Abstract
Interactions among neurons, microglia, and endothelial cells (ECs) -the principal components of the neurovascular unit (NVU)-are vital for maintaining central nervous system (CNS) homeostasis and are implicated in numerous neurological disorders. However, mechanistic insights into their crosstalk remain limited due to the lack of physiologically relevant in vitro models. In this study, we present an improved 3D vascularized tri-culture model that integrates human-induced neural stem cells (hiNSCs), human vascular organoids (hVOs), and microglia within a geometrically engineered silk fibroin scaffold. This platform effectively recapitulates critical features of the native CNS microenvironment, including spatial neurovascular patterning and cell-type-specific interactions. Within this model, hVOs significantly promoted neuronal differentiation of hiNSCs, resulting in extended axonal networks and improved neurovascular alignment. Microglial effects were found to be phenotype-dependent: both resting (M0) and pro-inflammatory (M1) microglia inhibited hiNSCs differentiation and vascular development, with M1 cells exerting the strongest suppressive influence. In contrast, anti-inflammatory (M2) microglia displayed the least inhibitory effect and even modestly supported neurovascular maturation. Mechanistic studies revealed that M2 microglia cooperate with hVOs via the stromal cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) signaling axis to promote neuronal differentiation. To our knowledge, this represents the first demonstration of SDF-1/CXCR4-mediated immune-neurovascular interaction within a human tri-culture system. Thereafter, this 3D vascularized co-culture model provides a physiologically relevant in vitro platform to investigate neuroimmune and neurovascular interactions. It holds broad potential for mechanistic studies in neurodevelopment and neurodegeneration, drug evaluation, and the development of regenerative therapies.