Csc37

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.

To accurately measure the thickness of carbon films, a portion of the carbon layer as evaporated on mica was cleaved off using adhesive tape (3M Scotch Crystal) leaving behind a step edge. This was imaged in constant force, DC mode using an oxide sharpened, contact mode silicon tip/cantilever (Mikromasch CSC37) in air at ambient conditions in an atomic force microscope (MFP3D, Asylum Research). After obtaining a 2D topographic image, the height was flattened using a two-dimensional, 2^nd order polynomial fit to the masked mica surface and rendered using a grayscale as indicated in the images (Supplementary Fig. 2d). A histogram of the height values was created, and the two peaks representing the substrate and carbon surfaces were fit with Gaussian functions (Supplementary Fig. 2e). The parameters of these fits were then used to measure the height and calculate the error in the measurement (std. dev.).