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.

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.

The generation of astrocytes from human induced pluripotent stem cells has been hampered by either prolonged diferentiation—spanning over two months—or by shorter protocols that generate immature astrocytes, devoid of salient matureastrocytic traits pivotal for central nervous system (CNS) modeling. We directed stable hiPSC-derived neuroepithelial stemcells to human iPSC-derived Astrocytes (hiAstrocytes) with a high percentage of star-shaped cells by orchestrating anastrocytic-tuned culturing environment in 28 days. We employed RT-qPCR and ICC to validate the astrocytic commitmentof the neuroepithelial stem cells. To evaluate the infammatory phenotype, we challenged the hiAstrocytes with the proinfammatory cytokine IL-1β (interleukin 1 beta) and quantitatively assessed the secretion profle of astrocyte-associatedcytokines and the expression of intercellular adhesion molecule 1 (ICAM-1). Finally, we quantitatively assessed the capacityof hiAstrocytes to synthesize and export the antioxidant glutathione. In under 28 days, the generated cells express canonicaland mature astrocytic markers, denoted by the expression of GFAP, AQP4 and ALDH1L1. In addition, the notion of a maturephenotype is reinforced by the expression of both astrocytic glutamate transporters EAAT1 and EAAT2. Thus, hiAstrocyteshave a mature phenotype that encompasses traits critical in CNS modeling, including glutathione synthesis and secretion,upregulation of ICAM-1 and a cytokine secretion profle on a par with human fetal astrocytes. This protocol generates amultifaceted astrocytic model suitable for in vitro CNS disease modeling and personalized medicine.