TISSUE NICHE-DERIVED SIGNALS LOCALLY PROGRAM HUMAN MICROGLIA PHENOTYPES IN AN ORGANOID MODEL OF THE DEVELOPING HUMAN BRAIN

Microglia, the resident immune cells of the human brain, acquire highly heterogeneous molecular and functional states that emerge dynamically during development. While this diversity has been characterized in animal models, its manifestation and functional relevance in the human brain remain poorly understood, largely due to the lack of suitable experimental systems. To address this gap, we developed a modular, fully iPSC-derived human forebrain assembloid platform that enables the integration and study of human microglia (hMG) within a physiologically relevant brain-like environment.
Using this model, we identified spatially defined microglial phenotypes that arise during early stages of human cortical development. iPSC-derived hMG displayed remarkable plasticity, adapting their morphology, behavior, and transcriptional profiles according to their spatial localization within the assembloid. Notably, we uncovered discrete microglial microdomains, including axon tract–enriched regions, where microglia exhibited distinct characteristics compared to those residing in soma-dense areas. These axon-associated microglia closely resembled axon tract–associated microglia (ATMs) previously described in the developing mouse brain, particularly at cortical boundary zones. Correlative light and electron microscopy, together with functional assays, suggested that these ATM-like hMG dynamically respond to local structural and cellular cues, adjusting to region-specific functional demands.
Taken together, our findings demonstrate that human microglia, despite originating from a common progenitor population, can diversify in response to microenvironmental signals. This assembloid-based platform provides a powerful framework to dissect microglial heterogeneity in human neurodevelopment and offers new opportunities to explore microglia-driven mechanisms underlying neurodevelopmental disorders and context-specific therapeutic interventions.