Fig. 1: 3D matrix stress relaxation is required for NPC stemness maintenance.
a Schematic of Dynamic HELP gels (Dynamic Fast or Dynamic Slow), which are composed of aldehyde- or benzaldehyde-modified hyaluronic acid (HA) and hydrazine-modified elastin-like protein (ELP) crosslinked through dynamic covalent bonds. b Schematic of Static HELP gels, which are composed of tetrazine-modified HA and norbornene-modified ELP crosslinked through static covalent bonds. c Shear storage moduli of rat brain and all three HELP gel formulations (N = 3 independent runs, data are means ± standard deviation). d Normalized representative stress relaxation profiles for rat brain and all three HELP gel formulations. e Stress relaxation half-lives (τ1/2) of Dynamic Fast and Dynamic Slow HELP gels (N = 3 independent runs, data are means ± standard deviation). ****p < 0.0001. f Representative maximum projection fluorescence images of NPCs encapsulated within all three HELP gel formulations after 7 days of culture with calcein AM labeled live cells (green) and ethidium homodimer-1 labeled dead cells (red). g Quantification of the relative metabolic activity of encapsulated NPCs over time, relative to the activity 1 hour post-encapsulation (N = 4 replicate hydrogels, data are means ± standard deviation). Day 1: Dynamic Fast vs. Static *p = 0.0397; Day 3: Dynamic Slow vs. Static ***p = 0.0004, Dynamic Fast vs. Static ****p < 0.0001; Day 7: Dynamic Fast vs. Static, Dynamic Slow vs. Static ****p < 0.0001. h Representative maximum projection fluorescence images of NPCs encapsulated within all three HELP gel formulations after 7 days of culture with labeled cells in active phases of the cell cycle (Ki67, white). Nuclei are counterstained with DAPI (blue). i Quantification of the percentage of Ki67+ cells after 7 days in culture (n = 6 randomized field of views, N = 3 replicate hydrogels, data are means ± standard deviation). ****p < 0.0001. j Quantification of the relative dsDNA content of encapsulated NPCs over time (N = 4 replicate hydrogels, data are means ± standard deviation) compared to suspension neurosphere cultures. ****p < 0.0001. k Representative maximum projection fluorescence images of NPCs encapsulated within all three HELP gel formulations after 7 days of culture labeled with the neural stem cell markers Nestin (red) and Sox2 (green). Nuclei are counterstained with DAPI (blue). l Quantification of Sox2 intensity after 7 days in culture (n = 75 single cells for Dynamic Fast, 50 single cells for Dynamic Slow, and 57 single cells for Static, data are means ± standard deviation). a.u., arbitrary units. ****p < 0.0001. m Quantification of the percentage of Sox2+ cells that are in clusters vs. single cells after 7 days in culture (N = 5 replicate hydrogels). Statistical analyses performed as one-way ANOVA with Tukey’s multiple comparisons test (c, i, m), two-tailed unpaired t test (e), two-way ANOVA with Tukey’s multiple comparisons test (g, j), and Kruskal-Wallis with Dunn’s multiple comparisons test (l). Source data are provided as a Source Data file.
