Fig. 3: The combination of TSA, pVc, and CP induces enhanced stemness and increased cellular diversity in organoids. | Nature Communications

Fig. 3: The combination of TSA, pVc, and CP induces enhanced stemness and increased cellular diversity in organoids.

From: A tunable human intestinal organoid system achieves controlled balance between self-renewal and differentiation

Fig. 3

a Brightfield and fluorescence images of organoids cultured for 1 week in indicated conditions. Representative images from three independent experiments. b, c Quantification of the percentage of LGR5-mNeonGreen cells (b) and relative fluorescence intensity (c) in organoids cultured in indicated conditions, n = 4 samples. Colony forming efficiency (d) and the number of cells (e) of organoids cultured in indicated conditions, n = 4 samples. f RT-qPCR quantification of cell markers expression levels, n = 3 samples. g Representative confocal images of LYZ, MUC2, and CHGA in organoids cultured in indicated conditions. Representative images from three independent experiments. h Proportions of secretory lineages in organoids cultured in indicated conditions, n = 4 samples. b–f and h, one-way ANOVA with Dunnett’s multiple comparisons test; data are presented as mean ± SD. i Schematic of experiments in (j–o). j Confocal images of EdU staining of organoids. k Quantification of the percentage of LGR5-mNeonGreen cells and relative fluorescence intensity in organoids cultured under the indicated conditions, n = 3 samples. l Brightfield and fluorescence images of organoids cultured in indicated conditions. m EdU and immunofluorescence staining of CHGA, MUC2, and LYZ in organoid cultures as indicated. EdU was added for 1 hour before staining. Representative images of (j, l, m) were from three independent experiments. n FACS gating strategy of LGR5-mNeonGreen cells. o Quantification of EdU, LYZ, MUC2 and CHGA positive cell percentage as in (m), n = 4 samples. k and o, two-tailed unpaired t-test; data are presented as mean ± SD. p Quantification of the percentage of LGR5-mNeonGreen cells in organoids cultured under the indicated conditions, n = 6 samples. q LGR5-mNeonGreen fluorescence images of organoids cultured in indicated conditions. Representative images from four independent experiments. r Proportions of LGR5-mNeonGreen cells in organoids cultured in conditions as in (q), n = 4 samples. (p and r), one-way ANOVA with Dunnett’s multiple comparisons test; data are presented as mean ± SD. Scale bars, (a and l), 200 μm; (g, j, m and q), 50 μm. Source data are provided as a Source Data file.

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