size and the density of the clonies were different; and BMSCs reached confluence 7-10 days later. Serial subcultivation BMSCs were no more formation of clonies, but distributed uniform fusiform.2.The cultivation of cardiomyocytes: change liquid 3h after, and cultivate in incubator. 24h after 95 percent cardiomyocytes beat rhythmically by phase contrast microscope.3.The result of GFP transfect BMSCs: After transfection, BMSCs still grow adherence, shuttle or polygon, still multiplication, but multiplication slowly. Under Olympus fluorescence phase contrast microscope, after 24h transfection cells can express green fluorescence, but quantity less and intensity lower; after 48h, green fluorescence change stronger.4.Change of BMSCs morphology and growth behavior after co-culture: after co-culture, BMSCs multiplication slowly, and easy become polygon or flat, along with the time of co-cultured prolong, most BMSCs generate cell disruption; after 24h many adherence nulli-dancing spindle cells adjoin pulsating cardiomyocytes; after 48h these spindle cells growth by clonies, and individual labeled BMSCs were observed myotubular structure by Olympus fluorescence phase contrast microscope. These BMSCs usually adjoin un-labeled pulsating cardiomyocytes. After 4 days part un-labeled BMSCs near to pulsating cardiomyocytes beat feeblely; at the same time, many BMSCs begin decease, to 64h most cells deceased. Conclusion1. Bone marrow stromal stem cells (BMSCs) could be isolated from bone marrow, and proliferated in vitro.2. GFP transfection BMSCs can express long and high efficient, and toxicity small to cell. It could be a method to filtration stem cells and a method to examine the functional state of stem cells.3. Bone marrow stromal stem cells (BMSCs) could be differentiated cardiomyocytes under co-cultured BMSCs and cardiomyocytes without any inducement.