Microglial dynamics and ferroptosis induction in human <scp>iPSC</scp> ‐derived neuron–astrocyte–microglia tri‐cultures

The dynamics of microglial activity within neuron-astrocyte-microglia tri-cultures derived from human induced pluripotent stem cells (iPSCs) present a complex interplay and offer an opportunity to obtain new insights into neuron-glia interactions. Iron-laden microglia, correlating with functional changes, represent a key pathological feature of Alzheimer's disease (AD). This study characterized the cellular crosstalk and transcriptional states of microglia in tri-cultures. Complement C3 can be detected in culture media when microglia are cocultured with neurons, and the addition of astrocytes in the coculture led to an increased amount of C3, indicating that the impact of glial interactions can be evaluated in this model system. We compared microglial gene expression profiles comprehensively in monoculture, coculture, and tri-culture settings. Single-cell RNA sequencing (scRNA-seq) revealed various microglial states with gene expression changes associated with endocytosis and neuron-related functions in tri-culture settings, suggesting that microglial behavior is profoundly impacted by the presence of neurons and astrocytes. We assessed microglial responses to iron overload combined with the ferroptosis inducer RSL3 (a GPX4 inhibitor) in tri-cultures. Microglial cell death was accompanied by ferritin heavy-chain expression, indicating microglia ferroptosis. scRNA-seq analyses highlighted alterations in pathways related to ferroptosis, stress response, and autophagy, indicating substantial shifts in microglial profiles upon iron perturbation. These findings underscore the necessity of using tri-cultures as a model to capture certain degrees of complex cellular interactions occurring in vivo. These results offer critical insights for establishing in vitro models for therapeutic development of neurodegenerative diseases, including AD.