Fig. 2: HAVDI/RGD co-presentation within fast stress-relaxing matrices permits NPC stemness maintenance as a distributed 3D culture.
a Schematic representation of the engineered peptides (RGD, HAVDI, RDG negative control) used in this study to emulate both cell–ECM and cell–cell interactions through integrin and N-cadherin receptors, respectively. b Amino acid sequences for the bioactive domains of RGD-, HAVDI-, and negative control RDG-ELP variants, as well as the elastin-like sequence used for all ELPs. c Shear storage moduli of Dynamic Slow HELP gel formulations with different ligand combinations (N = 3 independent runs, data are means ± standard deviation). d Stress relaxation half-lives (τ1/2) of Dynamic Fast and Dynamic Slow HELP gels with different ligand combinations (N = 3 independent runs, data are means ± standard deviation). RGD-RDG: ***p = 0.0002; RGD-HAVDI: ***p = 0.0006; HAVDI-RDG: ***p = 0.0005. NPCs encapsulated within either Dynamic Slow (e, f) or Dynamic Fast (g, h) HELP gels after 7 days of culture were immunolabeled for Nestin (red) and Sox2 (green) with nuclei counterstained (blue) (e, g) and analyzed for mRNA expression of Nestin and Sox2 (f, h) (N = 4 replicate hydrogels, data are means ± standard deviation). i Quantification of the percentage of neurospheres formed after 7 days in culture (N = 4 replicate hydrogels, data are means ± standard deviation). ****p < 0.0001. j Quantification of the diameter of neurospheres after 7 days in culture (n = 175 neurospheres for Dynamic Slow RGD-RDG, 99 neurospheres for Dynamic Slow RGD-HAVDI, 127 neurospheres for Dynamic Slow HAVDI-RDG, 47 neurospheres for Dynamic Fast RGD-RDG, 54 neurospheres for Dynamic Fast RGD-HAVDI, and 31 neurospheres for Dynamic Fast HAVDI-RDG, medians and quartiles denoted by dotted lines). ****p < 0.0001. k Quantification of the percentage of Sox2+ cells that are in clusters vs. single cells after 7 days in culture (N = 4 replicate hydrogels). ****p < 0.0001. Statistical analyses performed as one-way ANOVA with Tukey’s multiple comparisons test (c), two-way ANOVA with Tukey’s multiple comparisons test (d, f, h, i, k), and Kruskal-Wallis with Dunn’s multiple comparisons test (j). Source data are provided as a Source Data file.
