Integration and functionality of human iPSC-derived microglia in a chimeric mouse retinal model.

Introduction: Microglia, 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.

Methods: PLX5622 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.

Results: We 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.

Conclusion: This 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.