Figure 2.

Encapsulated culture promotes IEPC expansion. A, Confocal images of suspended and encapsulated spheroids showing EdU and Ki67 staining. Proliferating cells were quantified as the percentage of EdU+ or Ki67+ cells. Scale bar = 15 μm. n = 11 suspended spheroids, and n = 10 encapsulated spheroids. B, Bright‐field and fluorescence microscopy images of spheroids formed from suspended and encapsulated cells and expressing Lgr5‐EGFP. Scale bar = 500 μm. Quantification of the diameter of spheroids from (B); n = 22 suspended spheroids, and n = 18 encapsulated spheroids. Quantification of the percentage of Lgr5‐EGFP positive spheroids from (B) (n = 3); C, qRT‐PCR analysis showing the relative expression of cell cycle genes and markers of IEPCs, supporting cells, and hair cells. Results were normalized to GAPDH in the same sample and then normalized to the suspension group (n = 3). D, Confocal images showing Myosin7a and Sox2 immunofluorescent staining. Scale bar = 15 μm. Quantification of the percentage of Myosin7a/Sox2 double‐positive spheroids from (D) (n = 6), and quantification of the percentage of Myosin7a/Sox2 double‐positive cells per spheroid from (D) (n = 10 spheroids for each group). The data are presented as mean ± SEM; *P < .05; **P < .01 (unpaired two‐tailed Student's t test)