Experimental Study Of Three-dimensional Coculture Using Adult Human Mesenchymal Stem Cells And Neonatal Rat Cardiomyocytes

Objective:Diseases of the cardiovascular system remain a leadingcause of morbidity and mortality worldwide. Myocardial infarction oftenleads to the eternal damage of heart, even to heart failure. Cardiactransplantation has been the only available treatment that significantlylengthens and improves quality of life. A possible strategy to restore heartfunction after myocardial injury is to replace the damaged tissue withhealth tissue. Cardiac tissue engineering (CTE) provides a promisingapproach that creates new functional heart tissue. Ideally, cells for use inCTE should be pluripotent, possess the capacity to differentiate to thedesired cell type under appropriate stimuli, have a limited capacity tomultiply, and should be capable of functional integration into the hostmyocardium. The mesenchymal stem cells (MSCs) has many of thesecharacteristics, and as such may be highly suitable for CTE. The Objectiveof our study is to determine whether the cardic microenvironment caninduce hMSCs to differentiate into cardiomyocytes based on liquidcollagen. Methods: 1.The efficient and practical metheod for the hMSCs from adult bonemarrow samples isolation and culture in vitro was established after therepeated practices. The hMSCs were isolated by gradient densitycentrifugation with percoll solution from adult bone marrow samples andwere analyzed by flowcytometry. 2.The efficient and practical metheod for Newborn rat cardiacmyocytes : Cardiac myocytes were isolated from 1 to 2 day old newbornWistar rats. Hearts were rapidly excised and placed in cold DMEM, Theventricles were minced, rinsed with trypsin solution, and subjected to aseries of digestions in trypsin on a shaker to release cells. Supernatantwere collected and trypsin was blocked by addition of 10% FBS. cells werecollected and resuspended in culture medium to cast engineered cardiactissues. 3.Three-dimensional coculture using adult human mesenchymal stemcells and Neonatal rat cardiomyocytes: labeled with DAPI, hMSCs weremixed with neonatal rat cardiac myocytes and liquid collagen type I, andthen cast in circular molds to construct circular hMSCs/ cardiomyocyte/collagen strand. After 14 days of culture in circular molds,immunohistochemical staining and confocal laser scanning microscopy(CLSM) were used to analyze the phenotype of hMSCs. Results: Human MSC surface antigen profiles obtained by flowcytometry were positive for CD29, CD44, and negative for, CD33, CD45,and HLA-DR. DAPI-labeled hMSCs with blue nuclei were distributed extensively inthe strand. Troponin-T was positive not only immunohistochemically butalso immunofluorescently in the DAPI-labeled hMSCs. The laser confocalmicroscopy also showed positive staining of actin and actinnin, whichresembled that of the cadiomyocytes around them. Connexin 43 was alsopositive in the labeled hMSCs, which indicated the formation of gapjunctions. Conclusions : hMSCs have the potential to differentiate intocardiomyocytes and can form gap junctions under appropriate engineeredcardiac tissue microenvironment.