Functional analysis of gap junctional intercellular communication using differentiation of embryonic stem cells in vitro

1.ES细胞体外心肌细胞分化过程中Connexin的表达在心肌细胞出现跳动前后未分化ES细胞和胚状体中检测到C_x43和C_x45的转录本。相比之下，在未分化的 ES 细胞中未观察到 C_x40 转录本，并且在 3 天和 5 天的胚状体中几乎检测不到。随着与心脏特异性基因类似的跳动细胞的出现，C_x40 转录物显着增加。未分化的 ES 细胞、胚状体生长的预搏动细胞和 ES 细胞衍生的搏动心肌细胞中存在染料偶联。当染料被注射到跳动细胞中时，染料扩散仅限于邻近的跳动细胞。免疫荧光表明，C_x43 蛋白不仅定位于跳动细胞，还定位于周围非跳动细胞，而肌球蛋白重链 α/β 仅在跳动细胞中呈阳性。这些数据表明，多种连接蛋白的表达在ES细胞体外心肌细胞分化过程中受到差异性调节，并且C_x40表达可能与心肌细胞分化的早期阶段有关。2.C_x43缺陷型ES细胞的体外心肌细胞分化荧光黄染料转移实验测得C_x43缺陷型ES细胞几乎没有显示出染料偶联，但在体外存在心肌细胞分化，这与心肌细胞分化没有显着差异。野生型 ES 细胞的情况。 RT-PCR 显示，与野生型 ES 细胞一样，随着跳动细胞的出现，C_x40 和心脏特异性基因转录物的数量显着增加，而在 C_x43 缺陷细胞中未检测到 C_x43 转录物。这些数据表明C_x43对于体外ES细胞的心肌细胞分化不是必需的，并且其他连接蛋白例如C_x40和C_x45的表达不受C_x43表达缺失的影响。

1.Connexin expression during cardiomyocytic differentiation of ES cells in vitroThe transcripts for C_x43 and C_x45 were detected in undifferentiated ES cells and in embryoid bodies before and after the appearance of beating cardiomyocytes. In contrast, C_x40 transcripts were not observed in undifferentiated ES cells, and were barely detectable in 3-and 5-day-old embryoid bodies. C_x40 transcripts significantly increased with the appearance of beating cells similar to those of cardiac-specific genes. Dye coupling was present among undifferentiated ES cells, pre-beating cells of embryoid body outgrowth and ES cell-derived beating cardiomyocytes. When dye was injected into a beating cell, dye spread was restricted to neighboring beating cells. Immunofluorescence demonstrated that C_x43 protein was localized not only in beating cells but also in surrounding non-beating cells, whereas myosin heavy chain alpha/beta was exclusively positive in the beating cells. These data suggest that the expression of multiple connexins is differentially regulated during the cardiomyocytic differentiation of ES cells in vitro and that C_x40 expression may be linked to early stages in cardiomyocytic differentiation.2.Cardiomyocytic differentiation of C_x43-deficient ES cells in vitroC_x43-deficient ES cells showed little dye coupling measured by Lucifer Yellow dye transfer assay, but there was cardiomyocytic differentiation in vitro, which was not significantly different from that of wild-type ES cells. RT-PCR revealed that the amounts of C_x40 and heart-specific gene transcripts significantly increased with the appearance of beating cells as with wild-type ES cells, whereas C_x43 transcripts were not detected in the C_x43-deficient cells. These data indicate that C_x43 is not essential for cardiomyocytic differentiation of ES cells in vitro and that expression of other connexins such as C_x40 and C_x45 is not influenced by the absence of C_x43 expression.