Fig. 5. Actin polymerization is required for neurite outgrowth and neural maturation in stress-relaxing matrices.

(A) Representative maximum projection fluorescence images of hNPC neuritic extension (DAPI-labeled nuclei in magenta, TUBB3-labeled neurites in cyan) after 7 days in culture (initially seeded at 3 × 10⁴ cells/μl) within DYN Slow and DYN Fast with either the cell-binding RGD amino acid sequence (top row) or the scrambled RDG sequence (bottom row) within the cell-interactive domain of the ELP. (B) Higher-magnification representative maximum projection fluorescence images of neuritic extension after 7 days in culture within DYN Slow and DYN Fast with either the cell-binding RGD or the scrambled RDG sequence. (C) Quantification of TUBB3-expressing neurite area, normalized by cell number, over time across DYN Slow and DYN Fast within either RGD- or RDG-containing matrices (N = 5 replicate hydrogels). (D) Representative maximum projection fluorescence images of neuritic extension after 7 days in culture within all three gel formulations following daily treatment with either blebbistatin (BLEB) or latrunculin A (LatA). (E) Quantification of TUBB3-expressing neurite area, normalized by cell number, over time across all three gel formulations following daily treatment with either blebbistatin or latrunculin A (N = 3 replicate hydrogels, n = 5 fields of view). (F to M) mRNA expression of canonical neural differentiation markers for hNPCs encapsulated in DYN Fast following treatment with latrunculin A over 7 days in culture (N = 4 replicate hydrogels). Statistical analyses performed as two-way ANOVA with Tukey’s multiple comparisons test (C), two-way ANOVA with Dunnett’s multiple comparisons test (E), and two-way ANOVA with Šídák’s multiple comparisons test (F to M). Data plotted as mean ± SD where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.