Innate immunocompetent iNSpheroids: A hiPSC-derived 3D model to study the central nervous system captures an early CNS response to rAAV

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. Utilizing various scales of stirred-tank bioreactor systems, we produced iNSpheroids composed of neurons, astrocytes, and oligodendrocytes, alongside microglial cells (iMGL) to mimic the neuro-immune 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 a mild and transient response to rAAV9, mediated by the activation of inflammatory pathways (e.g., TNF-via NF-κB activation) in glial cell populations..