Mechanical Environment Afforded by Engineered Hydrogel Critically Regulates Survival of Neural Stem Cells Transplanted in the Injured Spinal Cord via Piezo1‐Mediated Mechanotransduction

Abstract Neural stem cell (NSC) transplantation is a promising therapeutic approach for spinal cord repair, but poor graft survival remains a critical challenge. This work reports that the mechanical properties of the transplantation environment play a crucial role in NSC survival in the injured spinal cord. While this previously developed engineered hydrogel effectively creates extracellular matrix preventing cystic cavity formation, NSCs transplanted as a complex with 10% hydrogel exhibits poor survival. Remarkably, increasing the hydrogel concentration to 16%, creating a fivefold stiffer matrix, significantly enhances NSC graft survival. Using in vitro models with controlled substrate stiffness, this work finds that NSCs on stiffer substrates display enhanced adhesion, complex morphology, and increased viability. Electrophysiological recordings in NSCs reveal pressure‐induced inward currents that are significantly reduced by Piezo1 inhibition. Pharmacological or siRNA inhibition of Piezo1 alters NSC morphology and reduces adhesion specifically on stiffer substrates. Importantly, CRISPR/Cas9‐mediated Piezo1 gene editing significantly reduces graft survival in vivo when transplanted with 16% hydrogel, confirming that Piezo1‐mediated mechanotransduction is essential for stiffness‐dependent NSC survival. These findings reveal a previously unrecognized mechanism governing graft survival and suggest that optimizing mechanical properties of biomaterial scaffolds or directly targeting Piezo1‐dependent mechanotransduction could substantially improve outcomes of cell‐based therapies for neurological disorders.