Addressing neuroinflammation in human induced pluripotent stem cell-derived central nervous system neurospheroids.

Growing evidence suggests that reactive astrocytes can acquire different functional subtypes, playing critical roles in neurological disorders. Human induced pluripotent stem cell (hiPSC)-derived three-dimensional (3D) CNS models have been proposed to study reactive astrocytes. Still, lack of reproducibility and immature/activated astrocyte phenotypes typical of these models limit their utility to address neuroinflammation. Here, we establish a robust hiPSC-derived 3D neuroinflammation model, exploring neurospheroid (iNSpheroid) differentiation in perfusion stirred-tank bioreactors to obtain neurons and glia co-cultures. iNSpheroids were challenged with TNF-α, IL-α, and C1q (TIC) cocktail. Transcriptome analysis revealed the upregulation of inflammatory modulators (e.g., and ) associated with TNF and NF-kB signaling. Secretome analysis showed increased secretion of inflammation-related cytokines (e.g., CCL2 and CXCL8) in TIC-stimulated iNSpheroids. Astrocytes displayed an impaired capacity for glutamate-glutamine recycling compared to the unstimulated control, indicating functional impairment. Together, these results demonstrate that astrocytes within iNSpheroids are functional and recapitulate canonical astrogliosis events, hallmarks of neuroinflammation.