Nanowired humam cardiac organoid derived exosomes for heart repair

项目摘要：外切体疗法在治疗急性心肌梗死方面前景显著，急性心肌梗死是心脏的主要病因 影响到美国600多万人的失败。外切体是由细胞分泌的纳米颗粒，以促进 细胞间信号通过其生物活性物质，如microRNAs(MiRNAs)。与基于单元的 治疗上，外切体具有明显的优势，包括低免疫原性，无肿瘤形成风险，以及 适合大规模生产和现成储存。在用于心脏的各种外切体中 再生医学中，人多能干细胞来源的心肌细胞外体(hPSC-CMS) 受到了极大的关注。虽然2D培养一直被认为是外显体生产的“黄金标准”， 最近的研究表明，3D培养可以促进修复前外切体的产生。为此，我们开发了 由hPSC-CMS，人原代心脏成纤维细胞， 内皮细胞、基质细胞和导电硅纳米线(e-SiNW)。与2D hPSC相比- CM培养，有机物提供了一个模拟心肌的微组织平台，而e-SiNW创造了一个 传导微环境促进外切体的生物发生和分泌。与无线网络相比 有机化合物(不含e-SiNW)，纳米线有机化合物表现出显著更高的改善能力 血管重建化，保护心肌，减轻梗死性心肌肥厚并恢复 它们的收缩功能。为了提高外切体的重复性，我们研制了等基因hPSC心脏 使用来自单个hPSC的hPSC-CMS、-CFBS(心脏成纤维细胞)和-ECs(内皮细胞)的有机类化合物 排队。该方案的目的是确定纳米线hPSC心脏的关键变量的影响 有机化合物(即大小、细胞组成和e-SiNW结构)对其外切体的生产和功能有影响。这个 该提议的中心假设是纳米线hPSC心脏器官提供导电， HPSC-CMS、-CFBS和-ECs的仿生环境以产生治疗心脏疾病的外切体 修理。这项提议的创新之处在于，我们将首次将电子纳米材料与 人类心脏有机化合物，以增强外切体的生产和功能。因此，我们将追求两个目标： 1)：确定细胞种植比例、大小和e-SiNW结构对纳米线等基因hPSC心脏的影响 有机化合物对外切体生产和功能的影响，2)：确定衍生的外切体的治疗效果 从经过优化的纳米线有机体中。拟议的研究具有重要意义，因为我们的目标是将当前的 利用最近的研究开发器官特异性治疗性外切体的外切体生产范式 纳米制造和工程化人类心脏有机体的研究进展。这些研究将为我们提供指导 设计和开发下一代组织工程构造的原则，可以提供 除了细胞替代疗法外，植入后持续的外切体产生。

Project Summary: Exosome therapy holds remarkable promise to treat acute infarction, a major cause of heart failure that affects over 6 million people in the US. Exosomes are nanoparticles secreted by cells to facilitate intercellular signaling through their bioactive cargos such as microRNAs (miRNAs). Compared to cell-based therapies, exosomes have distinct advantages including low immunogenicity, absence of tumorigenic risk, and amenable for large scale production and off-of-shelf storage. Among various exosomes used for cardiac regenerative medicine, exosomes from human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have received significant attention. While 2D culture has been considered as “gold standard” for exosome production, recent studies show 3D culture promote the production of pro-reparative exosomes. To this end, we developed nanowired human cardiac organoids, which are composed of hPSC-CMs, human primary cardiac fibroblasts, endothelial cells, stromal cells, and electrically conductive silicon nanowires (e-SiNWs). Compared to 2D hPSC- CM culture, organoids provide a myocardium mimetic microtissue platform, and the e-SiNWs creates a conductive microenvironment to enhance exosome biogenesis and secretion. Compared to the unwired organoids (without e-SiNWs), the nanowired organoids showed the significantly higher ability to improve vascularization, preserve myocardium, attenuate pathological hypertrophy of the infarcted hearts and recover their contractile function. To improve the reproducibility of exosomes, we developed isogenic hPSC cardiac organoids using hPSC-CMs, -cFBs (cardiac fibroblasts), and -ECs (endothelial cells) derived from a single hPSC line. The goal of this proposal is to determine the effects of key variables of the nanowired hPSC cardiac organoids (i.e., size, cell composition and e-SiNW structures) on their exosome production and functionality. The central hypothesis of the proposal is the nanowired hPSC cardiac organoids provide an electrically conductive, biomimetic environment for hPSC-CMs, -cFBs, and -ECs to produce therapeutically potent exosomes for cardiac repair. The proposal is innovative is that, for the first time, we will synergize electrical nanomaterials with human cardiac organoids to enhance exosome production and functionality. Accordingly, we will pursue 2 Aims: 1): Determine the effects of cell seeding ratio, size and e-SiNW structure of the nanowired isogenic hPSC cardiac organoids on exosome production and function, 2): Determine the therapeutic efficacy of the exosomes derived from the optimized nanowired organoids. The proposed studies are significant in that we aim to shift the current paradigm of exosome production to develop organ-specific therapeutic exosomes by leveraging the recent advances in nanofabrication and engineered human cardiac organoids. These studies will provide a guiding principle to design and develop next generation of tissue engineering constructs that can provide a source of sustained exosome production after implantation in addition to a cell replacement therapy.