Engineering Extracellular Vesicles of Human Brain Organoids for Stroke Therapy

项目摘要/摘要 目前，大约有400万美国人生活在中风的影响中，但没有 有效治疗，促进功能恢复。中风的受损部位尤其是 对神经元具有细胞毒性，因为它对活性氧物种和前体的敏感性很高。 发炎酶。最近人们意识到，细胞分泌体可能是 干细胞治疗期间的贡献者。细胞外小泡(EVS)，膜结合 微泡是细胞分泌体的活性成分。和一个主要贡献者 EVS的活性是microRNA的货物。自2013年以来，从人类衍生的脑有机化合物 诱导多能干细胞(IPSCs)是一种很有前途的方法，可以用来模拟三种干细胞-- 有维度的人脑组织。然而，目前对电动汽车治疗益处的认识 由IPSC衍生的脑器官分泌的物质是有限的。 本研究的目的是设计人脑器官的电动汽车。 IPSCs(INPCo)，并研究iNPCo分泌的EV对细胞存活、生物合成的影响 营养因子和细胞外基质与功能神经分化的体外和体外研究 活着。中心假说是iNPCo与幼稚的ipscs和单层神经前体细胞不同， 分泌携带脑特异性microRNA的EV，可以通过以下两种方式靶向缺血脑组织 提供神经保护，防止受伤，并促进受伤后的恢复；尤其是肝素- 透明质酸水凝胶胶囊将使iNPCo-EVS在体内持续递送 缺血环境，促进其治疗效果。基于这些假设，我们 提出三个目标：(1)目标1将检验iNPCo-Evs表达外体标记的假设 并在体外促进细胞在氧化应激下存活；(2)目标2将检验以下假设 INPCo-EVS中的microRNAs调节Wnt途径和营养因子的分泌 细胞外基质，以刺激体外神经发生；以及(3)Aim 3将测试以下假设 INPCo-EVS促进缺血性卒中神经原位分化和组织再生 模特。到目前为止，还没有对来自人类IPSCs的脑器官进行EV研究。 与以前的电动汽车研究相比，我们的研究的新颖性在于使用了三维大脑 区域特异性有机体系及可调肝素-透明质酸水凝胶包封剂 用于治疗性EV交付。这个项目将增进我们对旁分泌作用的理解。 神经再生的信号和建立一种变革性的方法来调节 细胞外微环境对减轻缺血相关神经病理的作用 通过新的疗法促进神经再生。

Project Summary/Abstract Currently, about 4 million Americans are living with the effects of stroke, but there is no effective treatment to improve functional recovery. The stroke-damaged site is especially cytotoxic to neurons because of the high susceptibility to reactive oxygen species and pro- inflammatory enzymes. It was recently realized that cellular secretome may be the major contributor during stem cell therapy. Extracellular vesicles (EVs), the membrane-bound microvesicles, represent an active component of the cell secretome. And a major contributor to the activity of EVs is the microRNA cargo. Since 2013, derivation of brain organoids from human induced pluripotent stem cells (iPSCs) has emerged as a promising approach for mimicking three- dimensional human brain tissue. However, current knowledge on the therapeutic benefits of EVs secreted by iPSC-derived brain organoids is limited. The objectives of this research are to engineer EVs of brain organoids derived from human iPSCs (iNPCo) and investigate the impacts of iNPCo-secreted EVs on the survival, biosynthesis of trophic factors and extracellular matrices, and functional neural differentiation in vitro and in vivo. The central hypothesis is that iNPCo, unlike naïve iPSCs and monolayer neural progenitors, secrete EVs carrying brain-specific microRNA cargo that can target ischemic brain tissue both by providing neuroprotection from injury and by promoting recovery after injury; in particular, heparin- hyaluronic acid hydrogel encapsulation will allow for the sustained delivery of iNPCo-EVs in the ischemic environment, promoting their therapeutic effects. Based on these hypotheses, we propose three aims: (1) Aim 1 will test the hypothesis that iNPCo-EVs express exosomal markers and promote cell survival under oxidative stress in vitro; (2) Aim 2 will test the hypothesis that the microRNAs in iNPCo-EVs regulate the Wnt pathway and the secretion of trophic factors and extracellular matrices to stimulate neurogenesis in vitro; and (3) Aim 3 will test the hypothesis that iNPCo-EVs promote in situ neural differentiation and tissue regeneration in an ischemic stroke model. To date, no EV study has been performed for brain organoids derived from human iPSCs yet. The novelty of our study in contrast to previous EV study is the use of three-dimensional brain region-specific organoid system and the tunable heparin-hyaluronic acid hydrogel encapsulation for therapeutic EV delivery. This project will advance our understanding of the effects of paracrine signaling on neural regeneration and establish a transformative approach to modulate extracellular microenvironment to attenuate ischemic-associated neuropathology toward the goal of promoting neural regeneration through novel therapeutics.