Optimization of a neuron-microglia co-culture model to explore cell-to-cell interaction dynamics

Stefania Vogiatzis, Martina Severa, Agostina Pietrantoni, Giada Cairo, Roberta Pia Falco, Caterina Veroni, Angela Lanciotti, Marilena P. Etna, Davide Antonio Abate, Lucia Nencioni, Marta Trevisan, Eliana M. Coccia, Luisa Barzon

Introduction The dialogue between the immune and nervous systems in the central nervous system (CNS) is a fundamental challenge in neuroscience and neuroimmunology. Microglia, the brain's resident immune cells, continuously communicate with neurons to maintain brain homeostasis, support development, and orchestrate responses to injury. Methods We adapted the CEBPA/SPI1 transcription factor overexpression approach to generate induced microglia-like cells (iMG) from human embryonic stem cells (hESCs), establishing a reproducible and standardized baseline framework for neuron-microglia co-culture. iMG characterization included assessment of key microglial markers (GPR34, MMP9, TMEM119, IBA1, P2Y12), phagocytic capability benchmarked against the human microglial cell line HMC3, and high-resolution scanning electron microscopy (SEM). iMGs were then co-cultured with hESC-derived NGN2-induced neurons (iNeu), with systematic optimization of seeding ratios and temporal parameters. Results iMGs expressed key microglial markers and demonstrated phagocytic capabilities comparable to HMC3 cells. In co-culture, iMGs exhibited remarkable phenotypic plasticity, with upregulation of both homeostatic and activation-associated microglial genes. Reciprocally, neurons showed increased expression of maturation and synaptic markers, indicating a bidirectional crosstalk. SEM revealed spatially variable iMG morphology — transitioning from ramified to amoeboid forms depending on proximity to neuronal structures. Discussion This contact-based in vitro system provides a versatile platform for investigating neuroimmune interactions, designed to be further integrated with additional CNS cell types, and applicable to the study of microglial activation, neuron-microglia signalling, and the impact of infections on CNS homeostasis.