Fig. 1: Optimized human intestinal organoid demonstrates enhanced stemness and increased cell diversity. | Nature Communications

Fig. 1: Optimized human intestinal organoid demonstrates enhanced stemness and increased cell diversity.

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

Fig. 1

a Schematic of screening strategy to optimize cultures of human intestinal organoids. b Schematic of the targeting strategy to generate LGR5-mNeonGreen reporter system. c Medium composition comparison among ES, IF, IL, and TpC culture systems. 1Noggin or BMP pathway inhibitor DMH1, 2R-Spondin1 conditioned media, 3WNT3a protein, WNT3a conditioned media or WNT surrogate. d Representative brightfield and fluorescence images of LGR5-mNeonGreen organoids cultured in IL patterning condition, IF condition or TpC condition. Representative images from three independent experiments. Scale bars, 200 μm. Quantification of LGR5-mNeonGreen proportion (e) and relative LGR5-mNeonGreen intensity (f) in IF, IL patterning and TpC organoids cultured for 4 weeks. n = 3 samples. Quantification of colony forming efficiency (g) and cell proliferation as indicated by the number of cells (h) in IF, IL patterning and TpC organoids cultured for ten days from single cells (8000 cells per well seeding). n = 4 samples. One-way ANOVA with Dunnett’s multiple comparisons test; data are presented as mean ± SD. i Proportion of secretory lineage cells in IF and TpC organoids quantified by positive staining of LYZ, MUC2, and CHGA, respectively. Two-tailed unpaired t-test; data are presented as mean ± SD; n = 3 samples. j Representative images of enterocytes (ALPI), goblet cells (MUC2), enteroendocrine (CHGA), and Paneth cells (LYZ) in TpC organoids. Representative images from five independent experiments. k Representative confocal images of LGR5-mNeonGreen and DEFA5 positive Paneth cells in TpC organoid. White arrowheads indicate Paneth cells adjacent to LGR5 intestinal stem cells, red arrowhead points out a double-positive cell. Representative images from four independent samples. l The growth and mNeonGreen expression of a single LGR5-mNeonGreen+ cell cultured in TpC condition over 13 days. Also showing Paneth (LYZ), goblet (MUC2), and enteroendocrine (CHGA) cells in the 13-day colonies detected by immunofluorescence staining. Representative images from three biological replicates. Scale bars, (j–l), 50 μm. Arrowheads indicate the emergence of mNeonGreen expression at the same location. Source data for this Figure are provided as a Source Data file.

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