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，对维持神经元健康和 对神经病理学有反应。然而，目前研究和利用长臂猿独特生物学的方法 人类的小胶质细胞目前受到严重限制。人小胶质细胞异种移植治疗免疫缺陷的研究 MICE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途径 体外培养。异种移植小胶质细胞(XMG)，由诱导性多能干细胞(IPSCs)产生 人类患者，可以被修改为产生独特的小胶质细胞活跃状态的报告和效应线， 对特定形式的神经病理作出反应而激活。一旦移植到人源化的MITRG小鼠体内 在维持人类表达模式的同时，XMG在中枢神经系统中定居，但它们是否会被证实仍有待验证 表现出与人脑组织相同的反应模式和活动。本附录的目的是 为建立XMGs作为体内研究人小胶质细胞的代用品的方法学做出贡献 小胶质细胞活性和对神经病理学反应的报告线的验证。功能和 使用钙离子报告的多光子成像将在活体内观察XMG的形态反应 活动，Salsa6f。XMGs和内源性小鼠钙信号模式的比较 小胶质细胞对激光诱导的脑组织微损伤的反应以验证XMG保留其人类 响应特征。XMG对β-淀粉样斑块的局部反应将使用脑- CD9的广泛组织学报道，它已被认为是小胶质细胞β-淀粉样蛋白反应的指标 州政府。使用光学清除技术iDISCO+，可以再现完整的小鼠大脑 透明的。这些被清除的大脑可以用来生成所有 XMG和β-淀粉样斑块遍布整个大脑。从这些渲染中，CD9的分布- 表达XMG的大脑将在对照组和β淀粉样蛋白表达的大脑中进行比较，以验证它们的有效性 作为接近β-淀粉样斑块的可靠记者。对这些成像方法和XMG的验证 记者专线可能会为研究人类小胶质细胞活动提供前所未有的途径。开发利用 靶向的小胶质细胞活性状态也使XMG有希望成为靶向递送的载体 效应器定位于神经病理受累区域，这些区域可能应用于药物发现和 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.