Figure 2.

Soft CGS promotes RhoA downregulation and neuronal conversion. A) Differentiation scheme for CGS‐based neuronal induction protocol. Human iPSCs initially preconditioned with N2B27 media supplemented with dual SMAD inhibition (Dorsomorphin and SB431542) for 7d were passaged onto CGSs. After passaging, the cells were maintained with N2B27 media without dual SMAD inhibitor. ICF, immunocytofluorescence (green arrow). Bar plots showing percentages of B) TUJ1⁺ and C) MAP2⁺ at 7d on CGSs of varying elasticity. D) Representative immunocytofluorescence analysis of RhoA (green)/YAP (red) and TUJ1 (green)/MAP2 (red) in iPSCs cultured on glass, stiff, and soft CGS. Cell nuclei were counterstained with DAPI (blue). Scale bars indicate 25 for RhoA/YAP and 50 µm for TUJ1/MAP2, respectively. E) qRT‐PCR analysis of Rhoa, an intracellular signal mediator, in iPSCs cultured on glass, stiff or soft CGS. Expression levels are normalized to GAPDH. F) Bar plot showing rigidity‐dependent nuclear co‐localization of YAP in iPSCs cultured for 2d on different substrates. B,C,E,F) Analyzed using a one‐way ANOVA, followed by Tukey's HSD post hoc test with ^* p < 0.05 and ^** p < 0.01. Values represent the mean of independent experiments (n = 4); error bars, SD.