Fig 2: Rps14 promotes the proliferation of cochlear progenitors postnatally via the Wnt signalling pathway. (A) Experimental design. (B) The QPCR analysis of Rps14 mRNA expression in AAV‐Rps14‐injected cochleae (n = 3). (C) EdU immunostaining in AAV‐transduced cochlear epithelia. EdU+/Sox2+ pillar cells were observed. AAV dose: 9 × 1010 GCs/cochlea. Sox2 (red) marks the supporting cells. Scale bar: 25 μm. (D) The number of EdU+ cells in (C) (n = 5). (E) Lgr5‐EGFP fluorescence signals in AAV‐HA, AAV‐Rps14‐ipsilateral and AAV‐Rps14‐contralateral cochleas. EGFP signals were captured under the same conditions. AAV dose: 4.5 × 1010 GCs/cochlea. Scale bar: 25 μm. (F) The QPCR analysis of Lgr5 expression in AAV‐mNeonGreen and AAV‐Rps14‐transduced cochleae (n = 3). (G) Lgr5+ cells were sorted and collected by the EGFP channel and then were cultured at 500 cells/well in a sphere‐forming assay. AAV‐mNeonGreen and AAV‐Rps14 were added to the culture medium from Day 2 to Day 4. (H) Brightfield images of AAV‐mNeonGreen and AAV‐Rps14‐overexpressing organoids after expansion for 5 days. AAV dose: 2 × 1010 GCs/well. Scale bar: 50 μm (I,J) The total numbers (I) (n = 4) and diameters (J) (n = 94) of spheres generated in (H). The results are shown as the mean ± SEM. The p‐value was calculated by Student's t‐test (*p < 0.05; **p < 0.01; ***p < 0.001; n.s. refers to no significance.). AAV, adeno‐associated virus; QPCR, quantitative real‐time polymerase chain reaction

Fig 3: Rps14 promotes hair cell reprogramming in postnatal cochleae. (A) Experimental design. (B) Representative Myosin7a immunostaining (magenta) in the apical, middle and basal turns of cochleae transduced by AAV‐mNeonGreen and AAV‐Rps14 at the same dose (9 × 1010 GCs per cochlea). Myosin7a (magenta) marks hair cells. Cochleae were harvested at P9 and P16 after microinjection with 1.5 μL of AAV stock solution in the left ear at P2. Scale bars, 50 μm. Yellow triangles indicate the ectopic hair cells. (C,D) The number of ectopic hair cells in P9 (C) (n = 4) and P16 (D) (n = 3) cochleae corresponding to (B). The results are shown as the mean ± SEM. The p‐value was calculated by Student's t‐test (*p < 0.05; **p < 0.01; n.s. refers to no significance.). AAV, adeno‐associated virus

Fig 4: Rps14 overexpression promotes cochlear progenitor expansion and hair cell production in three‐dimensional culture. (A) Experimental design of the three‐dimensional organoid culture. A single EdU pulse was given at Day 10, and EdU incorporation was analysed 1 h later. (B) Brightfield images of organoids overexpressing AAV‐mNeonGreen and AAV‐Rps14 after expansion. Scale bars: 100 μm. (C,D) The total numbers (C) (n = 3) and diameters (D) (n = 60) of organoids generated in (B). (E) Confocal images of control and Rps14‐overexpressing organoids. EdU (magenta) marks proliferated cells, AAV‐green marks transduced cells, and DAPI (grey) labels cell nuclei. Scale bars: 100 μm. (F) AAV transfection efficiency in the expansion assay. (G) The RNA expression level of Rps14 in the expansion assay. (H) The DAPI number per organoid in the expansion assay. (I) Average number of EDU+ cells generated by each organoid in (E) (n = 20). (J) The QPCR analysis of the differentially expressed genes in the expansion assay. (K) Experimental design of cochlear organoid culture in the differentiation assay. (L) Confocal images of control and Rps14‐overexpressing organoids. Myosin7a (red) marks hair cells, EdU (magenta) marks proliferating cells, AAV‐green marks transduced cells, and DAPI (grey) labels cell nuclei. Scale bars: 100 μm. (M) AAV transfection efficiency in the differentiation assay. (N) The RNA expression level of Rps14 in the differentiation assay. (O) Total numbers of Myosin7a+ cells in (L) (n = 3). All AAVs were used at a dose of 2 × 1010 GCs per well. (P) The number of Myosin7a + cells in each organoid in the differentiation assay. (Q) The QPCR analysis of the differentially expressed genes in the differentiation assay. The results are shown as the mean ± SEM. The p‐value was calculated by Student's t‐test. (*p < 0.05; **p < 0.01; ***p < 0.001; n.s. refers to no significance.). AAV, adeno‐associated virus; QPCR, quantitative real‐time polymerase chain reaction

Fig 5: Rps14 overexpression has limited effect on cochlear progenitor expansion and hair cell production in two‐dimensional culture. (A) Experimental design of the two‐dimensional organoid culture. AAV dose: 9 × 1010 GCs/cochlea. (B) Brightfield images of organoids overexpressing AAV‐mNeonGreen and AAV‐Rps14 after expansion. Scale bar: 50 μm. (C,D) The diameter (C) and the ratio (D) (n = 3) of three organoid morphologies after 5 days of culture. (E) The QPCR analysis of progenitor markers in two‐dimensional organoid culture. (F) The experimental design of cochlear organoid culture in the differentiation assay. AAV dose: 2 × 1010 GCs/well. (G) Confocal images of control and AAV‐Rps14‐overexpressing organoids. Myosin7a (red) marks hair cells, AAV‐green marks transduced cells and DAPI (grey) labels the cell nuclei. Scale bar: 100 μm. (H) Total Myosin7a+ cell counts in (G) (n = 3). Results are shown as the mean ± SEM. The p‐value was calculated by Student's t‐test. (I) The number of Myosin7a + cells in each organoid in (G). (J) The ratio of Myosin7a+ cells/DAPI per organoid in (G). Results are shown as the mean ± SEM. The p‐value was calculated by Student's t‐test. (*p < 0.05; **p < 0.01; ****p < 0.0001; n.s. refers to no significance). AAV, adeno‐associated virus; QPCR, quantitative real‐time polymerase chain reaction