Astrocyte induction of disease-associated microglia is suppressed by acute exposure to fAD neurons in human iPSC triple cultures

Advancements in human induced pluripotent stem cell (hiPSC) technology have enabled co-culture models for disease modeling in physiologically relevant systems. However, co-culturing protocols face challenges in usability and consistency. Here, we introduce a robust, reproducible hiPSC-derived co-culture system integrating astrocytes, neurons, and microglia. This model leverages cryopreserved cells, enabling co-cultures within 20 days post-thaw. Comparing monocultures and tri-cultures, we show how cell-cell interactions shape transcriptional and functional states across all three cell types. Neurons in tri-culture exhibit increased spine density and activity, while astrocytes and microglia show altered responses to proinflammatory stimulation. Surprisingly, astrocyte co-culture induces upregulation of disease-associated microglia (DAM) genes, including TREM2, SPP1, APOE, and GPNMB. Additionally, while familial Alzheimer’s disease neurons induce a prototypical inflammatory response in microglia, the DAM signature is significantly downregulated. Collectively, this study establishes a versatile human tri-culture model as a valuable resource for dissecting neuron-glia interactions and their role in neurodegenerative disease. For triple culture (TC) conditions, iNs and iAs were differentiated to day 20 and iMGs were differentiated to day 40. On day 20, iAs were dissociated and replated on top of iN cultures. On day 21 (day 40 of iMG differentiation), iMGs were dissociated and replated on top of iN-iA co-cultures. All three cell types were then co-cultured together for an additional six days, after which they were dissociated and analyzed by single-cell RNA-sequencing. For TC samples, four independent culture wells were combined for each culture condition. For monoculture (MC) conditions, cells were differentiated and cultured in parallel to triple cultures under the exact same culturing conditions. For MC samples, four independent monocultures for each cell type (iN, iMG, iA) were pooled.