Engineering Extracellular Vesicles of Human Brain Organoids for Stroke Therapy

点击翻译按钮获取中文摘要

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.