Figure 2. Maintenance of NPC stemness in hydrogels requires matrix degradation but does not depend on cytoskeletal tension or engineered matrix-adhesion ligand interactions.

(a) Schematic depicting a possible role for cytoskeletal tension in directing NPC stemness based on hydrogel degradability. If increased degradability permitted increased ligand clustering, which led to increased cytoskeletal tension and in turn an increase in NPC stemness, we would expect to see a decrease in NPC marker expression in high degradability hydrogels after inhibiting effectors of cytoskeletal tension. (b) However, no decrease in Nestin and Sox2 expression were observed after 7 days of culture in the presence of inhibitors in high degradability hydrogels. (c) Integrin-mediated engagement with the hydrogel matrix is altered by changing the amino acid sequence of the ELP cell-adhesion motif from an integrin-binding RGD sequence to a non-integrin-binding RDG sequence. (d) Expression of mRNA for the NPC markers Nestin and Sox2 in hydrogels with varying degradability, with or without integrin-binding RGD ligands, after 7 days in culture. Nestin and Sox2 expression is increased in hydrogels with increased degradability, irrespective of the presence of integrin-binding ligands. (e) Schematic depicting hypothesis that knocking down ADAM9 will lead to reduced hydrogel degradation, which will in turn lead to decreased NPC stemness. (f) Decreasing hydrogel degradation by shRNA-mediated ADAM9 knockdown decreases expression of Nestin and Sox2 after 3 days in culture in 3D hydrogels. ADAM9 knockdown does not alter expression of the NPC markers Nestin and Sox2 in 2D controls. In b, d, and f, data are presented as geometric means with 95% confidence intervals. n = 4. In b and d, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Bonferroni post-hoc test. In f, n.s. = not significant, **p<0.01, ****p<0.0001, two-tailed Student’s t-test.