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

本研究旨在探讨胚胎干细胞(ES细胞)来源的心肌细胞移植到梗死心肌的存活情况。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细胞在悬液中培养7d后，接种于培养皿中，然后解剖跳动的细胞簇，用于后续的分析和移植。通过心肌特异性基因的逆转录聚合酶链式反应、动作电位的记录和肌节肌球蛋白染色的免疫染色，这些细胞被鉴定为心肌细胞。体外研究表明，ES细胞来源的心肌细胞表达肌球蛋白轻链-2v和α-肌球蛋白重链基因，动作电位类似于窦房结细胞和心室肌细胞，肌节肌球蛋白染色阳性。将搏动簇注入梗死心肌与正常心肌交界处。移植后10天和30天观察EGFP表达、苏木精-伊红染色和肌球蛋白免疫染色。体内研究的结果是，移植后检测到绿色荧光蛋白表达的细胞，其中少数细胞肌节肌球蛋白阳性。提示ES细胞在体外可分化为心肌细胞，ES细胞来源的心肌细胞可移植到梗死区心肌内存活。

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.