Development and validation of a long-term co-maturation protocol for human stem cell-derived microglia and neuronal networks

Technologies using human induced pluripotent stem cells (hiPSCs) have been developed to model human brain cells with the goal to understand their function. To effectively study neuro-immune crosstalk and investigate microglial contributions to neuronal network development and function, both microglia and neurons should co-mature allowing for long-term interactions throughout their differentiation.

Here, we present a co-maturation protocol that robustly generates glutamatergic neuronal networks containing hiPSC-derived microglia. We validated the long-term co-cultures using single-cell transcriptomics, imaging and neuronal activity readouts. In this protocol, astrocytes were required for long-term survival of microglia and for their integration into neuronal networks.