Dai Yanmei, Qin Jian, Zhang Xiaogang, Zhang Xuehui, Chen Chuan, Liao Kangla
Department of Cardiology, First Affiliated Hospital, Chongqing Medical University, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 Apr;29(4):364-7.
To investigate the effect of electrical stimulation on the differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes in vitro.
Classical hanging-drop method was used to induce iPSCs from mice to form embryoid bodies (EBs) and vitamin C was contained in the medium through the induction period. According to whether or not electrical stimulation was used in the whole induction period, iPSCs were divided to electrical stimulation group and non-stimulation group. During the induction, dynamic morphological changes of the EBs were observed and photographed, the time point when beating EBs in each group appeared was recorded and the number of them was counted. The percentage of beating ones in all EBs was calculated as the differentiation rate of cardiomyocytes induced from iPSCs. Furthermore, expression of cardiac troponin T (cTnT) was observed by immunofluorescent staining under a confocal laser scanning microscope (CLSM), and mRNA expression levels of the related genes Oct-4, GATA-4 and α-MHC were analyzed by RT-PCR.
Compared with the non-stimulation group, beating cells in electrical stimulation group appeared in a shorter time, and the differentiation rate of cardiomyocytes was higher [(68.89 ± 5.09)% vs (52.22 ± 3.85)%, P<0.05]. c-TnT was expressed in the beating area of both groups, but the cells in the electrical stimulation group showed a more clear cytoskeleton. The mRNA level of Oct-4 decreased in a time-dependent manner in the whole period of induction and in the electrical stimulated group it decreased faster than the non-stimulation group (P<0.05). In addition, more GATA4 and α-MHC mRNA in electrical stimulation group were expressed than the non-stimulated group at the same point-in-time (P<0.05).
The electrical stimulation which simulates cardiac electrical microenvironment to some extent improved the differentiation of iPSCs into functional cardiomyocytes induced by vitamin C in vitro.
探讨电刺激对体外诱导多能干细胞（iPSCs）向心肌细胞分化的影响。
采用经典悬滴法从小鼠诱导iPSCs形成胚状体（EBs），诱导期间培养基中添加维生素C。根据整个诱导期是否施加电刺激，将iPSCs分为电刺激组和非刺激组。诱导过程中，观察并拍摄EBs的动态形态变化，记录每组出现跳动EBs的时间点并计数。计算跳动EBs在所有EBs中的百分比作为iPSCs诱导心肌细胞的分化率。此外，在共聚焦激光扫描显微镜（CLSM）下通过免疫荧光染色观察心肌肌钙蛋白T（cTnT）的表达，并通过RT-PCR分析相关基因Oct-4、GATA-4和α-MHC的mRNA表达水平。
与非刺激组相比，电刺激组跳动细胞出现时间更早，心肌细胞分化率更高[（68.89±5.09）%对（52.22±3.85）%，P<0.05]。两组跳动区域均有c-TnT表达，但电刺激组细胞的细胞骨架更清晰。诱导全过程中Oct-4的mRNA水平呈时间依赖性下降，电刺激组下降速度快于非刺激组（P<0.05）。此外，同一时间点电刺激组GATA4和α-MHC的mRNA表达量高于非刺激组（P<0.05）。
一定程度上模拟心脏电微环境的电刺激可提高体外维生素C诱导iPSCs向功能性心肌细胞的分化。
