Fig. 5

Organoids from EPCAM+ cells generate hair cell-like cells. a Schematic drawing of the experimental procedure. EPCAM+ organoids were expanded for 2 weeks with or without CHIR99021, then co-cultured with EPCAM− cells in transwell inserts. EPCAM− cells were derived in parallel from the same sample. The medium was supplemented twice with LY411575 or with vehicle during the 2-week differentiation period as indicated. b Organoid derived from sample E1229 and immunostained at the end of the differentiation protocol for MYO7A, SOX2, and EPCAM. Scale bar = 100 μm. The merged high magnification image is a confocal projection; the single channels are shown as individual confocal sections. Scale bar = 50 μm. c Organoid derived from sample E1246 (+CHIR99021) and immunostained at the end of the differentiation protocol for MYO7A. F-actin bundle was labeled with phalloidin. Consecutive magnifications are shown. Scale bars = 100μ m (left and middle), and 10 μm (right). d Images of MYO7A positive areas in organoids untreated/treated with CHIR99021 during the expansion phase and untreated/treated with LY411575 during the differentiation phase, as indicated. Scale bar = 100 μm. e Quantification of the number of hair cells-like cells (HC) per organoid at the end of the differentiation protocol for the different culture conditions as indicated. Three to four organoids per condition per sample were analyzed. Five human fetal samples of ≈week 11 were used for this experiment (E1245, E1246, E1229, E1248, E1253). Box plots showing sample distribution and median. Whiskers represent minimum and maximum values. Untreated: 6.45 ± 6.6 (n = 11); CHIR99021: 9.09 ± 19.84 (n = 11); LY411575: 10.46 ± 10.11(n = 13); CHIR99021 + LY411575: 2.53 ± 1.995 (n = 15). Values are mean ± s.d.