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

Abstract

ABSTRACT 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.