Fig. 1. Substrate stiffness-induced biphasic enhancement of iN reprogramming efficiency.

(A) Experimental timeline for substrate stiffness-induced iN reprogramming. (B) Immunofluorescent mages of Tubb3⁺ iN cells derived on glass or PAAm gels of varying stiffness on day 7. Scale bar, 200 μm. DAPI, 4′,6-diamidino-2-phenylindole. (C) Reprogramming efficiency of BAM-transduced fibroblasts that were cultured on glass and PAAm gels of varying stiffness (n = 4 independently prepared gel surfaces). On day 7, the cells were fixed and stained for Tubb3, followed by immunofluorescence microscopy to quantify Tubb3⁺ iN cells. (D) Relative Ascl1 mRNA expression in BAM-transduced fibroblasts seeded on glass and PAAm gels of varying stiffness at day 1 following Dox activation (n = 3). (E) Fibroblasts transduced with BAM and an Ascl1 promoter–GFP construct were seeded on glass and PAAm gels of varying stiffness for 24 hours and activated by Dox for another 24 hours. The cells were fixed and observed by immunofluorescence microscopy, showing that 20-kPa PAAm gel induced more Ascl1 promoter–GFP⁺ cells at day 1, as quantified in the bar graph (n = 3). (F) Representative images of Tubb3⁺ cells expressing mature neuronal markers, NeuN, MAP2, and synapsin at 21 days after cells were cultured on 20-kPa gels. Scale bars, 100 μm. (G) Representative trace showing spontaneous changes in membrane potential in response to current injection from iNs derived on 20-kPa gels. In (C) to (E), statistical significance was determined by a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test (N.S.: not significant, *P ≤ 0.05 and ***P ≤ 0.001). In (C) to (E), bar graphs show means ± SD.