Engineering Extracellular Vesicles of Human Brain Organoids for Stroke Therapy

工程化人脑类器官细胞外囊泡用于中风治疗

• 批准号: 10589782
• 负责人: Yan Li
• 金额: $ 36.44万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-15 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10589782
• 关键词: 3-Dimensional; Adult; American; Anabolism; Animals; Anti-Inflammatory Agents; Attenuated; Behavior; Binding; Biodistribution; Brain; Brain Ischemia; Brain region; Caspase; Cell Survival; Cells; Cellular Structures; Clinical; Confocal Microscopy; Derivation procedure; Development; Distal; Electron Microscopy; Electrophysiology (science); Embryo; Encapsulated; Engineering; Environment; Enzymes; Exhibits; Extracellular Matrix; Goals; Heparan Sulfate Proteoglycan; Heparin; Heparitin Sulfate; Histologic; Histology; Human; Hyaluronic Acid; Hydrogels; In Situ; In Vitro; Inflammatory; Injections; Injury; Ischemia; Ischemic Brain Injury; Ischemic Stroke; Knowledge; Label; Liquid Chromatography; Magnetic Resonance Imaging; Membrane; Mesenchymal Stem Cells; MicroRNAs; Middle Cerebral Artery Occlusion; Monitor; Morphology; Nerve Regeneration; Neurons; Neurophysiology - biologic function; Organoids; Outcome; Oxidative Stress; Paracrine Communication; Pathway interactions; Predisposition; Prosencephalon; Proteins; Proteomics; Rattus; Reactive Oxygen Species; Recovery; Recovery of Function; Research; Rodent; Signal Pathway; Signal Transduction; Site; Stroke; Structure; System; TSG101 gene; Technology; Testing; Therapeutic; Therapeutic Effect; Transfection; Transplantation; Undifferentiated; Vesicle; WNT Signaling Pathway; Western Blotting; Work; analog; beta catenin; brain tissue; cytotoxic; effective therapy; exosome; extracellular; extracellular vesicles; hindbrain; improved; in vivo; in vivo imaging; induced pluripotent stem cell; injury recovery; microvesicles; monolayer; nanoparticle; nerve stem cell; neural; neurodevelopment; neurogenesis; neuropathology; neuroprotection; non-invasive imaging; non-invasive monitor; novel therapeutics; overexpression; pharmacologic; receptor expression; stem cell therapy; stem cells; stroke model; stroke therapy; tandem mass spectrometry; therapeutic miRNA; tissue regeneration; tissue repair; transcriptome; uptake; virtual

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.

项目概要/摘要 目前，大约有 400 万美国人受到中风的影响，但没有 有效治疗以改善功能恢复。尤其是中风受损的部位 由于对活性氧和亲氧物质高度敏感，对神经元具有细胞毒性 炎症酶。最近人们认识到细胞分泌组可能是主要的 干细胞治疗期间的贡献者。细胞外囊泡（EV），膜结合 微泡代表细胞分泌组的活性成分。并且做出了主要贡献者 EV 的活性是 microRNA 货物。自2013年以来，从人类大脑中提取类器官 诱导多能干细胞（iPSC）已成为模仿三体细胞的有前途的方法 三维人类脑组织。然而，目前对 EV 治疗益处的了解 iPSC 衍生的脑类器官分泌的数量有限。 这项研究的目的是设计源自人类的大脑类器官的 EV iPSC (iNPCo) 并研究 iNPCo 分泌的 EV 对存活、生物合成的影响 营养因子和细胞外基质的作用，以及体外和体内的功能性神经分化 体内。中心假设是 iNPCo 与原始 iPSC 和单层神经祖细胞不同， 分泌携带大脑特异性 microRNA 货物的 EV，可以通过以下方式靶向缺血脑组织： 提供神经保护免受损伤并促进损伤后的恢复；特别是肝素- 透明质酸水凝胶封装将允许 iNPCo-EV 在体内持续输送 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-EV 表达外泌体标记物的假设 并促进细胞在体外氧化应激下的存活； (2) 目标 2 将检验假设 iNPCo-EV 中的 microRNA 调节 Wnt 通路和营养因子的分泌 细胞外基质刺激体外神经发生； (3) 目标 3 将检验以下假设： iNPCo-EVs 促进缺血性中风的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。 与之前的 EV 研究相比，我们研究的新颖之处在于使用了三维大脑 区域特异性类器官系统和可调肝素-透明质酸水凝胶封装 用于治疗性 EV 输送。该项目将增进我们对旁分泌作用的理解 神经再生信号传导并建立一种变革性的调节方法 细胞外微环境可减轻缺血相关的神经病理学，从而实现目标 通过新疗法促进神经再生。