Nanowizard 4

HLO treated with 0, 200, 400, 800 μM OA were used for stiffness measurement with an AFM (NanoWizard IV, JPK Instruments). The AFM head with a silicon nitride cantilever (CSC37, k = 0.3 N/m, MikroMasch) was mounted on a fluorescence stereo microscope (M205 FA, Leica) coupled with a Z-axis piezo stage (JPK CellHesion module, JPK Instruments), which allows the indentation measurement up to the depth of ~ 100 μm. As a substrate for organoids, a fibronectin-coated dish was used. The tissue culture dish (φ = 34 mm, TPP Techno Plastic Products) was first incubated with a 1 μg/mL fibronectin solution (Sigma) at 4 °C for overnight. Then, the tissue culture dish was washed twice by distilled water and dried for 1 hour. Thereafter, HLO incubated with OA for 5 days were deposited to the fibronectin-coated dish and incubated for 1 hour at 37 °C. The sample dish was then placed onto the AFM stage, and force-distance curves in a 14×14 matrix in a 25×25 μm square were measured from each HLO. Due to technical issues for fixing the position of the FGF19-treated Wolman iPSC-sHLO, Figure4G assessed healthy PSC-sHLO to enhance the robustness. Finally, Young’s moduli (E, Pa) of organoids were determined by fitting the obtained force-distance curves with the modified Hertz model (Sneddon, 1965). Post hoc analysis was carried out by Dunn-Holland-Wolfe test for significance test.