大尺寸功能性人心肌补片的构建及治疗心肌梗死的机制研究

Cardiovascular diseases such as myocardial infarction (MI) are the main causes of death in China. Transplantation of human induced pluripotent stem cells (hiPSCs)-derived cardiomyocytes combined with other differentiated cardiac cells can significantly improve the viability of ischemic cardiomyocytes, restore the function of the lesion heart, and improve the blood supply and myocardial remodeling, which brings hope for the treatment of myocardial infarction. However, recent studies in non-human primates have shown that direct intramyocardial injection of such cardiomyocytes will increase the incidence of arrhythmias. Therefore, using cardiac muscle patch to repair heart may be a better approach. Currently, only relatively thin and small patches can be produced due to technology limitations, and the structure and function are relatively immature, which limit its application in clinic. We have accumulated some experience in stem cell therapy for MI in large animal model: We have successfully mastered the skill to differentiate hiPSCs to cardiac cells such as cardiomyocytes, endothelial cells, smooth muscle cells, fibroblasts and pericytes with the high purity; We can design cardiac muscle patches of relatively large size, which showed protective effects in MI pig; Exosomes extracted from the patch has a significant protective effect of ischemia, hypoxic cardiomyocytes. In order to build functional large-size cardiac muscle patches which further meet the clinical needs and also to clarify its protective mechanism, the purpose of this project is : 1) explore the best formula for large-size myocardial patches that meets the clinical needs using hiPSCs differentiated cardiomyocytes and other cardiac cells, combined with effective material scaffolds; 2) explore the best methods to promote structure and function maturation of myocardial patch using multiple stimulation method, pre-built vascularization, and 3D printing technology; 3) verify the patch’s protective effect on myocardial infarction in large animal model; 4) and explore its paracrine characteristics including exosomes and its protective mechanism of myocardial infarction after myocardial infarction, which provide theoretical basis for clinical treatment of myocardial infarction.

心肌梗死等心血管疾病是我国居民死亡的主要原因。人诱导多能干细胞(hiPSCs)分化的心脏细胞构建的心肌补片移植为治疗心肌梗死带来了新的希望。目前的技术只能制备出相对薄和尺寸小的补片，并且结构和功能不成熟，从而限制了其在临床上的使用。我们已经成功掌握从hiPSCs分化得到高纯度的各种心脏细胞; 能够设计出一定尺寸的心肌补片，在心梗猪上观察到了其保护作用；并发现从补片中提取的外泌体具有明显保护心肌的作用。为了进一步构建符合临床需求，功能性大尺寸人心肌补片，并阐明其保护机理，本课题旨在：1）利用hiPSCs分化的心肌细胞联合其他辅助细胞，探索构建大尺寸心肌补片的最佳配方；2）应用多重促成熟刺激方法，预建血管化，结合3D打印技术，探讨促进大尺寸心肌补片结构和功能成熟的方法；3）在猪等心肌梗死模型上验证其对心梗心肌的保护作用；4）并解析其旁分泌包括外泌体作用特征和其对心梗后受损心肌的保护机制。

心肌梗死等心血管疾病是我国居民死亡的主要原因，致死率远超癌症等其他疾病。人诱导多能干细胞分化的心脏细胞(hiPSC-CCs)构建的心肌补片移植为治疗心肌梗死带来了新的希望。但目前的技术只能制备出相对尺寸小的补片，缺少大动物研究，机制不清，并且心肌补片中细胞在缺血区域的低驻留率也影响了其移植的治疗效果，从而推迟了在临床上的使用。为了解决以上问题，本课题进行了以下研究，并获得了一系列成果：1）建立了从hiPSCs定向分化得到各种类型心脏细胞（心肌细胞、内皮细胞、平滑肌细胞等）的方法，并掌握了各种促心脏细胞成熟的技术，明确了血纤蛋白为构建大尺寸心肌补片所需的生物材料。2）发现装载神经生长因子(NGF)-聚乳酸-羟基乙酸（PLGA）纳米颗粒的心脏补片可增强移植后心肌补片在宿主心脏上的驻留，改善小鼠心梗后的心脏收缩功能，增加心肌细胞存活和血管生成，和减少心室重构。3）我们用hiPSC-CMs制备了个临床应用相关尺寸大小的心肌补片（2x4 cm），动态培养7天刺激其成熟。移植这类心肌补片到心梗猪的心脏上，可显著减少猪心脏的梗死面积和改善心功能，增加心肌梗死边缘区的微血管和小动脉的密度，减少心肌的凋亡，具有一定的治疗猪心梗的作用，但并不引起心律失常发生的显著变化。4）分析了主要hiPSCs分化的心脏细胞中外泌体特征和表达谱，发现hiPSCs分化的内皮细胞外泌体 (hiPSC-EC-Exo) 可以通过维持心肌细胞钙稳态促进小鼠心梗后的心功能的改善，和hiPSCs分化的心肌细胞外泌体 (hiPSC-CM-Exo) 可以通过促进血管新生促进小鼠心梗后的心肌修复。这些发现为改善干细胞的移植治疗效果提供了新的见解，并且为以后心肌补片临床研究提供了基础研究支持。

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