Fig. 1: Organoid growth and patterning modulation through matrix actuation.

a Schematic representation of the hNTO differentiation protocol. Floor plate induction by RA-SAG treatment, with organoid phenotype categorized based on scattered, patterned or absence of FOXA2 expression. Representative images of hNTOs at days 0, 3, 5, and 11 (n = 3). b Actuation of hNTOs. Hydrogel droplets (shown in blue for visualization) in stretchable membrane attached to the arms of an equibiaxial actuation device. Stretching is imposed simultaneously with RA-SAG treatment (days 3–5), with membrane surface area reaching 100% area increase at day 5 and maintained thereafter. c Increase in hNTO size by stretching at day 5 until day 11 (n = 3 for a total of 50 hNTOs per datapoint, statistical analysis was determined by two-way ANOVA with Fisher’s LSD test for multiple comparisons (day 5 p < 0.0001, day 7 p < 0.0001, day 9 p < 0.0001, day 11 p < 0.0001). Representative brightfield images, 3D confocal reconstructions and corresponding optical sections (n = 3). d Quantification of FP induction and pattering events in FOXA2+ hNTOs (n = 5 for unstretched (303 hNTOs) and n = 6 for stretched (480 hNTOs) conditions, statistical analysis was determined by unpaired two-sided t-test (p < 0.0001)). e Quantification of FOXA2 dynamics from day 7 (n = 2 for a total of >20 hNTOs per day). f Quantification of organoid size for stretched and unstretched conditions, binned for scattered and patterned hNTOs (hNTOs from d, statistical analysis was determined by unpaired two-sided t-test with Welch’s correction (scattered p < 0.0001, patterned p < 0.0001)). g Patterned hNTO frequency as a function of binned organoid size for stretched and unstretched conditions (hNTOs from d). h Area ratio (AR) quantifying the ratio between FOXA2+ domain area and total organoid area, for scattered and patterned hNTOs within each condition (hNTOs from d, statistical analysis was determined by unpaired two-sided t-test with Welch’s correction (unstretched p < 0.0001, stretched p < 0.0001)). Error bars are SEM, scalebars 50 µm.