Fig. 3

NGF enhances the functions of intestinal stem cells in organoid cultures. a Representative images show the intestinal organoids cultured with or without recombinant NGF at day 3 (D3) and day 6 (D6) in culture (scale bars: 100 μm). b The size of organoids cultured with or without recombinant NGF (*p < 0.05, **p < 0.01; n = 20 organoids per group, two-tailed t test). c The efficiency in organoid formation under the culture condition with/without recombinant NGF (*p < 0.05, **p < 0.01; n = 3 per group, two-tailed t test). d Whole-mount confocal imaging for the EGFP expression (green) in Lgr5-EGFP⁺ organoids cultured with/without NGF. Nuclei were counterstained by DAPI (blue) (scale bars: 50 μm for panels a, b; 100 μm for panels c, d). e Western blotting analyses for Lgr5 expression in organoids with/without NGF. f qPCR analyses for the expression of stem cell marker genes (Lgr5, Sox9, and Olfm4) in organoids cultured with/without NGF (*p < 0.05, **p < 0.01; n = 3, two-tailed t test). g The enteroendocrine cells in organoids cultured with/without NGF were stained with chromogranin A (Chga) (red) and visualized by whole-mount confocal imaging. The lower panels show the higher magnification of boxed areas in the upper panels. The density for Chga⁺ in organoids was quantified in h. (*p < 0.05; n = 20 organoids, two-tailed t test) (scale bars: 50 μm for upper panels, 20 μm for lower panels). i Whole-mount immunofluorescent co-staining for serotonin/5-HT (red) and DAPI (blue) in organoids cultured with/without NGF (scale bars: 50 μm). j Quantification for the density of 5-HT⁺ EC cells in organoids cultured with/without NGF (**p < 0.01; n = 20 organoids, two-tailed t test). k Confocal imaging shows immunostaining for lysozyme (red) in organoids cultured with/without NGF (scale bars: 20 μm). l The Paneth cell frequency per bed was presented by the average number of lyz⁺ cells per bud in organoids (**p < 0.01; n = 20 organoids, two-tailed t test). All data represent the mean ± SEM