基于人诱导多能干细胞的心脏类器官模型构建研究

Study of Cardiac Organoids Model Construction Based on Human Induced Pluripotent Stem Cells

采用人诱导多能干细胞(hiPSC)构建基于类器官的药物心脏毒性体外替代模型。首先筛选了hiPSC的优化分化代次,并使用免疫荧光染色法鉴定干细胞多能性。将hiPSC分别以不同的细胞密度接种到超低吸附96孔板,加入含有生长因子和信号通路抑制剂的培养基进行诱导分化,采用光学显微镜记录形态变化及搏动特征。收集分化过程中不同时间点的心脏类器官,采用实时逆转录定量聚合酶链反应(RT-qPCR)对心脏类器官组成细胞特异性基因的相对表达量进行检测。结果显示,复苏后的hiPSC生长状态良好,边缘清晰且细胞多能性优异。密度为每孔5000个细胞分化形成的心脏类器官形态最优,培养8~10 d可趋于相对稳定,培养18 d内可维持相对稳定状态。RT-qPCR结果显示,心肌细胞及其他心脏组成细胞,如内皮细胞、平滑肌细胞、成纤维细胞等的特异性基因均有表达。该研究建立的心脏类器官模型,能更真实、更准确地模拟体内心脏的生物学特性和功能,为后续在研发早期采用心脏类器官模型对药物进行潜在心脏毒性研究奠定了基础。

In this study,human induced pluripotent stem cells(hiPSCs)were used to construct an in vitro organoid-based substitution model for drug cardiotoxicity.The hiPSCs were cultured to determine the optimal differentiation generation,and pluripotency was identified using immunofluorescence staining.The hiPSCs were seeded into ultra-low adsorption 96-well plates at different cell densities,and the medium containing growth factors and signaling pathway inhibitors were added for inducing differentiation,and the morphological changes and pulsatile characteristics were recorded by light microscopy.Cardiac organoids at different time points in the differentiation process were collected,and real-time reverse transcription-quantitative polymerase chain reaction(RT-qPCR)was adopted to detect the relative expression of cell-specific genes of cardiac organoids.The results showed that the resuscitated hiPSCs grew well with clear edges and excellent cell pluripotency.The morphology of the differentiated cardiac organoids at a density of 5000 cells per well was the best,and trended to be a relatively stable state for 8-10 d culturing,and a relatively stable state could be maintained within 18 d.RT-qPCR results showed that specific genes of cardiomyocytes and other cardiac components,such as endothelial cells,smooth muscle cells,and fibroblasts,were expressed.The establishment of cardiac organoid models in this study can more realistically and accurately simulate the biological characteristics and functions of the heart in vivo,which lays a foundation for the subsequent use of cardiac organoid models for potential cardiotoxicity studies of drugs in the early stage of research and development.