Electrical integration, maturation and proarrhythmic risk of induced pluripotent stem cell-derived cardiomyocytes after transplantation in infarcted mouse hearts

最先进的心肌梗死治疗不能使失去的心肌再生。利用多能干细胞衍生的心肌细胞进行细胞治疗被认为是克服这一障碍的有希望的策略。潜在的心律失常风险是对心脏细胞治疗的重要安全问题。先前的研究对不同来源的移植细胞的促或抗心律失常作用提供了相互矛盾的结果，但不包括诱导多能干细胞衍生的心肌细胞（iPSCM），这被认为是最有前途的细胞类型之一。本项目的目的是研究iPSCM在梗死小鼠心脏移植后的细胞持久性、功能整合、成熟和心律失常的潜力。将在α -肌球蛋白重链启动子控制下表达增强绿色荧光蛋白和嘌呤霉素抗性的小鼠iPSCM注射到冠状动脉结扎诱导心肌梗死后的成年小鼠心脏中。将比较不同发展阶段iPSCM的疗效和安全性。另一个实验组将是iPSCM和间充质干细胞群，因为有证据表明，与纯iPSCM相比，移植物的持久性更高。在对照动物身上注射生理盐水。移植后6天、6周和3个月，通过右心室电生理导管进行程序性刺激。外部心电图记录将显示诱发性心室颤动发作的次数和持续时间。同时，将进行左心室的体内多电极阵列记录，以显示兴奋的扩散。之后，将心脏切除，并准备活的心室组织切片（150µm厚），以便在移植的iPSCM和周围宿主心肌细胞中使用玻璃微电极进行场电位的体外多电极阵列测量和动作电位记录。这些实验将为移植细胞的电整合和成熟提供信息。初步实验已经证明了计划项目的可行性。预期的结果将揭示iPSCM是否适合应用于心脏细胞治疗，基于它们的电整合和心律失常电位的质量。在项目中实施的四种电生理方法允许单细胞整合和成熟与组织和器官水平上的心律失常风险直接相关，因为所有测量都是用相同的准备进行的。这将使心律失常发生机制的全面评估，这将是迈向更高疗效和安全性的心脏细胞治疗的重要一步。

State-of-the-art therapy of myocardial infarction does not result in regeneration of lost myocardium. Cell therapy using pluripotent stem cell-derived cardiomyocytes is regarded as promising strategy to overcome this hurdle. A potential arrhythmic risk is an important safety concern against cardiac cell therapy. Previous studies provided conflicting results on pro- or antiarrhythmic effects of transplantated cells of different origin, but did not include induced pluripotent stem cell-derived cardiomyocytes (iPSCM), which are regarded as one of the most promising cell types. The aim of this project is to investigate cell persistence, functional integration, maturation and arrhythmic potential of iPSCM after transplantation in infarcted mouse hearts. Murine iPSCM expressing enhanced green fluorescent protein and a puromycin resistance under control of the alpha-myosin heavy chain promoter will be injected into adult mouse hearts after induction of myocardial infarction by coronary artery ligation. iPSCM at different developmental stages will be compared regarding efficacy and safety. Another experimental group will be clusters of iPSCM and mesenchymal stem cells, since there is evidence of a higher persistence of the graft as compared with pure iPSCM. Saline will be injected in control animals. 6 days, 6 weeks and 3 months after transplantation, programmed stimulation will be applied by a right ventricular electrophysiological catheter. External ECG recordings will reveal number and duration of induced ventricular fibrillation episodes. Simultaneously, in vivo multi electrode array recordings of the left ventricle will be performed to display excitation spread. Afterwards, hearts will be resected and viable ventricular tissue slices (150 µm thick) will be prepared to enable in vitro multi electrode array measurements of field potentials as well as action potential recordings with glass microelectrodes in transplanted iPSCM and surrounding host cardiomyocytes. These experiments will provide information on electrical integration and maturation of transplanted cells. Preliminary experiments have already demonstrated the feasibility of the planned project.The expected results will reveal, if iPSCM are suitable for application in cardiac cell therapy based on their quality of electrical integration and arrhythmic potential. The four electrophysiological methods implemented in the project allow a direct correlation of single cell integration and maturation with the arrhythmic risk on tissue and organ levels, since all measurements are performed with the same preparations. This will enable a thorough evaluation of mechanisms of arrhythmogenesis, which will be an important step towards a higher efficacy and safety of cardiac cell therapy.