Mechanical environment afforded by engineered polymer hydrogel critically regulates survival of neural stem cells transplanted in the injured spinal cord via Piezo1-mediated mechanotransduction

Neural stem cell (NSC) transplantation is a promising therapeutic approach for spinal cord repair, but poor graft survival remains a critical challenge. Here, we demonstrate that the mechanical properties of the transplantation microenvironment play a crucial role in NSC survival in the injured spinal cord. While our previously engineered imidazole-poly(organophosphazene) (I-5) hydrogel effectively prevented cavity formation by promoting extracellular matrix remodeling, NSCs transplanted with 10% hydrogel exhibited poor survival. Remarkably, increasing the hydrogel concentration to 16%, which created a 5-fold stiffer matrix, significantly enhanced NSC graft survival and synaptic integration. Using in vitro models with controlled substrate stiffness, we found that NSCs on stiffer substrates displayed enhanced adhesion, complex morphology, and increased viability. Importantly, we identified the mechanosensitive ion channel Piezo1 as the key molecular mediator of these stiffness-dependent behaviors. CRISPR/Cas9-mediated Piezo1 gene editing in NSCs significantly reduced graft survival in vivo when transplanted with 16% hydrogel, confirming that Piezo1-mediated mechanotransduction is essential for NSC survival in the injured spinal cord. Our findings reveal a previously unrecognized mechanism governing graft survival in the injured spinal cord and suggest that optimizing the mechanical properties of biomaterial scaffolds or targeting Piezo1-dependent mechanotransduction could substantially improve outcomes of cell-based therapies for neurological disorders.