人多能干细胞源心血管前体细胞治疗心肌梗死的旁分泌作用和机制

Myocardial infarction (MI) is a leading causes of death among cardiovascular diseases. To limit post-MI cell death, there is a urgent need to improve existing therapies by a better understanding of the mecanistic origins of heart development, failur, and regeneration. Recently studies showed that cardiac stem cells or cardiovascular progenitors (CVPCs) hold great promise for the cell-based therapy of MI, however, the exact mechanistic and functinal outcome implications of those cells, especially of human embryonic stem cells-derived CVPCs (hESC-CVPCs), remain largely unknown, while the latter can provide unlimited resource and have strong cardiovascular lineage differentiation potential to meet the demand of basic and application studies. MI-associated with an inflammatory reaction is a prerequisite for healing and scar formation, and the secreted soluble factors are key players during this process. More recently, secreted membrane vesicle exsomes, acting as a potential intercellular and distant organ communicators, are been recognized as new candidates in the tissue repair. However, it is unknown what soluble factors are secreted by hESC-CVPCs, whether hESC-CVPCs secrete exsomes, and what do hESC-CVPC-secreted soluble factors and exsomes contribute to the inflammatory reaction and cardiac repair after MI. We recently developed a highly efficient approach for the induction of hESC-CVPCs, and found that hESC-CVPC not only limited the infarct size, but they improved the post-MI cardiac performance, decreased neutrophil and macrophage infiltration, and changed abundance of proinflammatory cytokines even at 3 days of transplantation. Moreover, the hESC-CVPC conditional media contains exsomes and hundreds of secreted factors, and the media enhanced cardiomyocyte survival under oxidative stress. Thus, we proposed that hESC-CVPCs may improve the cell survial and cardiac function during early MI via their secreted soluble factors and exsomes, and inflammatory regulation. To test this hypothesis, in the present study we aim to investigate the following questions: 1) how is the effect of hESC-CVPCs in the improvement of cell survial and cardiac function after MI? 2) what is the effect of hESC-CVPCs on the immune response in MI hearts and how these immune responses are regulated by the hESC-CVPCs during the early stage of MI? and 3)what kind of soluble factors and exsomes are secreted by hESC-CVPCs and what are the roles of exsomes and major soluble factors in the regulation of immune response, cell survival, myocardial regeneraton, and cardiac performance at the early stage of MI. The achievement will provide new insight into a comprehensive understanding of the paracrine role of hESC-CVPCs and their contribution to cell survival, immune regulation, and cardiac repair after MI. The findings related to the secreted soluble factors and exsomes by CVPCs could also bridge a major gap in the knowledge of the repair mechanism after myocardial injury.

如何减轻心肌梗死后损伤、促进心肌再生和功能重建是心血管领域亟需解决的重大科学问题。近年心血管前体细胞（CVPCs）移植和外泌体治疗心梗的初步研究展示了令人兴奋的前景，但对可提供无限供源的多能干细胞源的CVPCs旁分泌特征和其改善心梗的机制尚不清。我们新近建立了人胚胎干细胞高效分化为CVPCs（hCVPCs)的方法，移植这些细胞3天后小鼠心梗后心功能即有改善，并伴有心梗早期炎症细胞侵润减少和炎症因子丰度改变；其培养液含大量分泌因子和外泌体；并可改善损伤心肌细胞存活，提示hCVPCs有可能通过分泌因子和外泌体减轻心梗损伤。为验证此设想，本项目旨在探讨：1) hCVPC移植治疗心梗疗效如何? 2) hCVPCs如何调节心梗早期炎症反应？3) hCVPC分泌因子和外泌体的特征及其对心梗的作用和机制是什么？研究发现将揭示hCVPCs旁分泌新作用和调控心梗炎症、心肌再生新机制，为其心梗干预提供依据。

如何减轻心肌梗死（心梗）后损伤、促进心肌功能重建是国际心脏学领域研究热点和治疗学面临的巨大挑战，近年来自人类多能干细胞（hPSCs）的心血管前体细胞（CVPCs）移植和外泌体治疗心梗的初步研究展示了令人兴奋的前景，但多能干细胞源CVPCs 的旁分泌特征和其改善心梗的机制尚不清。我们建立了人胚胎干细胞源心血管前体细胞（hPSC-CVPCs）高效分化体系，揭示了P2受体激活物ATP对hESCs自我更新和细胞周期的影响，发现嘌呤能受体P2受体（P2R）介导的钙信号在hESCs及其分化的CVPCs中存在明显差异，hESCs及分化的CVPCs对嘌呤或嘧啶类核苷酸如腺苷三磷酸和尿苷三磷酸的刺激表现出不同的反应性。移植hPSC-CVPCs对心梗小鼠及非人灵长类食蟹猴心功能具有的保护作用，并明显缩小心梗小鼠的疤痕面积。研究发现hESCs-CVPCs移植到急性心梗小鼠心脏后，明显减少心梗早期炎症细胞侵润和降低炎症因子丰度。为解析细胞移植对心梗后急性炎症反应的调控作用，我们系统描绘了小鼠长时程缺血/再灌注损伤后心室、淋巴结以及血液中免疫细胞及其细胞因子的变化图谱；发现hESCs-CVPCs通过促进巨噬细胞M2型极化，抑制M1型极化，进而抑制心梗后炎症反应，减少心肌细胞凋亡。进一步分析筛选得到了具有保护作用的hESCs-CVPCs分泌因子，其显著改善损伤心肌细胞的细胞活力，参与细胞移植对巨噬细胞极化的调控；心肌注射NT3可显著改善心梗小鼠心功能、促进血管形成。此外，建立了外泌体的分离纯化技术，并明确了hESCs-CVPCs外泌体对内皮成管和心肌细胞抵抗损伤的促进作用。这些发现为移植细胞的质量控制提供依据，进一步明确了hESCs-CVPCs的心肌修复作用，并为急性心梗细胞移植的微环境调控机制提供了新见解，丰富了研究hESCs-CVPCs心梗治疗作用机制的手段，为进一步机制研究提供了保障。