A novel platform for the investigation of human microglia

研究人类小胶质细胞的新平台

基本信息

批准号：
10337872
负责人：
Mathew Mark Blurton-Jones
金额：
$ 15.63万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-03-01 至 2022-08-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10337872
关键词：
3-Dimensional Amyloid beta-Protein Anatomy Biological Markers Brain Calcium Calcium Signaling Cell Transplantation Cells Characteristics Collaborations Coupled Development Diagnostic Disease Progression Environment Exhibits Fluorescence Foundations Functional disorder Future Gene Expression Glycoproteins Goals Health Histology Human Human Biology Imaging Techniques Immune Immune response Immunodeficient Mouse In Vitro Injury Investigation Lasers Light Maintenance Methodology Methods Microglia Microscopy Morphology Mus Nerve Degeneration Nervous System Trauma Neurodegenerative Disorders Neurons Optics Pathology Patients Pattern Pharmaceutical Preparations Proxy Recovery Reporter Role Scanning Senile Plaques Signal Transduction Site Structure System Techniques Therapeutic Tissues Translating Transplantation Validation Visual Xenograft Model Xenograft procedure abeta deposition brain tissue disorder risk drug development drug discovery environmental chemical imaging modality in vivo in vivo imaging induced pluripotent stem cell method development migration multiphoton imaging multiphoton microscopy nerve injury neuropathology new technology novel novel strategies pathogen practical application relating to nervous system response response to injury synaptic pruning targeted delivery tool vector

项目摘要

Abstract Microglia are the primary immune cells of the CNS and are critical to maintaining neuron health and responding to neuropathology. However, current methods for studying and harnessing the unique biology of human microglia is currently severely limited. Xenotransplantation of human microglia into immunodeficient mice is a promising new approach that provides extensive functional and visual access to human microglia in vitro. Xenotransplanted microglia (XMGs), generated from induced pluripotent stem cells (iPSCs) derived from human patients, can be modified to generate reporter and effector lines for unique microglial active states that activate in response to specific forms of neuropathology. Once transplanted into humanized MITRG mice these XMGs colonize the CNS while maintaining human expression patterns, but it remains to be verified if they exhibit the same response patterns and activity as in human brain tissue. The purpose of this supplement is to contribute to establishing the methodology for using XMGs as a proxy for studying human microglia in vivo and validation of reporter lines for both microglial activity and responses to neuropathology. Functional and morphological XMG responses will be observed in vivo using multiphoton imaging of a reporter for calcium activity, Salsa6f. Calcium signaling patterns will be compared between XMGs and endogenous mouse microglia reacting to laser-induced microlesions in brain tissue to verify that the XMGs retain their human response characteristics. Localized responses of XMGs to β-amyloid plaques will be evaluated using brain- wide histology of a reporter for CD9, which has implicated as an indicator for the microglial β-amyloid response state. Using an optical clearing technique, iDISCO+, it is possible to render complete intact mouse brains transparent. These cleared brains can be used to produce highly detailed three-dimensional renders of all XMGs and β-amyloid plaques throughout the whole brain. From these renders, the distribution of CD9- expressing XMGs will be compared between control and β-amyloid-expressing brains in order to validate them as a reliable reporter for proximity to β-amyloid plaques. Validation of these imaging methods and XMG reporter lines may provide for unprecedented access into studying human microglial activity. Exploitation of targeted microglial active states also gives XMGs promising potential as vectors for targeted delivery of effectors localized to neuropathology afflicted regions which may have applications for drug discovery and therapeutics.

抽象的小胶质细胞是中枢神经系统的主要免疫细胞，对于维持神经元健康和然而，目前研究和利用神经病理学独特生物学的方法。人类小胶质细胞目前受到严重限制。小鼠是一种有前途的新方法，它为人类小胶质细胞提供了广泛的功能和视觉途径体外异种移植小胶质细胞 (XMG)，由衍生自诱导多能干细胞 (iPSC) 产生。人类患者，可以进行修改以生成独特的小胶质细胞活性状态的报告基因和效应细胞系一旦移植到人源化 MITRG 小鼠体内，这些药物就会对特定形式的神经病理学产生反应。 XMG 在维持人类表达模式的同时定殖中枢神经系统，但是否有待验证表现出与人类大脑组织相同的反应模式和活动。该补充剂的目的是有助于建立使用 XMG 作为体内研究人类小胶质细胞的替代方法验证小胶质细胞活性和对神经病理学反应的报告系。将使用钙报告器的多光子成像在体内观察形态学 XMG 反应将比较 XMG 和内源性小鼠之间的 Salsa6f 活性。小胶质细胞对激光诱导的脑组织微损伤做出反应，以验证 XMG 保留其人类特征 XMG 对 β-淀粉样斑块的局部反应将使用脑评估。 CD9 报告基因的广泛组织学，表明它是小胶质细胞 β-淀粉样蛋白反应的指标使用光学清除技术 iDISCO+，可以呈现完整完整的小鼠大脑。这些清晰的大脑可用于生成所有内容的高度详细的三维渲染。 XMG 和 β-淀粉样蛋白斑块遍布整个大脑从这些图中可以看出 CD9- 的分布。表达 XMG 的大脑将在对照大脑和表达 β-淀粉样蛋白的大脑之间进行比较，以验证它们作为接近 β-淀粉样斑块的可靠报告器验证这些成像方法和 XMG。报告系可能为研究人类小胶质细胞活动提供前所未有的途径。靶向小胶质细胞活性状态也赋予 XMG 作为靶向递送载体的巨大潜力效应器定位于神经病理学受影响区域，可能可用于药物发现和疗法。