Differentiation Of Bone Marrow Mesenchyma Stem Cells Into Cardiomyogenic Cells With Pacemaking Function

Background and ObjectivesSick sinus syndrome is caused by the sinus node dysfunction resulting in a variety of arrhythmias. Its pathogenesis is yet unclear, and there is no effective prevention and treatment up till now. The placement of artificial electronic cardiac, pacemaker is current main treatment. However, it is not the best treatment, and it has many limitations, such as limited battery life, needing a permanent pacemaker implanted and the lack of response to the autonomic nervous system. For these reasons, ideal pacemaker should be the biological pacemaker which considering physiological function of heart and adaptability of human body. Mesenchymal stem cells (MSCs) are stromal cells from bone marrow, with multiple differentiated potential, which are easy to be isolated and cultured. MSCs become one of priorities as cell replacement therapy because they can be differentiated into neural cells, muscle cells, fat cells and so on. MSCs can be used for autologous transplantation because there is no immune rejection problem. Animal experiments show that BMSCs transplanted into the damaged area of sinus node can be differentiated into sinus node-like cells and significantly improve the function of sinus node pacemaker, which indicates that the myocardial microenvironment have an induced effect on the differentiation of BMSCs. we observe the effectiveness of different concentrations of sinus-node cell lysate on BMSCs induced in vitro. Our aim is to observe whether the cTnI and HCN2 expressed by BMSCs after induced is positive or not. In terms of cell level, we discuss the effectiveness of sinus node's microenvironment on the function of differentiation and induction of BMSCs and explore its optimal concentration..Materials and methods1. The neonatal SD rats sinus node cells were cultured and purified with the method of differential attachment and 5-bromodeoxyuridine (BrdU) treatmen. The cultured cells were identified by morphology and electrophysiological properties2. The cultured cells were made into sinus node lysate by repeated freezing and thawing.3. The rat MSCs were isolated by gradient centrifugation method and expanded in vitro and were induced with different concentration of lysate of the sinus node cells. The group in ordinary medium was chosen as control group.4 The expression of protein cTnI and HCN2 were analyzed by immuno-histochemistry, and the cells function were proved with patch clamp techniques.Results1 Three different types of cells were observed among the cultured cells of sinus node, which were spindle cells, triangular cells and irregular cells. The spindle cells took up the greatest proportion, and the configuration and structure were accorded with the character of pacemaker cell of sinus node cells. Triangle cells isolated from sinus node were similar with triangle cells of atrial muscle.2 Different concentrations of sinus-node cell lysate can also induce the MSCs to differentiate into cardiac cells, which express the cardiocyte-specic protein and HCN2, and a hyperpolarization inward current can be tested in the differentiated cells by patch clamp technique. To some extent, the higher the concentration of sinus-node cell lysate is, the higher rates of differentiation are.Conclusions 1 The culture method of differential attachment and BrdU-treatment can significantly increase the proportion of spindle cells and is a reliable method of primary culture of the isolated sinus node cells. 2 Lysate of the sinoatrial cell can induce the MSCs to differentiate into cardiac cells with pacemaking function in vitro, which establishes theoretical foundation for the choice of seed cells on biological pacemaker.