Engineering Extracellular Vesicles of Human Brain Organoids for Stroke Therapy

项目总结/摘要 目前，约有400万美国人生活在中风的影响下，但没有 有效治疗，促进功能恢复。中风受损的部位 细胞毒性的神经元，因为高敏感性的活性氧和亲， 炎症酶最近认识到，细胞分泌组可能是主要的 干细胞治疗中的作用。细胞外囊泡（EV），膜结合的 微泡代表细胞分泌组的活性组分。也是 EV的活性是microRNA货物。自2013年以来，从人类脑类器官的衍生 诱导多能干细胞（iPSC）已经成为一种有前途的方法，用于模拟三种- 三维人脑组织然而，目前关于EV治疗益处的知识 由iPSC衍生的脑类器官分泌的蛋白质是有限的。 本研究的目的是工程化来自人类的脑类器官的EV iPSC（iNPCo），并研究iNPCo分泌的EV对iPSC（iNPCo）的存活、生物合成 的营养因子和细胞外基质，和功能性神经分化，在体外和体内 vivo.核心假设是iNPCo与幼稚iPSC和单层神经祖细胞不同， 分泌携带脑特异性microRNA货物的EV，其可以靶向缺血性脑组织， 提供免受损伤的神经保护和促进损伤后的恢复;特别是，肝素- 透明质酸水凝胶包封将允许iNPCo-EV的持续递送， 缺血环境，促进其治疗效果。基于这些假设，我们 提出三个目的：（1）目的1将检验iNPCo-EV表达外泌体标记的假设 并促进细胞在体外氧化应激下的存活;（2）目的2将检验以下假设： iNPCo-EV中的microRNA调节Wnt途径和营养因子的分泌， 细胞外基质刺激体外神经发生;和（3）目的3将检验假设， iNPCo-EV促进缺血性卒中中原位神经分化和组织再生 模型迄今为止，尚未对源自人类iPSC的脑类器官进行EV研究。 与以前的EV研究相比，我们的研究的新奇在于使用了三维大脑 区域特异性类器官系统和可调肝素-透明质酸水凝胶包封 用于治疗性EV输送。这个项目将促进我们对旁分泌的影响的理解 神经再生的信号传导，并建立一种变革性的方法来调节 细胞外微环境以减轻缺血相关的神经病理学 通过新疗法促进神经再生。

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.