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人诱导多能干细胞分化的心肌细胞电生理鉴定

张弘源^1,2, 张婷婷^1,2, MachukiJeremiahOng’achwa¹, 吴立娟^1,2, 孙红^1,*

¹徐州医科大学生理机能与损伤实验室，徐州 221004；²徐州医科大学心血管病研究所，徐州 221004

摘要

本文旨在研究人诱导多能干细胞分化的心肌细胞(human induced pluripotent stem cell-derived cardiomyocytes, hiPSC-CMs)的电生理特性。采用时序性短暂激活/短暂抑制Wnt信号通路的方法将未分化的IMR90-4细胞系定向诱导分化为心肌细胞。用免疫荧光染色法和流式细胞术检测心肌肌钙蛋白T (cardiac troponin T, cTnT)蛋白表达，计算hiPSC-CMs的分化率。用膜片钳技术记录hiPSC-CMs的动作电位，根据动作电位的表现对心肌细胞进行分类，并进一步分析电生理特征。结果显示，hiPSC-CMs的纯度大于95%。根据动作电位的表现，hiPSC-CMs可分为心房肌样细胞、心室肌样细胞和窦房结样细胞。其中，心室肌样细胞的动作电位时程(action potential duration, APD)、动作电位幅度(action potential amplitude, APA)和最大去极化速率(dV/dtmax)均大于心房肌样细胞和窦房结样细胞，窦房结样细胞的dV/dtmax低于心室肌样细胞和心房肌样细胞。以上结果表明hiPSC-CMs纯度高，并且分化出的三种不同类型的心肌细胞具有和成熟心肌细胞相似的动作电位特征。

关键词： 人诱导多能干细胞; 心肌细胞; 动作电位; 电生理

分类号：R337.5

Electrophysiological identification of human induced pluripotent stem cell- derived cardiomyocytes

ZHANG Hong-Yuan^1,2, ZHANG Ting-Ting^1,2, Machuki Jeremiah Ong’achwa¹, WU Li-Juan^1,2, Sun Hong^1,*

¹Department of Physiology, Xuzhou Medical University, Xuzhou 221004, China；²Institute of Cardiovascular Disease Research, Xuzhou Medical University, Xuzhou 221004, China

Abstract

The present study was aimed to characterize the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). IMR90-4 cells were induced to differentiate into cardiomyocytes by temporal modulation of regulators of canonical Wnt signaling. The protein expression of cardiac troponin T (cTnT) was detected by immunofluorescence staining and flow cytometry, and the differentiation rate of hiPSC-CMs was calculated. The action potentials (APs) of hiPSC-CMs were recorded by patch clamp and used to classify different types of cardiomyocytes. The electrophysiological characteristics of hiPSC-CMs were further analyzed. The results showed that the cTnT positive rate of hiPSC-CMs was above 95%. hiPSC-CMs were differentiated into 3 types of cardiomyocytes based on the properties of AP: ventricular-, atrial- and nodal-like cells. In comparison with the other two types of cells, the APs of ventricular-like cells exhibited longer duration, higher amplitude and higher dV/dtmax. The nodal-like cells had the lowest dV/dtmax among all the three types. These results indicate that hiPSC can be differentiated into the cardiomyocytes with high purity and the differentiated hiPSC-CMs have similar electrophysiological characteristics to adult cardiomyocytes.

Key words: human induced pluripotent stem cell; cardiomyocyte; action potential; electrophysiology

收稿日期：2017-09-11　　录用日期：2018-01-11

通讯作者：孙红　　E-mail: sunh@xzhmu.edu.cn

DOI: 10.13294/j.aps.2018.0040

引用本文：

张弘源, 张婷婷, MachukiJeremiahOng’achwa, 吴立娟, 孙红. 人诱导多能干细胞分化的心肌细胞电生理鉴定[J]. 生理学报 2018; 70 (3): 281-286.

ZHANG Hong-Yuan, ZHANG Ting-Ting, Machuki Jeremiah Ong’achwa, WU Li-Juan, Sun Hong. Electrophysiological identification of human induced pluripotent stem cell- derived cardiomyocytes. Acta Physiol Sin 2018; 70 (3): 281-286 (in Chinese with English abstract).