Human iPSC-based coculture model reveals neuroinflammatory crosstalk between microglia and astrocytes
Authors: Tujula Iisa , Hyvärinen Tanja , Lotila Johanna , Rogal Julia , Voulgaris Dimitrios , Sukki Lassi , Tornberg Kaisa , Korpela Katri , Jäntti Henna , Malm Tarja , Herland Anna , Kallio Pasi , Narkilahti Susanna , Hagman Sanna 
Published: January 2025

Background:Microglia and astrocytes have been implicated as central mediators of neuroinflammatory processes in several neurodegenerative diseases. However, their intricate crosstalk and contributions to pathogenesis remain elusive, highlighting the need for innovativein vitroapproaches for investigating glial interactions in neuroinflammation. The aim of this study was to develop advanced human-based glial coculture models to explore the inflammatory roles and interactions of microglia and astrocytesin vitro.Methods:We utilized human induced pluripotent stem cell (iPSC)-derived microglia and astrocytes cultured both in conventional culture dishes and in a compartmentalized microfluidic chip coculture platform. This novel platform features separate compartments for both cell types, enabling the creation of fluidically isolated microenvironments with spontaneous migration of microglia toward astrocytes through interconnecting microtunnels. To induce inflammatory activation, glial cultures were stimulated with lipopolysaccharide (LPS), a combination of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), or interferon-γ (IFN-γ) for 24 hours. The glial activation and crosstalk were analyzed with immunocytochemistry, the secretion of inflammatory factors from the culture media was measured, and microglial migration was quantified.Results:Microglia–astrocyte cocultures were successfully generated in both conventional cultures and the microfluidic chip platform. Inflammatory stimulation with LPS and TNF-α/IL-1β elicited cell type-specific responses in microglia and astrocytes, respectively. Notably, the levels of secreted inflammatory mediators were altered under coculture conditions, revealing significant glial crosstalk. Utilization of our microfluidic coculture platform facilitated the study of microglial migration and glial activation within distinct inflammatory microenvironments. Microglia migrated efficiently toward the astrocyte compartment, and the chemoattractant adenosine diphosphate (ADP) notably increased microglial migration within this platform. Furthermore, inflammatory stimulation of the microfluidic chip cocultures successfully recapitulated glial crosstalk, revealing unique responses. This crosstalk was associated with elevated levels of complement component C3 in the cocultures, emphasizing the intricate interplay between microglia and astrocytes under inflammatory conditions.Conclusions:Our results depict an elaborate molecular crosstalk between inflammatory microglia and astrocytes, providing evidence of how glial cells orchestrate responses during neuroinflammation. Importantly, we demonstrate that the microfluidic coculture platform developed in this study for microglia and astrocytes provides a more functional and enhanced setup for investigating inflammatory glial interactionsin vitro.