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Generation of iPSC-based human-mouse microglial brain chimeras to study senescence of human microglia.

ABSTRACT: Here, we provide a step-by-step protocol for generating human induced pluripotent stem cell (hiPSC)-based microglial mouse brain chimeras. In addition, we detail steps for intracerebral injection of pathological tau and magnetic cell isolation of human microglia from chimeric mouse brains for single-cell RNA sequencing. Human microglia developed in chimeric mouse brains recapitulate the pathophysiology of microglia in human brain tissue, offering unprecedented opportunities to study human microglial senescence in vivo. For complete details on the use and execution of this protocol, please refer to (Jin et al., 2022b).

SUBMITTER: Jin M

PROVIDER: S-EPMC9667309 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Generation of iPSC-based human-mouse microglial brain chimeras to study senescence of human microglia.

Jin Mengmeng M Ma Ziyuan Z Jiang Peng P

STAR protocols 20221112 4

Here, we provide a step-by-step protocol for generating human induced pluripotent stem cell (hiPSC)-based microglial mouse brain chimeras. In addition, we detail steps for intracerebral injection of pathological tau and magnetic cell isolation of human microglia from chimeric mouse brains for single-cell RNA sequencing. Human microglia developed in chimeric mouse brains recapitulate the pathophysiology of microglia in human brain tissue, offering unprecedented opportunities to study human microg ...[more]

PMID: 36595906

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Project description:Our group was the first to describe direct antagonism of the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway by dengue virus (DENV) in human cells, and here, we report new findings on the characterization of the interaction between the DENV nonstructural protein 2B (NS2B)-NS3 (NS2B3) protease complex and STING. We demonstrate interactions between NS2B and the transmembrane domains of human STING and between NS3 and a portion of the cytoplasmic C-terminal domain of human STING. One significant obstacle we face today in the DENV field is the lack of small animal models available that can effectively recapitulate DENV pathogenesis in the early events of infection. The existing mouse models are either immunocompromised mice lacking interferon (IFN) receptors or "humanized" mice reconstituted with human stem cells. However, both approaches fail to capture important aspects of human pathogenesis because they lack critical innate immunity components or have deficiencies in immune cell development or maintenance. As an important step toward developing an immunocompetent mouse model for DENV, we have generated two chimeric human-mouse STING constructs that have promise in retaining both cleavability by NS2B3 and signaling capacity in the mouse. IMPORTANCE This article characterizes the interaction between human STING and DENV viral protease complex NS2B3 by constructing serial deletion mutants of STING. Our findings suggest that DENV nonstructural protein NS2B interacts with the transmembrane domains and NS3 with the C-terminal cyclic dinucleotide binding domain of human STING. Furthermore, as there exists no ideal immunocompetent murine model that can simultaneously support robust DENV replication and recapitulate the clinical manifestation of dengue disease observed in humans, we expressed and characterized two promising human-mouse chimeric STING constructs that can be used for developing a relevant transgenic mouse model to study dengue in the future. Both constructs can activate normal IFN responses in the overexpression system and be cleaved under infection conditions. We believe our findings offer a roadmap to the further development of a murine model that can greatly facilitate antiviral discoveries and vaccine research for DENV.

Project description:IntroductionMicroglia, the resident immune cells of the central nervous system, play a pivotal role in maintaining homeostasis, responding to injury, and modulating neuroinflammation. However, the limitations of rodent models in accurately representing human microglia have posed significant challenges in the study of retinal diseases.MethodsPLX5622 was used to eliminate endogenous microglia in mice through oral and intraperitoneal administration, followed by transplantation of human induced pluripotent stem cell-derived microglia (hiPSC-microglia, iMG) into retinal explants to create a novel ex vivo chimeric model containing xenotransplanted microglia (xMG). The number and proportion of xMG in the retina were quantified using retinal flat-mounting and immunostaining. To evaluate the proliferative capacity and synaptic pruning ability of xMG, the expression of Ki-67 and the phagocytosis of synaptic proteins SV2 and PSD95 was assessed. The chimeric model was stimulated with LPS, and single-cell RNA sequencing (scRNA-seq) was used to analyze transcriptomic changes in iMG and xMG. Mouse IL-34 antibody neutralization experiments were performed, and the behavior of xMG in retinal degenerative Pde6b-/- mice was examined.ResultsWe demonstrated that xenotransplanted microglia (xMG) successfully migrated to and localized within the mouse retina, adopting homeostatic morphologies. Our approach achieved over 86% integration of human microglia, which maintained key functions including proliferation, immune responsiveness, and synaptic pruning over a 14-day culture period. scRNA-seq of xMG revealed a shift in microglial signatures compared to monoculture iMG, indicating a transition to a more in vivo-like phenotype. In retinal degenerative Pde6b-/- mice, xMG exhibited activation and migrated toward degenerated photoreceptors.ConclusionThis model provides a powerful platform for studying human microglia in the retinal context, offering significant insights for advancing research into retinal degenerative diseases and developing potential therapeutic strategies. Future applications of this model include using patient-derived iPSCs to investigate disease-specific microglial behaviors, thereby enhancing our understanding of microglia-related pathogenesis.

Dataset Information

Generation of iPSC-based human-mouse microglial brain chimeras to study senescence of human microglia.

Publications

Generation of iPSC-based human-mouse microglial brain chimeras to study senescence of human microglia.

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