Embryonic stem cell (ES cell)-derived cardiomyocyte transplantation into the infarcted myocardium

将胚胎干细胞（ES细胞）来源的心肌细胞移植到梗塞心肌中

• 批准号: 13671401
• 负责人: TANIGUCHI Shigeki
• 金额: $ 2.37万
• 依托单位: NARA MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671401/
• 关键词: embryonic stem cell; cardiomyocyte; cell transplantation; myocardial infarction; 移植

The purpose of this study was to investigate the survival of embryonic stem cell (ES cell)-derived cardiomyocytes transplanted into the infarcted myocardium.In in vitro study, undifferentiated mouse ES cells carrying the enhanced green fluorescent protein (EGFP) were cultured in hanging drops for 7 days and plated onto dishes, and then beating clusters were dissected and used for the following analyses and transplantation. These cells were identified as cardiomyocytes by reverse transcription-polymerase chain reaction of cardiac-specific genes, recording of action potential, and immunostaining of sarcomeric myosin staining. As a result of in vitro study, ES cell-derived cardiomyocytes were shown to have myosin light chain-2v and alpha-myosin heavy chain genes expression, action potential like one from a sinus nodal cell and a ventricular myocyte, and positive for sarcomeric myosin staining.In in vivo study, a myocardial infarction rat model was made by the ligation of the left coronary artery. Beating clusters were injected into the border zone between the infarcted and normal myocardium. Ten and 30 days after transplantation EGFP expression, hematoxylin-eosin staining and immunostaining of sarcomeric myosin were investigated. As a result of in vivo study, EGFP-expressed cells were detected after transplantation, and a few of them were positive for sarcomeric myosin. No lymphocytic infiltration was observed.These results suggested that ES cells differentiated into cardiomyocytes in vitro and ES cell-derived cardiomyocytes could be transplanted into the infarcted myocardium and survive in vivo.

本研究的目的是研究胚胎干细胞（ES细胞）来源的心肌细胞移植到梗塞心肌后的存活情况。在体外研究中，携带增强型绿色荧光蛋白（EGFP）的未分化小鼠ES细胞在悬滴中培养7天并铺在培养皿上，然后解剖打浆簇并用于以下分析和移植。通过心脏特异性基因的逆转录聚合酶链反应、记录动作电位以及肌节肌球蛋白染色的免疫染色，将这些细胞鉴定为心肌细胞。体外研究结果显示，ES细胞来源的心肌细胞具有肌球蛋白轻链2v和α-肌球蛋白重链基因表达，具有与窦房结细胞和心室肌细胞类似的动作电位，并且肌球蛋白呈阳性染色。在体内研究中，通过结扎左冠状动脉制作心肌梗死大鼠模型。将跳动簇注入梗塞心肌和正常心肌之间的边界区域。移植后10天和30天，研究了EGFP表达、苏木精-伊红染色和肌节肌球蛋白的免疫染色。体内研究结果显示，移植后检测到表达EGFP的细胞，其中少数细胞肌节肌球蛋白呈阳性。没有观察到淋巴细胞浸润。这些结果表明ES细胞在体外分化为心肌细胞，并且ES细胞衍生的心肌细胞可以移植到梗塞心肌并在体内存活。