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.

项目摘要/摘要 目前，大约有400万美国人生活在中风的影响下，但没有 有效的治疗方法以改善功能恢复。尤其是中风损坏的网站 细胞毒性对神经元的细胞毒性，因为对活性氧和促氧的敏感性很高 炎症酶。最近意识到蜂窝秘密组可能是主要的 干细胞疗法期间的贡献者。细胞外蔬菜（EV），膜结合 微泡，代表细胞分泌组的活性成分。以及主要的贡献者 电动汽车的活性是microRNA货物。自2013年以来，人类的脑官派生 诱导的多能干细胞（IPSC）已成为模仿三的有前途的方法 尺寸的人脑组织。但是，当前有关电动汽车治疗益处的知识 由IPSC衍生的脑器官分泌的是有限的。 这项研究的目的是设计源自人类的脑器官的电动汽车 IPSC（INPCO）并研究了INPCO分泌的电动汽车对生物合成的影响 营养因子和细胞外属性，以及在体外和IN的功能中性分化 体内。中心假设是INPCO与IPSC和单层神经祖细胞不同， 携带脑特异性microRNA货物的秘密电动汽车，可以针对缺血性脑组织 通过受伤和促进受伤后的恢复提供神经保护作用；特别是肝素 - 水解酸水凝胶封装将允许持续递送INPCO-EV在 缺血环境，促进其治疗作用。基于这些假设，我们 提案三个目标：（1）目标1将检验Inpco-evs表达外泌体标记的假设 并在体外促进细胞存活； （2）AIM 2将检验以下假设 INPCO-EV中的microRNA调节Wnt途径以及营养因素和分泌 细胞外矩阵在体外刺激神经发生； （3）AIM 3将检验以下假设。 INPCO-EVS在缺血性中风中促进原位神经分化和组织再生 模型。迄今为止，尚未针对从人IPSC衍生的脑器官进行EV研究。 与先前的EV研究相反，我们的研究的新颖性是使用三维大脑 区域特异性的类器官系统和可调的肝素 - 冰盖封装 用于热电动汽车传递。该项目将促进我们对旁分泌的影响的理解 对神经回病的信号传导，并建立一种变革性的方法来调节 细胞外微环境可减弱与目标的缺血性相关神经病理学 通过新疗法促进神经发生的。