Transwell permeable support membranes

For staining and imaging, organoids were allowed to adhere ON onto transwell-permeable support membranes (Corning) in 12-well plates coated with 100% Matrigel. The next day, they were fixed with 4% PFA (10 min). Cells were permeabilized with 3% Triton-X 100 for 10 min and incubated ON with blocking buffer containing 2% BSA and 0.01% Triton-X 100. Primary antibodies were incubated ON at 4°C (1:200 dilution). Anti-Sox2 (mouse, BD Bioscience), anti-Pou4f3/Brn-3c (mouse, Santa Cruz Biotechnology) anti-Myo7a (rabbit, Enzo Life Sciences) and anti-p27/Kip1 (rabbit, Abcam). Organoids were then rinsed and incubated ON with the secondary antibodies as described above. For FM1-43 uptake, organoids were first incubated with Hoechst (10 μg/ml), 30 min at 37°C, then a brief incubation FM1-43 (5 μM; Life Technologies) for 30 s, and washed twice before fixation (4% PFA). To image organoids, inserts were placed face down in a 24 well glass bottom plate (MatTek) and imaged on a Zeiss LSM710 laser scanning microscope (5 μm Z-stacks intervals). Compiled Z-projections were processed (FIJI, ver2.0) and hair cells quantified on individual planes with manual cell counter plugin, by evaluation of cytosolic expression and nuclear exclusion of Myo7a. Organoid volumes were estimated from the sum of the product of the area of the organoid (at each plane) and the Z stack height.

Organoids expanded for 2 weeks were plated on transwell-permeable support membranes (Corning) in 12-well plates. 100 µl of 50% Matrigel was used to pre-coat each insert for 30 min before plating the organoids. This coating facilitated the immobilization of the organoids at the bottom of the insert. EPCAM-sorted cells were cultured in the lower compartments at 70%–100% confluency directly on plastic. Supplemented DMEM-F12 medium was further supplemented when indicated, with 1 μM LY411575 and left unchanged for 7 days. The medium was then refreshed by replacing 50% of the medium with fresh one (DMEM-F12 with B27 and N2, lacking growth factors) and re-adding LY411575 (1 μM) every 3–4 days. Organoids were fixed and immunostained for characterization as described above.