Fig 2: Organoid formation by cochlear cell groups at different cell densities(A–C) Fluorescent reporter gene expression in P2 organ of Corti vibratome sections of Sox2-GFP (A), lunatic fringe (Lfng)-GFP (B), and Fgfr3-iCreERT2/Ai14tdTomato/Sox2-GFP (C) mouse strains. Scale bars: 50 µm. Schematic drawings illustrate the different cell groups for each mouse line.(D–F) FACS plots (left: dot plots; right: zebra plots) and gates applied for cell isolation: Sox2-positive gate (G_Sox2) (D), Lfng-positive gate (G_Lfng) (E), and gates 1–4 (G1–G4) defined by different levels of GFP and tdTomato expression using Fgfr3-iCreERT2, Ai14-tdTomato, and Sox2-GFP triple-transgenic mice (F). We expected that G_Sox2 represents all supporting cells and some GER cells. G_Lfng should consist of DCs, OPCs, IPHs, and IBCs. G1–G4 were expected to discriminate among all Sox2-GFP-positive supporting cells, with G1 and G2 representing enriched GER populations and G3 and G4 representing DCs and pillar cells.(G) Boxplot presenting the number of organoids generated after 7 days in suspension culture at low cell density (2.5 cells/µL media) from G_Sox2 and G_Lfng cells. The mean ± SD is shown next to each boxplot. ***p < 0.001, two-tailed unpaired Student’s t test; n = 7 for each group.(H and I) Boxplots presenting the number of organoids from the cells in G1–G4 generated after 7 days in low cell density culture (2.5 cells/µL media) (H) and higher cell density culture (10 cells/µL media) (I). The mean ± SD is shown next to each boxplot. ***p < 0.001, **p < 0.01, *p < 0.05, two-tailed unpaired Student’s t test; n = 11 (G1), n = 11 (G2), n = 11 (G3), and n = 10 (G4) in low cell density plots (H). n = 7 (G1), n = 6 (G2), n = 6 (G3), and n = 4 (G4) in higher cell density plots (I).(J and K) Colorimetric cell viability assays of G1–G4 cells after 7 days of culture at low density (2.5 cells/µL media) (J) and higher density (10 cells/µL media) (K). Shown are the means ± SD of four experiments. **p < 0.01, *p < 0.05, two-tailed unpaired Student’s t test.(L) Colorimetric cell viability assays performed after 7-day culture of cells from G1. The absorbance at 490 nm was compared between wells that received at the onset of the experiment 2,000 cells at higher cell density (10 cells/µL media) and the combined cells harvested from four individual wells that received 500 cells each (also a total of 2,000 cells), but at low cell density (2.5 cells/µL media). Shown are the means ± SD of triplicate experiments. The data are representative of three independently performed experiments. **p < 0.01, two-tailed unpaired Student’s t test.DC, Deiters’ cell; GER, greater epithelial ridge; HeC, Hensen’s cells; IBC, inner border cell; IPC, inner pillar cell; IPH, inner phalangeal cell; ns, not significant; OPC, outer pillar cell.

Fig 3: Emx1-Cre Cic knockout mice have a diminished pool of adult hippocampal neural progenitor cells. (A–C) Representative images of the dentate gyrus four hours post EdU injection showing staining for EdU (red) and DAPI (blue). EdU staining is mainly found in the neurogenic subgranular zone. Scale bars = 200 µm. (D–F) Representative images of the subgranular zone (SGZ) showing staining for Ki67 (green), SOX2 (magenta), and DAPI (blue). Yellow line in (D) indicates the boundary between the SGZ and the granule cell layer (GL). Yellow arrowheads in (F) point to cells double positive for SOX2 and Ki67. Scale bars = 50 µm. Quantifications of EdU+ cells (G), SOX2+ cells (H), Ki67+ SOX2+ cells (I), and the percentage of proliferating SOX2+ neural progenitor cells (J) are shown at the bottom. N = 4?7 animals per group. Box-and-whisker plots show all data points; centre lines represent medians, box limits represent interquartile ranges, and whiskers represent minimum to maximum data ranges. Statistical analyses were performed with one-way ANOVA with Tukey’s post hoc test. *P < 0.05.

Fig 4: Organoids demonstrate varied induction of apoptotic and senescence activity in response to clinical therapies. A Apoptotic staining of pGBM001 organoids treated with chemotherapeutics for Cleaved caspase 3, scale bar = 100um. B Double immunolabeling of pGBM001 organoids for markers of stemness (SOX2) and apoptosis (Cleaved caspase 3), and graph of percent of SOX2+, Cleaved Caspase 3+ and SOX2+, Cl.Casp.3+ double positive cells, scale bar = 50um. C Graph for flow cytometric analysis of pGBM001 organoids for senescence marker C12FDG and DNA (apoptosis) marker To-Pro3. * indicates p < 0.05, ** p < 0.001, *** p < 0.0001, ns non-significant.

Fig 5: Analyzing origin of newly generated SGNs.(A) Timeline for investigating the origin of new SGNs. (B) Marker+ cells in the SG of Ctrl and on days 3 and 7. On day 3, ß-III tubulin+Sox2+ cells and ß-III tubulin+Sox10+ cells were found (arrowheads). (Scale bars, 50 µm.) (C) Total cell and ß-III tubulin+ neuron number per hbSG of Ctrl and on days 1 to 28. (D) Sox2+Sox10-, Sox2+Sox10+, and Sox2-Sox10+ cell numbers per hbSG of Ctrl and on days 1 to 28. (E and F) ß-III tubulin+Sox2+ and ß-III tubulin+Sox10+ cell number per hbSG of Ctrl and on days 1 to 28. (C–F) n = 3 animals each; error bars are mean ± SEM; *P = 0.05, **P = 0.01, ***P = 0.001; 1-way ANOVA and Dunnett’s multiple-comparison test compared with the control. (G) Sox2+ cell proportion among ß-III tubulin+Sox10+ cells per hbSG of Ctrl and on days 1 to 28 (n = 3 animals each; error bars are mean ± SEM; 1-way ANOVA and Tukey’s multiple-comparison test). (H) Sox10- cell proportion among ß-III tubulin+Sox2+ cells per hbSG of Ctrl and on days 1 to 28 (n = 3 animals each; error bars are mean ± SEM; ***P = 0.001; 1-way ANOVA and Tukey’s multiple-comparison test). (I) Ki67+, Sox2+, and Sox10+ cells in the SG on days 3 and 7. (Scale bars, 50 µm.) (J) Sox2+Sox10-, Sox2+Sox10+, and Sox2-Sox10+ cell number in Ki67+ cells per hbSG of Ctrl and on days 1 to 28 (n = 3 animals each; error bars are mean ± SEM; *P = 0.05, **P = 0.01, ***P = 0.001; 1-way ANOVA and Tukey’s multiple-comparison test compared with the control).