Innate Immunocompetent hiPSC‐Derived Neurospheroids Capture Early CNS Responses to rAAV

Gene therapies using adeno-associated viruses (AAVs) for central nervous system (CNS) disorders face challenges because host immune responses are not represented in classical preclinical models. Here, we present a human-induced pluripotent stem cell (hiPSC)-derived innate immunocompetent 3D CNS model that recapitulates neuroinflammatory hallmarks, serving as a platform for preclinical gene therapy development. By utilizing various scales of stirred-tank bioreactor systems, we generated neurospheroids (iNSpheroids) composed of neurons, astrocytes, and oligodendrocytes, alongside microglial cells (iMGLs) to mimic the neuroimmune axis. These systems enabled large-scale production of iNSpheroids and subsequent miniaturization for co-culture experiments and screening of inflammatory stimuli, while maintaining a highly controlled environment. The iMGL-iNSpheroids demonstrated active neuron-microglia crosstalk and exhibited distinct inflammatory responses to a series of neuroinflammatory factors. iMGL-iNSpheroids mounted an early response to rAAV9, which is underscored by the activation of inflammatory pathways (e.g., TNF-via NF-κB activation) in glial cell populations. This model offers a valuable tool to dissect neuroinflammatory mechanisms, accelerating gene therapy development.