Fig. 3
LESC-like phenotype can be controlled through fine modulation of the mechanical properties of collagen substrates. a Schematic representation of the collagenase treatment method used to modulate the stiffness of compressed collagen gels. High-density, plastic-compressed collagen gels were softened with collagenase type-I solution in well-defined areas (ring-shaped, semi-circular, or entire gel surface) for up to 60 min. b Analysis of compressed collagen gel density after collagenase treatment. The regions corresponding to collagenase-treated collagen gels showed increased transparency under bright-field imaging (upper left panel; scale bar, 5 mm) and lower collagen density compared to untreated regions, as indicated by the lower collagen-I detection by immunofluorescence confocal microscopy (upper right panel; scale bar, 50 µm). The confocal Z-scans (lower panel) demonstrated that the difference in signal intensity between treated and untreated areas was not restricted to the surface, indicating that the collagenase in solution acted through the entire depth of the compressed collagen maintaining a defined treatment zone. c Average frequency ± S.D. of the elastic modulus, E (MPa), of treated (orange) and untreated gels (blue bars) calculated from three independent experiments using force–distance spectroscopy (n = 3). The frequency histograms of treated and untreated gels were used to calculate Gaussian curves by non-linear regression (orange and blue areas, respectively), with corresponding E = 0.7 ± 0.4 and 4.8 ± 3.5 MPa. d Effects of substrate stiffness on the expression of CK3 (differentiation) and CK15 (LESC protein marker) in cells grown for 4 weeks on treated and untreated regions of collagen gels (green staining) after normalisation for total cell number (red staining), and represented as average ± S.D. from three independent experiments (n = 3; ** and *** corresponds to p < 0.01 and 0.001 after one-way ANOVA, respectively). Source data are provided as a Source Data file. e Creation of a pseudo-limbus. Cells growing on ring-shaped softened areas (week 0) expressed higher levels of CK15 compared to the high CK3-positive cells growing on the untreated (stiffer) central region of the collagen gels, up to 4 weeks in culture (scale bar, 5 mm)
