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. Overall design: NSCs were isolated from cerebral cortices of 1–day-old neonatal Sprague-Dawley rats. After tissue pieces of dissociation, the mixed cortical cells were first passed through a 100 µm mesh of nylon cell strainer, then plated into tissue culture flasks. RNA-seq library preparation. Total RNA was isolated from each sample using the RNAmini kit (Qiagen, Germany). RNA quality was examined by gel electrophoresis and with Qubit (Thermo, Waltham, MA, USA). Strand-specific libraries were constructed using the TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA), through reverse transcription using S1000TM Touch Thermal Cycler (Bio Rad) at 53°C (45 min) - 85°C (5 min) - 4°C cycle. The generated cDNA was amplified, and quality estimated using Agilent 4200 (performed by CapitalBio Technology, Beijing). The sequences of libraries were finally obtained using the Illumina Novaseq6000 sequencer with pair-end 150 bp (PE150).