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

为探讨胚胎干细胞（embryonic stem cell，ES cell）来源的心肌细胞移植到梗死心肌后的存活情况，将携带增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）的未分化小鼠ES细胞悬滴培养7 d后接种于培养皿上，分离搏动的细胞团，用于后续分析和移植。通过逆转录-聚合酶链反应检测心肌特异性基因、记录动作电位和肌节肌球蛋白免疫染色鉴定这些细胞为心肌细胞。体外研究的结果显示，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，动作电位类似于来自窦房结细胞和心室肌细胞的动作电位，并且肌节肌球蛋白染色阳性。将搏动簇注射到梗死和正常心肌之间的边界区。移植后10天和30天，观察肌节肌球蛋白的苏木精-伊红染色和免疫组化染色。作为体内研究的结果，移植后检测到EGFP表达的细胞，并且其中一些细胞对肌节肌球蛋白呈阳性。这些结果表明，ES细胞在体外可分化为心肌细胞，ES细胞源性心肌细胞可移植到梗死心肌中并在体内存活。

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.