Fig. 4: NPCs in neurospheres exhibit limited expansion due to hypoxia and cell death, whereas distributed NPC cultures maintain viability and proliferation over long-term culture.

a Representative maximum projection (left and center) and single z-plane cross-section (right) fluorescence images of NPCs encapsulated within Dynamic Fast and Dynamic Slow RGD-HAVDI HELP gels after 14 days of culture labeled with Nestin (red) and cleaved caspase-3 (yellow), with nuclei counterstained (blue). b Quantification of cleaved caspase-3 area, normalized by cell number, after 14 days in culture. Each data point represents the average cleaved caspase-3 area per nucleus from one confocal z-stack containing several cells or neurospheres (N = 3 replicate hydrogels, data are means ± standard deviation). ***p = 0.0001. c LDH cytotoxicity assay, normalized to metabolic activity, after 14 days in culture (N = 4 replicate hydrogels, data are means ± standard deviation). ***p = 0.0009. d Representative brightfield and fluorescence images of NPCs encapsulated within Dynamic Fast and Dynamic Slow RGD-HAVDI HELP gels after 14 days of culture labeled with a fluorogenic hypoxia probe (red). e Quantification of fluorescence intensity of cultures stained with hypoxia probe. Each data point represents the fluorescence intensity from one confocal z-stack containing several cells or neurospheres (N = 3 replicate hydrogels, data are means ± standard deviation). ****p < 0.0001. f Quantification of the relative metabolic activity of encapsulated NPCs at days 7, 10, and 14, relative to the activity 1 hour post-encapsulation (N = 4 replicate hydrogels, data are means ± standard deviation). ****p < 0.0001. Statistical analyses performed as two-tailed unpaired t test (b, c, e) and two-way ANOVA with Šídák’s multiple comparisons test (f). Source data are provided as a Source Data file.