(672d) Maintenance of Neural Progenitor Cell Stemness in 3D Hydrogels Requires Matrix Remodeling

While neural progenitor cells (NPCs) hold significant therapeutic promise, relatively little is known about the material properties required to maintain NPC stemness and regenerative potential. Previous studies using 2D substrates have suggested that matrix stiffness can bias NPC differentiation, but studies in 3D materials have conflicting results about the relative importance of stiffness. To determine the material properties required to maintain NPC stemness, we designed an elastin-like protein (ELP) hydrogel system in which matrix stiffness and degradability are co-varied independently of hydrogel swelling, microstructure, and nutrient diffusivity. NPCs cultured in gels with low stiffness and high degradability significantly improved their maintenance of stemness compared to cells in gels with high stiffness and low degradability. Quantifiable indicators of stemness included increased proliferation rate, enhanced metabolic activity, expression of the NPC markers Nestin and Sox2, and efficient differentiation into neurons and astrocytes. To assess if these changes in stemness were due to matrix stiffness, encapsulated NPCs were treated with small molecule inhibitors of cytoskeletal tension and classical mechanotransduction pathways. Interestingly, inhibition of mechanosensing resulted in no changes in NPC behavior relative to uninhibited controls. Furthermore, preventing integrin-mediated matrix engagement by using non-cell-adhesive ELPs also had no effect on NPC phenotype. These results indicate that the observed changes in NPC stemness were independent of matrix stiffness. To investigate the role of NPC-mediated matrix remodeling, antibody arrays and protease inhibitor screens were used to identify a disintegrin and metalloprotease 9 (ADAM9) as the protease primarily responsible for ELP degradation by NPCs. Reducing ADAM9 activity with small molecule inhibitors or with shRNA-mediated knockdown resulted in decreased hydrogel degradation and a significant reduction in NPC stemness. These results suggested that matrix remodeling, and not matrix stiffness, was the required material parameter for maintaining NPC stemness. To test the generalizability of this result, a second engineered biomaterial was designed using poly(ethylene glycol) crosslinked with ADAM9-cleavable peptides to create a family of hydrogels with independent tuning of stiffness and ADAM9-mediated degradability. When ADAM9-degradability was held constant, no differences in Nestin or Sox2 expression were observed regardless of the gel stiffness. In stark contrast, Nestin and Sox2 expression were significantly degreased in gels with 0% compared to 100% ADAM9-cleavable crosslinks, even in the softest gels. Taken together, these results have identified matrix degradation as a previously unknown requirement for NPC stemness maintenance in 3D hydrogels. Furthermore, these data demonstrate that this effect is independent of classical mechanosensing through cytoskeletal contractility, which was previously shown to play a critical role in NPC-matrix interactions in 2D. In the future, biomaterials that exploit ADAM9-mediated remodeling may increase the regenerative potential of NPC therapies.