A novel platform for the investigation of human microglia

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.