Department of Histology and Embryology, Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Nagano, Japan.
Ann Anat. 2010 Sep 20;192(5):314-21. doi: 10.1016/j.aanat.2010.07.001. Epub 2010 Aug 5.
Bone marrow stromal cells (BMSCs) secrete soluble factors and display varied cell-biological functions. To confirm the ability and efficiency of BMSCs to induce embryonic stem cells (ESCs) into cardiomyocytes, mouse embryoid bodies (EBs) were co-cultured with rat BMSCs. After about 10 days, areas of rhythmically contracting cells in more solid aggregates became evident with bundle-like structures formed along borders between EB outgrowth and BMSC layer. ESC-derived cardiomyocytes exhibited sarcomeric striations when stained with troponin I (Trop I), organized in separated bundles. Besides, the staining for connexin 43 was detected in cell-cell junctions, which demonstrated that ESC-derived cardiomyocytes were coupled by gap junction in culture. The related genes of cardiomyocytes were found in these beating and no-beating EBs co-cultured with BMSCs. In addition, an improved efficiency of cardiomyocyte differentiation from ESC-BMSC co-culture was found in the serum-free medium: 5-fold up-regulation in the number of beating area compared with the serum medium. Effective cardiac differentiation was also recognized in transfer filter assay and in condition medium obtained from BMSC culture. A clear increase in the expression of cardiac genes and TropI protein confirmed further cardiac differentiation by BMP4 and Retinoic Acid (RA) treatment. These results demonstrate that BMSCs can induce cardiomyocyte differentiation from ESCs through soluble factors and enhance it with BMP4 or RA treatment. Serum-free ESC-BMSC co-culture represents a defined in vitro model for identifying the cardiomyocyte-inducing activity from BMSCs and, in addition, a straightforward experimental system for assessing clinical applications.
骨髓基质细胞（BMSCs）分泌可溶性因子，并表现出多种细胞生物学功能。为了确认 BMSCs 将胚胎干细胞（ESCs）诱导为心肌细胞的能力和效率，将鼠胚体（EBs）与大鼠 BMSCs 共培养。大约 10 天后，在更密集的聚集物中出现了具有节律性收缩的细胞区域，并且沿着 EB 外生和 BMSC 层之间的边界形成了束状结构。当用肌钙蛋白 I（Trop I）染色时，ESC 衍生的心肌细胞表现出肌节条纹，排列成分离的束。此外，在细胞-细胞连接处检测到连接蛋白 43 的染色，表明 ESC 衍生的心肌细胞在培养中通过缝隙连接偶联。在与 BMSCs 共培养的搏动和非搏动 EBs 中发现了心肌细胞的相关基因。此外，在无血清培养基中发现 ESC-BMSC 共培养的心肌细胞分化效率得到了提高：与血清培养基相比，搏动面积的数量增加了 5 倍。在转移滤器测定和 BMSC 培养获得的条件培养基中也观察到了有效的心脏分化。心脏基因和 TropI 蛋白的表达明显增加，进一步证实了 BMP4 和视黄酸（RA）处理的心脏分化。这些结果表明，BMSCs 可以通过可溶性因子诱导 ESCs 分化为心肌细胞，并通过 BMP4 或 RA 处理增强其分化能力。无血清 ESC-BMSC 共培养代表了一种用于鉴定 BMSCs 诱导心肌细胞活性的明确的体外模型，此外，还代表了一种用于评估临床应用的简单实验系统。
