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. 2020 Aug 21;117(36):22225–22236. doi: 10.1073/pnas.2000417117

Fig. 1.

Fig. 1.

Cochlear epithelial cells from stage P5 mice fail to expand and produce hair cells in organoid culture. Organoid cultures were established from cochlear epithelia cells obtained from Atoh1-nGFP transgenic mice, stages P2 and P5. Atoh1-nGFP marks nascent hair cells. (A) Experimental design. (B) Brightfield (BF) images of P2 and P5 cochlear organoid cultures after 5 d of expansion. (C) Organoid forming efficiency in P2 (blue) and P5 (red) cultures in B (n = 8). (D) Organoid diameters in P2 (blue) and P5 (red) cultures in B (n = 8). (E) Cell proliferation in P2 and P5 organoids. A single EdU pulse was given at 5 d of expansion and EdU incorporation (red) was analyzed 1 h later. SOX2 (green) marks supporting cells/prosensory cells and Hoechst (blue) labels cell nuclei. (F) Percentage of EdU+ cells in P2 and P5 organoids in E (n = 8). (G) BF and green fluorescent images (Atoh1-nGFP) of P2 and P5 organoid cultures after 10 d of differentiation. (H) Percentage of Atoh1-nGFP+ organoids in G (n = 6). (I) Confocal images of P2 and P5 organoid cultures after 10 d of differentiation. Newly formed hair cells express Atoh1-nGFP (green) and MYO7A (magenta). (J) Percentage of MYO7A+ hair cells per organoid in I (n = 6). Graphed are individual data points and mean ± SD. Individual data points in D, F, and J represent the average value per animal. n = animals analyzed per group. All data are from two independent experiments. Two-tailed, unpaired Student’s t tests were used to calculate P values.