诱导多能干细胞分化的心肌细胞电生理特点  

Electrophysiological characteristics of cardiomyocytes differentiated from induced pluripotent stem cells

作　　者：熊挺淋 应梦慧 张丽莎 张晓刚[3] 杨燕 Xiong Tinglin;Ying Menghui;Zhang Lisha;Zhang Xiaogang;Yang Yan(Department of Cardiology,Nanchong Central Hospital,Second Clinical Medical College,North Sichuan Medical College,Nanchong 637000,Sichuan Province,China;Sichuan Academy of Medical Sciences,Sichuan Provincial People’s Hospital,Chengdu 610000,Sichuan Province,China;First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China;Sichuan Chidingshengtong Biological Technology Co.,Ltd.,Chengdu 610041,Sichuan Province,China)

机构地区：[1]南充市中心医院心内科·川北医学院第二临床医学院,四川省南充市637000 [2]四川省医学科学院·四川省人民医院,四川省成都市610000 [3]重庆医科大学附属第一医院,重庆市400016 [4]四川驰鼎盛通生物科技有限公司,四川省成都市610041

出　　处：《中国组织工程研究》2022年第7期1063-1067,共5页Chinese Journal of Tissue Engineering Research

基　　金：四川省科学技术厅重点研发项目(2019YFS0314),项目负责人:杨燕。

摘　　要：背景:诱导多能干细胞定向分化后移植治疗解决了免疫排斥及伦理问题,其向心肌细胞的定向分化为心肌细胞再生研究提供了可能。目的:观察诱导多能干细胞分化的心肌细胞电生理功能。方法:体外培养诱导多能干细胞,采用直接悬浮培养法及分化培养基诱导形成拟胚体,观察细胞生长情况,记录心肌细胞的形成时间、数目和收缩率;使用Axon Axopatch 200B单探头超低噪声膜片钳放大器及MED64多电极阵列系统检测心肌细胞的电生理特点,以及观察分化心肌细胞对异丙肾上腺素的反应性;免疫荧光检测分化心肌细胞中心肌肌钙蛋白T的表达。结果与结论:①诱导多能干细胞生长特点与胚胎干细胞相似,形似鸟巢,呈集落样生长;②在分化培养基培养下,悬浮培养5 d后诱导多能干细胞聚集而形成大小不一的拟胚体,在拟胚体贴壁生长期间,最高将近10%的菌落出现跳动的心肌细胞,第20天和第25天时的搏动率明显高于其他时间点,差异有显著性意义(P<0.05);③诱导多能干细胞分化的心肌细胞观察到了心室、心房和窦房结样细胞的动作电位;④异丙肾上腺素可以提高诱导多能干细胞分化的心肌细胞自发搏动频率;⑤细胞免疫荧光提示诱导多能干细胞分化的心肌细胞中肌钙蛋白T呈阳性表达;⑥结果表明,诱导多能干细胞分化的心肌细胞具有窦房结样、心房样和心室样的动作电位,且具有肾上腺素信号传导。BACKGROUND:Transplantation therapy after the directional differentiation of induced pluripotent stem cells can solve the immune elimination and ethical problems,and directional differentiation of induced pluripotent stem cells into cardiomyocytes provides the possibility of cardiomyocyte regeneration research.OBJECTIVE:To detect the electrophysiological function of cardiomyocytes differentiated from induced pluripotent stem cells.METHODS:By culturing induced pluripotent stem cells in vitro,direct suspension culture and differentiation medium were used to induce induced pluripotent stem cells to form embryoid bodies.Cell growth was observed and formation time,number and contraction rate of cardiomyocytes were recorded.The electrophysiological characteristics of cardiomyocytes were detected by Axon Axopatch 200B single probe ultra-low noise patch clamp amplifier and MED64 multi-electrode array system.The responsiveness of differentiated cardiomyocytes to isoproterenol was observed.Cardiac troponin T expression levels in differentiated cardiomyocytes were detected by immunofluorescence.RESULTS AND CONCLUSION:(1)The growth characteristics of induced pluripotent stem cells were similar to embryonic stem cells,resembling a bird’s nest and growing like a colony.(2)In the differentiation medium culture,induced pluripotent stem cells aggregated to form embryoid bodies of different sizes after 5 days of suspension culture.During the adherent growth of embryoid bodies,up to 10%of the colonies appeared beating cardiomyocytes,and the beating rate on the 20^(th) and 25^(th) days was significantly higher than other time points(P<0.05).(3)Action potentials of ventricular,atrial and sinoatrial nodular cells were observed in induced pluripotent stem cells differentiated cardiomyocytes.(4)Isoproterenol could increase the spontaneous beat frequency of cardiomyocytes differentiated from induced pluripotent stem cells.(5)Cellular immunofluorescence indicated that cardiomyocytes differentiated from induced pluripotent stem cells were

关 键 词：干细胞 诱导多能干细胞 心肌细胞 胚胎干细胞 拟胚体 分化 动作电位 电生理 异丙肾上腺素 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]