利用人诱导多能干细胞构建心脏类器官方法的研究  

Generation of cardiac organoids from human induced pluripotent stem cells

作　　者：董竹君 张璟 李扬[1] 李玉琳[1] DONG Zhujun;ZHANG Jing;LI Yang;LI YULIN(Department of Vascular Biology,Beijing Anzhen Hospital,Capital Medical University,Beijing Institute of Heart,Lung and Blood Vessel Diseases,Beijing 100029,China)

机构地区：[1]首都医科大学附属北京安贞医院-北京市心肺血管疾病研究所,教育部心血管重塑相关疾病重点实验室科研基地建设-心血管重大疾病防治协同创新中心,100029

出　　处：《心肺血管病杂志》2024年第6期635-642,共8页Journal of Cardiovascular and Pulmonary Diseases

基　　金：国家自然科学基金(82171835)。

摘　　要：目的:探索人诱导多能干细胞(human induced pluripotent stem cells,hiPSCs)分化为心脏类器官的方法。方法:首先通过免疫荧光染色验证hiPSCs多能性标志物的表达;之后将hiPSCs培养为拟胚体(embryoid bodies,EBs),利用双向调控WNT(wingless and Int-1,WNT)信号通路法诱导EBs向心脏类器官分化,记录分化过程中的形态变化。最后,通过免疫荧光染色、实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)和微电极阵列(multi-electrode array,MEA)技术检测心脏类器官的细胞成分、基因表达谱、电生理特性和收缩舒张功能,验证心脏类器官分化方案的有效性。结果:免疫荧光染色显示hiPSCs中OCT4和SSEA4高表达。利用双向调控WNT信号的方法成功诱导hiPSCs产生跳动的心脏类器官。免疫荧光染色表明心脏类器官中cTNT阳性心肌细胞比例为(59.3±12.8)%,非心肌细胞比例为(40.7±12.8)%。qRT-PCR结果表明其中非心肌细胞包括平滑肌细胞、成纤维细胞及内皮细胞。与分化15D相比,分化30D的类器官中心肌成熟相关基因和心脏各项功能相关基因表达水平更高。MEA检测结果显示,随分化时间增加,类器官逐渐具备类似在体心脏的电生理特征,跳动频率减小[分化15d时跳动(58±5)vs.30d(38±5)次/min],收缩幅度增加15d时收缩幅度[(8.57±3.7)vs.30d(16.4±5.9)%]。结论:通过该方法能使hiPSCs分化出成熟且有功能的心脏类器官。Objective:To explore the method for differentiating human induced pluripotent stem cells(hiPSCs)into cardiac organoids.Methods:First,the expression of pluripotency markers in hiPSCs is validated using immunofluorescence(IF).Subsequently,hiPSCs are cultured into embryoid bodies(EBs),and differentiated into cardiac organoids through bidirectional modulation of the Wingless and Int-1(WNT)signaling pathway,with morphological changes recorded during the differentiation process.Finally,the cellular composition,gene expression profile,electrophysiological properties,and contractile functions of the cardiac organoids are assessed using IF,quantitative real-time polymerase chain reaction(qRT-PCR),and multielectrode array(MEA)techniques to validate the effectiveness of the cardiac organoid differentiation protocol.Results:The IF results indicated high expression of OCT4 and SSEA4 in hiPSCs,indicating that these cells possess pluripotency.Furthermore,hiPSCs were successfully differentiated into cardiac organoids through bidirectional modulation of the WNT signaling pathway.The IF results showed that the induced cardiac organoids consisted of(59.3±12.8)%cTNT-positive cardiomyocytes and(40.7±12.8)%non-cardiomyocytes.qRT-PCR analysis showed that the non-cardiomyocytes included smooth muscle cells,fibroblasts,and endothelial cells.Genes associated with cardiac maturation exhibited higher expression in organoids at day 30 compared to day 15.MEA analysis demonstrated that,as differentiation progressed,the cardiac organoids gradually acquired electrophysiological characteristics similar to those of in vivo hearts,with the beating frequency decreasing[(58±5)beats per minute at 15 days vs.(38±5)beats per minute at 30 days],and the contraction amplitude increasing[(8.6±3.7)at 15 days vs.(16.4±5.9)%at 30 days].Conclusions:This method enables the differentiation of hiPSCs into mature and functional cardiac organoids.

关 键 词：诱导多能干细胞 心脏类器官 拟胚体 

分 类 号：R54[医药卫生—心血管疾病]