Physical crowding in 3D niche regulates neural stem cell differentiation via Cx43-mediated gap junction communication

Neural stem cells (NSCs) maintain central nervous system (CNS) homeostasis through self-renewal and differentiation into neurons and glia. Although physical crowding shapes the NSC niche during CNS development, its role in fate determination remains poorly understood. We investigated how NSC crowding influences intercellular junctions and lineage specification in 2D and 3D environments. While crowding promotes neuronal differentiation in both environments, robust junctional remodeling occurred only in 3D. Specifically, 3D crowding uniquely upregulated connexin 43 (Cx43)-mediated gap-junction assembly. Pharmacological Cx43 inhibition selectively attenuated 3D crowding-induced neuronal differentiation, demonstrating that gap-junction signaling is essential for fate determination in 3D. These findings highlight that the regulatory influence of NSC crowding is dimension-dependent and mediated through Cx43 gap-junction communication. By elucidating how biophysical context integrates with intercellular signaling to guide NSC behavior, this study offers mechanistic insights into stem cell biology and informs biomimetic 3D culture systems and regenerative strategies for neural tissue repair.