A novel platform for the investigation of human microglia

摘要 小胶质细胞是中枢神经系统的主要免疫细胞，对维持神经元健康至关重要， 神经病理学反应然而，目前研究和利用这种独特生物学的方法， 人类小胶质细胞目前受到严重限制。人小胶质细胞在免疫缺陷小鼠体内的异种移植 小鼠是一种很有前途的新方法，它为人类小胶质细胞提供了广泛的功能和视觉通路， 体外异种移植的小胶质细胞（XMG），由源自以下的诱导多能干细胞（iPSC）产生： 人类患者的小神经胶质细胞，可以被修饰以产生用于独特的小神经胶质细胞活性状态的报告细胞系和效应细胞系， 对特定形式的神经病理学做出反应。一旦移植到人源化的MITRG小鼠中，这些 XMG在维持人类表达模式的同时定殖于CNS，但它们是否能在CNS中表达仍有待验证。 表现出与人脑组织相同的反应模式和活动。本补充文件的目的是 有助于建立使用XMG作为体内研究人类小胶质细胞的代理的方法， 验证报告细胞系的小胶质细胞活性和对神经病理学的反应。功能和 将使用钙的报告物的多光子成像在体内观察形态学XMG应答 活动，Salsa 6 f.将在XMG和内源性小鼠之间比较钙信号传导模式。 小胶质细胞对脑组织中激光诱导的微损伤作出反应，以验证XMG保留了其人 响应特性XMG对β-淀粉样蛋白斑块的局部反应将使用脑- CD 9报告基因的广泛组织学研究，该报告基因是小胶质细胞β-淀粉样蛋白反应的指示物 状态使用光学透明技术iDISCO+，可以使小鼠大脑完整无损 透明.这些被清除的大脑可以被用来制作高度详细的三维渲染， XMG和β-淀粉样斑块遍布整个大脑。从这些渲染图中，CD 9- 将在对照和表达β-淀粉样蛋白的脑之间比较表达XMG的脑以验证它们 作为接近β-淀粉样蛋白斑块的可靠报告者。这些成像方法和XMG的验证 报道细胞系可以提供前所未有的研究人类小胶质细胞活性的途径。剥削 靶向的小胶质细胞活性状态也给予XMG作为靶向递送 定位于受神经病理学影响的区域的效应物，其可用于药物发现， 治疗学

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.