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Human cortical organoids with engineered microglia-like cells [scRNA-Seq]

ABSTRACT: Microglia play vital roles in the emergence and preservation of a healthy brain microenvironment with their impaired functions highlighted in neurodevelopmental and neurodegenerative disorders. However, investigating the microglia function in health and disease states has been challenging due to the lack of easily accessible human models. Here, we develop a method to generate functional microglia inside human cortical organoids (hCOs) from human embryonic stem cells (hESCs) and apply this system to dissect the role of microglia under inflammation induced by amyloid- (A). The overexpression of the myeloid-specific transcription factor PU.1 generated microglia-like cells in hCOs, producing mhCOs (microglia-containing hCOs) and engrafted in the mouse brain. Single-cell transcriptomics reveals that mhCOs acquire a microglia cell cluster with an intact complement/chemokine system. Functionally, microglia in mhCOs protect parenchyma from cellular and molecular damage caused by A. A-induced expression of genes associated with apoptosis, ferroptosis, and Alzheimer’s disease (AD) stage III genes was attenuated in mhCOs. Finally, we determined the function of AD-associated genes highly expressed in microglia in response to A by using pooled CRISPRi coupled with single-cell RNA sequencing in mhCOs. Together, mhCOs represent an innovative platform to investigate neurodegenerative disorders and serve to develop therapeutics in the future.

ORGANISM(S): Homo sapiens

PROVIDER: GSE175719 | GEO | 2021/12/21

REPOSITORIES: GEO

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Project description:Microglia are innate immune cells of the brain that perform phagocytic and inflammatory functions in disease conditions. Transcriptomic studies of acutely-isolated microglia have provided novel insights into their molecular and functional diversity in homeostatic and neurodegenerative disease states. State-of-the-art mass spectrometric methods can comprehensively characterize proteomic alterations in microglia in neurodegenerative disorders, potentially providing novel functionally-relevant molecular insights that are not provided by transcriptomics. However, proteomic profiling of adult primary microglia in neurodegenerative disease conditions has not been performed. We performed quantitative proteomic analyses of purified CD11b+ acutely-isolated microglia adult mice in normal, acute neuroinflammatory (LPS-treatment) and chronic neurodegenerative states (5xFAD model of Alzheimer’s disease [AD]) using tandem mass tag mass spectrometry. Differential expression analyses were performed to characterize specific microglial proteomic changes in 5xFAD mice and identify overlap with LPS-induced pro-inflammatory changes. Our results were also contrasted with existing proteomic data from wild-type mouse microglia and from existing microglial transcriptomic data from wild-type and 5xFAD mice. Neuropathological validation studies of select proteins were performed in human AD and 5xFAD brains. Of 4,133 proteins identified, 187 microglial proteins were differentially expressed in the 5xFAD mouse model of AD pathology, including proteins with previously known (Apoe, Clu and Htra1) as well as previously unreported relevance to AD biology (Cotl1 and Hexb). Proteins upregulated in 5xFAD microglia shared significant overlap with pro-inflammatory changes observed in LPS-treated mice. Several proteins increased in human AD brain were also upregulated by 5xFAD microglia (Aβ peptide, Apoe, Htra1, Cotl1 and Clu). Cotl1 was identified as a novel microglia-specific marker with increased expression and strong association with AD neuropathology. Apoe protein was also detected within plaque-associated microglia in which Apoe and Aβ were highly co-localized suggesting a role for Apoe in phagocytic clearance of Aβ. We report the first comprehensive comparative proteomic study of adult mouse microglia derived from acute neuroinflammatory and AD models, representing a valuable resource to the neuroscience research community. We highlight shared and unique microglial proteomic changes in acute neuroinflammatory, aging and AD mouse models in addition to identifying novel roles for microglial proteins in human neurodegeneration.

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Human cortical organoids with engineered microglia-like cells [scRNA-Seq]

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