Morphological, Electrophysiological and Molecular Analysis of the Cardiomyocytes Defferentiated From Bone Marrow Mesenchymal Stem Cells

我们从小鼠骨髓基质细胞中分离出一种心肌细胞系。细胞形态呈成纤维细胞样，但约30%的细胞在5-氮胞苷处理后形态发生改变；1周后与相邻细胞连接，2周后形成肌管样结构，开始自发跳动，3周后同步跳动。表达ANP和BNP。电镜显示心肌细胞样超微结构，包括典型的肌瘤和心房颗粒。这些细胞有几种类型的动作电位；窦结样和心室细胞样动作电位。所有细胞均具有较长的动作电位持续时间或平台期，相对较浅的静息膜电位，以及类似于心脏起搏器的舒张后期缓慢去极化。对肌球蛋白重链、肌球蛋白轻链、α-肌动蛋白等收缩蛋白基因的同工型分析表明，5-氮胞苷处理前其肌肉Nkx2.5/Csx、GATA4、TEF-1、MEF-2C mRNA表达，处理后表达MEF-2A、MEF-2D。使用成人组织作为心肌细胞的来源使得这个系统特别适合于心脏病基因治疗策略的发展。这一新细胞系为心肌细胞分化的研究提供了一个强有力的模型。

We have isolated a cardiomyogenic cell line from murine bone marrow stromal cells. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in approximately 30% of the cells; they connected with adjoining cells after 1 week, formed myotube-like structures and began spontaneous beating after 2 weeks, and beat synchronously after 3 weeks. The expressed ANP and BNP. Electron microscopy revealed a cardiomyocyte-like ultrastructure including typical sarcomeres and atrial granules. These cells had several types of action potentials; sinus node-like and ventricular cell-like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain and α-actin, indicated that their muscle Nkx2.5/Csx, GATA4, TEF-1 and MEF-2C mRNA before 5-azacytidine treatment, and expressed MEF-2A and MEF-2D after treatment. The use of adult tissues as a source of cardiomyocytes makes this system particularly appropriate for the development of gene therapy strategies for heart disease. This new cell line provides a powerful model for the study of cardiomyocytes differentiation.