The Role of Exosomes in Mesenchymal Stem Cell-Mediated Enhancement of Cardiac Contractility

项目摘要 非缺血性心肌病的一种新兴疗法涉及人间充质干细胞的递送 细胞（hMSCs）。临床试验证明对心肌收缩力有适度的益处，强调需要更好地 了解和利用hMSCs-心肌细胞（hCMs）相互作用组的潜在机制。 最近关于hMSC介导的心脏治疗的研究表明，旁分泌信号通过分泌 是减少心脏纤维化和增强血管生成的关键介质。此外，hMSC 已显示旁分泌因子通过改变心肌细胞离子通道/泵活性来影响收缩性。 然而，这些发现未能确定hMSC分泌蛋白组的关键组分，以增强收缩性。 我们建议利用三维人类工程心脏组织（hECTs）作为体外模型， 研究hMSC外来体在增强心脏收缩力中的作用。 本实验室最近发现hMSCs主要通过旁分泌增强hECT的收缩力 信号传导，抵消hMSC-hCM异细胞偶联的不利风险。重要的是，我们发现， hMSC分泌组的外泌体组分对于hMSC旁分泌介导的细胞内分泌是必要的和充分的， hECT收缩性的增强。此外，通过利用系统生物学方法和整合hECT， 收缩功能的结果与外泌体的miRNA数据，我们预测外泌体的miRNA-21作为一个领先的候选者 负责hMSC旁分泌信号传导的有利收缩作用。我们后来用qPCR验证， miRNA-21水平在补充有hMSC外来体和hMSC总调节的hECT中增加。 这激发了我们的中心假设，即外泌体miRNA-21在hMSC中起关键作用。 旁分泌介导的人工程心脏组织收缩性能的增强。 在检验这一假设时，我将直接讨论一个特别感兴趣的NHLBI主题（HL-142）， 外泌体作为旁分泌介质在心血管疾病中的作用。在目标1中，我将确定 外泌体miRNA-21在hMSC介导的hECT收缩性增强中的作用：1）用 衍生自具有miRNA-21抑制剂的hMSC的外来体（子目的1A）;和2）配制的类胡萝卜素纳米颗粒 将miRNA-21模拟物递送到hECT中（子目标1B）。在目标2中，我将测试hMSC外泌体在以下方面的作用： 使用获得性（子目标2A）和遗传性（子目标2B）非- 缺血性心力衰竭 总的来说，该项目旨在将研究框架置于临床背景下，并提供严格的多方面研究。 组织工程、系统生物学、电生理学、干细胞生物学等学科培训， 生物化学作为一个坚实的基础上建立我的职业生涯作为一个未来的物理学家科学家。

PROJECT SUMMARY An emerging therapy for non-ischemic cardiomyopathy involves the delivery of human mesenchymal stem cells (hMSCs). Clinical trials document modest benefits on cardiac contractility, underscoring a need to better understand and exploit the underlying mechanisms governing hMSCs-cardiomyocyte (hCMs) interactome. Recent studies on hMSC-mediated heart therapies demonstrated that paracrine signaling—via secreted factors—is a crucial mediator of reduced cardiac fibrosis and enhanced angiogenesis. Moreover, hMSC paracrine factors have been shown to impact contractility by altering cardiomyocyte ion channel/pump activity. However, these findings fail to identify the key components of the hMSC secretome for enhancing contractility. We propose utilizing three-dimensional human engineered cardiac tissues (hECTs) as an in vitro model to investigate the role of hMSC exosomes in enhancement of cardiac contractility. Our lab recently discovered that hMSCs enhance hECT contractile force predominantly through paracrine signaling, counteracting adverse risks of hMSC-hCM heterocellular coupling. Importantly, we discovered that the exosomal component of the hMSC secretome is necessary and sufficient for hMSC-paracrine mediated enhancement of hECT contractility. Furthermore, by utilizing a systems biology approach and integrating hECT contractile function results with exosomal miRNA data, we predicted exosomal miRNA-21 as a lead candidate responsible for the favorable contractile effects of hMSC paracrine signaling. We later validated with qPCR that miRNA-21 levels are increased in hECTs supplemented with hMSC exosomes and hMSC total conditioned media relative to control, motivating our central hypothesis that exosomal miRNA-21 plays a key role in hMSC paracrine-mediated enhancement of human engineered cardiac tissue contractile performance. In testing this hypothesis, I will directly address an NHLBI topic of special interest (HL-142) by studying the role of exosomes as paracrine mediators in cardiovascular disease. In Aim 1, I will identify the role of exosomal miRNA-21 in hMSC-mediated enhancement of hECT contractility by: 1) treating hECTs with exosomes derived from hMSCs with miRNA-21 inhibitor (Sub-aim 1A); and 2) formulated lipidoid nanoparticle delivery of miRNA-21 mimic into hECTs (Sub-aim 1B). In Aim 2, I will test the role of hMSC exosomes on recovery of contractility using in vitro hECT models of acquired (Sub-aim 2A) and genetic (Sub-aim 2B) non- ischemic heart failure. Overall, the project is designed to frame the research within a clinical context, and provide a rigorous multi- disciplinary training in tissue engineering, systems biology, electrophysiology, stem cell biology, and biochemistry as a solid foundation on which to build my career as a future physician-scientist.