Extended Data Fig. 15. Characterization of stem cells in RANK–RANKL-stimulated human intestinal organoids.
a, Uniform manifold approximation and projection (UMAP) of 2,265 human intestinal epithelial cells. Data were taken from Fujii et al. 43. Cells are colour coded according to epithelial cell-type annotation based on unsupervised clustering. b, Violin plots show single cell log-normalized expression of RANK and RANKL in each intestinal cell-type. Each dot represents an individual cell. c, Quantitative RT–PCR analyses to compare expression levels of anti-apoptotic genes, stem cell signature genes, and BMP signalling genes in human duodenal organoids. Data represent the relative expression of the indicated genes in rhRANKL (500 ng/ml) stimulated duodenal organoids compared to control (no RANKL) organoids (set at 1). rhRANKL stimulation was for 12 and 96 h. n = 4 (control), n = 4 (RANKL). d, Representative images (left) and quantified areas (right) of human duodenal organoids cultured for 2 days without (control; n = 83) and with recombinant human RANKL (rhRANKL; 500 ng/ml; n = 54) in growth-factor-reduced medium (GFR medium) lacking EGF, IGF-1, and FGF-2. Scale bars, 100 μm. Each dot represents an organoid, assessed in three independent experiments. e, RANKL-induced proliferation of duodenal organoids. Organoids were left untreated (control) or treated with rhRANKL (500 ng/ml) and proliferation determined using the MTT assay. Each plot represents an MTT OD, pooled from two independent experiments. n = 20 (control), n = 20 (RANKL). f, Representative cell cycle FACS plots (left) and quantification of S-phase and subG1 + G1 entry (right) assessing EdU labelled human duodenal organoids cultured in GFR medium and stimulated with rhRANKL for 24 and 72 h. Dots represent individual organoids, assessed in three independent experiments. n = 14 (control, 24hrs), n = 16 (control, 72hrs), n = 18 (rmRANKL, 24hrs), n = 18 (rmRANKL, 72hrs). g, Representative images of anti-OLFM4 immunostaining (green) of human duodenal organoids cultured in GFR medium in the absence (control) and presence of rhRANKL (500 ng/ml) for 24 and 96 h. Organoids were counterstained with DAPI (blue) to detect nuclei and Phalloidin (magenta) to detect filamentous actin. Scale bars, 50 μm. h, Representative images of OLFM4+ stem cells in human intestinal organoids cultured in the presence of rhRANKL (500 ng/ml) without (control, DMSO solvent) or with the BMP inhibitor (BMPi) LDN193189 (1.6μM) for 96 hrs. Organoids were stained with DAPI (blue) to detect nuclei and Phalloidin to detect filamentous actin. Scale bars, 50 μm. i, Proposed function of RANK–RANKL in the small intestine during pregnancy and lactation. During pregnancy and lactation, RANK–RANKL signalling promotes intestinal stem cell proliferation and differentiation as well as intestinal cell survival, ultimately resulting in massive villous expansion. Villous expansion facilitates nutritional uptake, which is important for nourishment of offspring as well as their transgenerational metabolic health. The figure was created with BioRender.com. Data are mean ± s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Two-tailed Student’s t-test (c-f). More details on statistics and reproducibility can be found in the Methods.