Radiation-induced neurocognitive dysfunction after brain radiotherapy is a growing concern among the increasing numbers of long-term cancer survivors, particularly in children. This dysfunction significantly impacts memory, learning, and overall quality of life. Neural stem and progenitor cells (NSPCs) play a vital role in maintaining neurogenesis and plasticity, processes essential for memory formation and cognitive resilience. Currently, no effective treatments exist, highlighting the urgent need for strategies to mitigate these effects. One potential contributing factor to this dysfunction is the depletion or dysregulation of NSPCs following radiation. Here, we developed an in vitro microfluidic neurogenic niche setup to investigate how non-irradiated NSPCs respond to the inflammatory secretome produced by irradiated human fetal astrocytes (HFA) and human brain microvascular endothelial cells (HBMEC). NSPCs viability was dose-dependently affected when exposed to conditioned media from irradiated cells. Notably, NSPCs exposed to conditioned media from cells irradiated at 2 Gy and 8 Gy exhibited increased expression of SOX9 and S100B, respectively, suggesting a shift toward a gliogenic fate. Our findings suggest that this microfluidic model is valuable for exploring radiation-induced neurocognitive dysfunction and identifying potential therapeutic targets.

Background: Microglia and astrocytes are central mediators of neuroinflammation in several neurodegenerative diseases. Their intricate crosstalk and contributions to pathogenesis remain elusive, highlighting the need for innovative in vitro approaches for investigating glial interactions in neuroinflammation. This study aimed to develop advanced human-based glial coculture models to explore the inflammatory interactions of microglia and astrocytes in vitro. Methods: Human induced pluripotent stem cell (iPSC)-derived microglia and astrocytes were cultured both in conventional culture dishes and in a microfluidic coculture platform. This platform features separate compartments for both cell types, enabling the creation of distinct 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 h. Glial activation and interactions 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 generated in both conventional cultures and the microfluidic platform. Inflammatory stimulation with LPS and TNF-α/IL-1β elicited cell type-specific responses in microglia and astrocytes, respectively. LPS stimulation of cocultures induced lower secretion of several inflammatory mediators, suggesting dampening of microglial inflammatory responses when cocultured with astrocytes. Notably, inflammatory interaction between glial cells was demonstrated by increased level of IL-10 after TNF-α/IL-1β stimulation in cocultures compared with monocultures. The microfluidic coculture platform enabled the parallel study of microglial migration, glial activation and phagocytic function, thereby facilitating the investigation of glial responses within distinct inflammatory microenvironments. Furthermore, glial inflammatory responses and interactions were demonstrated in the controlled microenvironments of the microfluidic coculture platform. The inflammatory coculture environment was associated with elevated levels of complement component C3, emphasizing the intricate interplay between microglia and astrocytes. Conclusions: Our results depict an elaborate inflammatory interaction between iPSC-derived microglia and astrocytes via reciprocal molecular signaling. Importantly, the microfluidic coculture platform established in this study provides a more functional and advanced setup for investigating inflammatory glial interactions in vitro.

Neurological conditions conquer the world; they are the leading cause of disability and the second leading cause of death worldwide, and they appear all around the world in every age group, gender, nationality, and socioeconomic class. Despite the growing evidence of an immense impact of perturbations in neuroenergetics on overall brain function, only little is known about the underlying mechanisms. Especially human insights are sparse, owing to a shortage of physiologically relevant model systems. With this perspective, we aim to explore the key steps and considerations involved in developing an advanced human in vitro model for studying neuroenergetics. We discuss biological and technological strategies to meet the requirements of a predictive model, aiming at providing a guide and inspiration for future in vitro models of neuroenergetics.