Nanowired humam cardiac organoid derived exosomes for heart repair

项目概述：外泌体疗法在治疗急性梗死方面具有显着的前景，急性梗死是心脏病的主要原因。 影响美国600多万人的故障。外泌体是由细胞分泌的纳米颗粒， 通过它们的生物活性货物如microRNA（miRNAs）的细胞间信号传导。与基于细胞的 在治疗中，外泌体具有明显的优势，包括低免疫原性，没有致瘤风险， 适合于大规模生产和现货储存。在用于心脏的各种外泌体中， 再生医学，来自人多能干细胞衍生的心肌细胞（hPSC-CM）的外泌体具有 受到了极大的关注。虽然2D培养被认为是外泌体生产的“黄金标准”， 最近的研究表明，3D培养促进了前修复外泌体的产生。为此，我们开发了 人心脏类器官，其由hPSC-CM，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，类器官提供了心肌模拟微组织平台，而e-SiNWs创建了一个 传导微环境以增强外泌体生物发生和分泌。与未连接的相比 在类器官（没有e-SiNW）中，被取代的类器官显示出显著更高的改善能力。 血管化，保护心肌，减轻梗死心脏的病理性肥大， 收缩功能。为了提高外泌体的可重复性，我们开发了同基因hPSC心脏 使用来源于单个hPSC的hPSC-CM、-cFB（心脏成纤维细胞）和-EC（内皮细胞）的类器官 线本提案的目的是确定移植的hPSC心脏的关键变量的影响， 类器官（即，大小、细胞组成和e-SiNW结构）对它们的外泌体产生和功能性的影响。的 该提议的中心假设是， hPSC-CM、-cFB和-EC的仿生环境，以产生用于心脏的治疗有效的外泌体 修复.该提案的创新之处在于，我们将首次将纳米电材料与 人类心脏类器官以增强外泌体产生和功能。因此，我们将追求两个目标： 1）：确定细胞接种比例、大小和e-SiNW结构对所述经扩增的同基因hPSC心脏的影响 类器官对外来体产生和功能的影响，2）：确定来源于类器官的外来体的治疗功效。 优化的类器官。拟议的研究是重要的，因为我们的目标是改变目前的 外泌体生产的范例，以开发器官特异性治疗外泌体， 纳米纤维和工程人类心脏类器官的进展。这些研究将提供指导 设计和开发下一代组织工程构建体的原则， 植入后持续的外泌体产生以及细胞替代疗法。

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.