During differentiation, neural stem cells (NSCs) encounter diverse cues from their niche, including not only biophysical cues from the extracellular matrix (ECM) but also cell-cell communication. However, it is still poorly understood how these cues cumulatively regulate mechanosensitive NSC fate commitment, especially in 3D matrices that better mimic in vivo systems. Here, we develop a click chemistry-based 3D hydrogel material system to fully decouple cell-cell and cell-ECM interactions by functionalizing small peptides: the HAVDI motif from N-cadherin and RGD motif from fibronectin. The hydrogel is engineered to range in stiffness from 75 Pa to 600 Pa. Interestingly, HAVDI-mediated interaction shows increased neurogenesis, except for the softest gel (75 Pa). Moreover, the HAVDI ligation attenuates the mechanosensing state of NSCs, exhibiting restricted cytoskeletal formation and RhoA signaling. Given that mechanosensitive neurogenesis has been reported to be regulated by cytoskeletal formation, our finding suggests that the enhanced neurogenesis in the HAVDI-modified gel may be highly associated with the HAVDI interaction-mediated attenuation of mechanosensing. Furthermore, NSCs in the HAVDI gel shows higher β-catenin activity, which has been known to promote neurogenesis. Our findings provide critical insights into how mechanosensitive NSC fate commitment is regulated as a consequence of diverse interactions in 3D microenvironments.
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