INTRODUCTION: In the mammalian brain, neural stem cells (NSCs) reside in specialized microenvironments within the subgranular zone (SGZ) and subventricular zone (SVZ), where they possess distinct differentiation potentials. Three-dimensional neural spheroid and organoid models differ in their degree of tissue self-organization. Organoids exhibit complex regional architecture, whereas spheroids provide uniform and reproducible cellular assemblies that are particularly well suited for controlled intervention studies and the dissection of specific cell-to-cell interaction mechanisms.

AIM(S): Here, we developed a 3D neural spheroid-based model derived from primary mouse postnatal NSCs undergoing multilineage differentiation, resulting in functional neuronal networks and myelination.

METHOD(S): Spheroids were generated from primary mouse postnatal SVZ- and SGZ-derived NSCs to assess niche-specific differentiation potential. Immunocytochemical analysis was used to confirm the presence of mature astrocytic, neuronal, and oligodendrocytic markers. Neuronal activity was assessed using calcium imaging in response to glutamate and histamine.

RESULTS: Immunocytochemical staining revealed the colocalization of myelin sheets with axons and the maintenance of niche-dependent lineage marker expression. Calcium imaging revealed functional neuronal network activity patterns and propagating astrocytic calcium waves across spheroids, indicative of network-level glial signalling.

CONCLUSIONS: This platform enables the investigation of cell-to-cell interaction and communication mechanisms that are inaccessible in vivo or oversimplified in 2D in vitro systems, providing a simple yet reproducible tool for both basic and applied research.

FINANCIAL SUPPORT: Work was financed by the National Science Centre, Poland, 2025/57/N/NZ5/03530, and 2021/43/B/NZ7/01162