Physical Crowding in 3D Niche Regulates NSCs of Cell Junction and Differentiation

Neural stem cells (NSCs) maintain central nervous system (CNS) homeostasis through self-renewal and differentiation into neurons and glia. While physical crowding dynamically regulates NSCs niches during CNS development, yet its role in regulating NSCs fate remains unclear. Herein, we systematically investigated how NSCs crowding influences intercellular junction formation and differentiation in 2D and 3D niches. Using RNA sequencing, RT-qPCR, Western blotting, and immunofluorescence, we found that cell crowding promotes neuron differentiation in both systems. Notably, in 3D cultures, crowding robustly upregulated cell-cell junction genes and enhanced junction-mediated communication. These findings reveal that the 3D environment provides essential biophysical contexts for NSCs, in which cell crowding synergistically enhances differentiation through coordinated cell-cell and cell-ECM signaling. This study offers new insights into the role of physical niche cues in NSCs biology and provides valuable guidance for the design of biomimetic 3D culture platforms and regenerative strategies for neural tissue engineering.神经干细胞（Neural stem cells, NSCs）通过自我更新以及向神经元和神经胶质细胞分化来维持中枢神经系统（Central nervous system, CNS）的稳态。尽管在中枢神经系统发育过程中，物理拥挤可动态调控神经干细胞微环境，但其在调控神经干细胞命运中的作用仍不明确。本研究系统探究了细胞拥挤如何在二维（2D）和三维（3D）微环境中影响神经干细胞的细胞间连接形成与分化。通过RNA测序、实时定量聚合酶链反应（RT-qPCR）、蛋白质印迹法（Western blotting）及免疫荧光染色，我们发现细胞拥挤在两种培养体系中均能促进神经元分化。值得注意的是，在三维培养体系中，细胞拥挤显著上调了细胞间连接相关基因的表达，并增强了连接介导的细胞通讯。本研究揭示，三维环境为神经干细胞提供了必要的生物物理环境，在此环境中细胞拥挤通过协同调控细胞-细胞及细胞-细胞外基质信号通路，协同增强神经干细胞的分化。本研究为物理微环境线索在神经干细胞生物学中的作用提供了新的见解，并为仿生三维培养平台的设计以及神经组织工程的再生策略提供了有价值的指导。