Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Representative images of anti-RANK immunostaining in mouse jejunal intestinal organoids from Rank WT and Rank Δ Vil mice. DAPI is shown to visualize nuclei. Scale bars, 50 μm. b , Proliferation assay in organoids without (control) or in the presence of recombinant mouse RANKL (rmRANKL; 50 ng/ml) as determined by an MTT assay at the indicated time points. Each plot represents MTT OD, pooled from at least two independent experiments. Group numbers at 2 days: n = 56 (control), n = 56 (rmRANKL); 3 days: n = 50 (control), n = 50 (rmRANKL); 5 days: n = 31 (control), n = 31 (rmRANKL) and 7 days: n = 32 (control), n = 31 (rmRANKL). c , Numbers of buds per intestinal organoid cultured in ENR medium with/without rmRANKL. Data were combined from three independent experiments. Group numbers at 3 days: n = 22 (control), n = 59 (rmRANKL); 4 days: n = 57 (control), n = 102 (rmRANKL) and 5 days: n = 32 (control), n = 66 (rmRANKL) per group. d , Representative images of jejunal organoids at passage 0 and passage 1 cultured in the absence (control) or presence of the indicated concentrations of rmRANKL. Scale bars, 100 μm. e , Ratios of organoid numbers after prolonged passaging in the absence (control) and presence of rmRANKL. Numbers of organoids were counted at each passage. Data from two independent experiments are shown. n = 8 (control), n = 8 (10 ng/ml RANKL), n = 8 (50 ng/ml RANKL), n = 8 (500 ng/ml RANKL). f , Total cell numbers, OLFM4 positive cells per organoid and ratios of OLFM4 positive cell in relation to the total cell number per each jejunal organoid cultured in ENR medium with/without rmRANKL. Data were combined from two independent experiments. 2 days: n = 31 (control), n = 37 (rmRANKL); 5 days: n = 29 (control), n = 29 (rmRANKL) per group. g , Gating strategy for detecting Lgr5-eGFP + cells using fluorescence-activated cell sorting (FACS). Viability was determined using the viability-dye described in the . FSC, forward scatter; SSC, side scatter. h , Representative FACS histograms of Lgr5 high cells and CD44 + cells isolated from Lgr5-eGFPiresCreER/+ jejunal organoids cultured without (control) and with rmRANKL (50 ng/ml) for the indicated times. Numbers of Lgr5 high cells and CD44 + cells among total viable organoid cells are indicated for each group. Data are representative of at least two independent experiments. i , Quantification of Lgr5 high , Lgr5 high CD44 + , and CD44 + cells per well in Lgr5-eGFPiresCreER/+ jejunal organoids cultured without (control) or with rmRANKL (50 ng/ml) for the indicated times. n = 36 (Control, 24hrs), n = 36 (RANKL, 24hrs), n = 21 (Control, 48hrs), n = 24 (RANKL, 48hrs). Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. One-way analysis of variance (ANOVA) with Tukey’s post hoc test ( b,c,f ); Two-tailed Student’s t-test ( i ). More details on statistics and reproducibility can be found in the .

Article Snippet: The medium was replaced every 48–72 h. Once organoids were established, they were further cultured in an expansion medium composed of advanced DMEM/F12 supplemented with penicillin–streptomycin, 10 mM HEPES, GlutaMAX, N2 (Life Technologies), B27 (Life Technologies), 1 mM N -acetylcysteine (Sigma-Aldrich), R-spondin1 (conditioned medium from 293T-HA-RspoI-Fc cells, 10% final volume), 100 ng ml −1 NOGGIN (Peprotech), 10 nM human gastrin I (Sigma-Aldrich), 500 nM A83-01 (Tocris), WNT3A (conditioned medium from WNT-producing L-cell line, 50% final volume), 50 ng ml −1 mouse recombinant epidermal growth factor (EGF; Peprotech), 100 ng ml −1 human insulin-like growth factor-1 (IGF-1; BioLegend), and 50 ng ml −1 human recombinant fibroblast growth factor-basic (FGF-2; Peprotech) .

Techniques: Immunostaining, Proliferation Assay, Control, Recombinant, MTT Assay, Cell Culture, Passaging, Fluorescence, FACS, Isolation, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , RANK and EPCAM intestinal staining in Rank WT and Rank Δ vil mice. Scale bars, 25 μm. b , Left, representative images of jejunal organoids cultured without (control) and with recombinant mouse RANKL (rmRANKL; 50 ng ml −1 ) for 3 days. Scale bars, 100 μm. Right, quantification of organoid areas after culture with or without rmRANKL for 3 days. n = 185 (control) and n = 222 (rmRANKL) from three independent experiments. c , Representative 3D images of Lgr5-eGFP;Ires-cre ER/+ organoids. Scale bars, 50 μm. d , Representative images of OLFM4 staining of control and rmRANKL-treated organoids. Scale bars, 50 μm. e , Single-cell log-normalized expression of the indicated anti-apoptotic genes ( y axis) in each cell type ( x axis) (control jejunal organoids versus organoids cultured with 50 ng ml −1 rmRANKL for 12 h). f , Organoids treated with or without rmRANKL (50 ng ml −1 ) were irradiated and cultured in WENR medium with a ROCK inhibitor (Y-27632; 10 μM) (see ) for 7 days. The numbers of surviving organoids from three independent experiments are shown. n = 22 (control and rmRANKL). g , Single-cell log-normalized expression of Bmp2 and the BMP targeted genes Id2 and Id3 ( y axis) in each cell type ( x axis). h , The ratio of organoid numbers cultured in the presence of rmRANKL (50 ng ml −1 ), DMSO (control) or with the BMP inhibitor (BMPi) LDN193189 (0.5 μM). The ratio of organoid numbers in the control + RANKL group was normalized to the control group, whereas the ratio of organoid numbers in the iBMP + RANKL group was normalized to the iBMP group. Data are combined from two experiments. n = 10 for each group shown. i , Representative images of rmRANKL-treated Lgr5-eGFP;Ires-cre ER/+ organoids that were cultured with recombinant mouse NOGGIN. Scale bars, 50 μm. In the indicated images, phalloidin stains actin filaments and DAPI stains nuclei. Data are mean ± s.e.m. Statistical analysis was performed using two-tailed Student’s t -tests ( b and f ), two-sided Wilcoxon rank-sum tests between samples, adjusted using Benjamini–Hochberg correction ( e and g ) and one-way analysis of variance (ANOVA) with Tukey’s post hoc test ( h ); **** P < 0.0001. Further details on statistics and reproducibility are provided in the .

Article Snippet: The medium was replaced every 48–72 h. Once organoids were established, they were further cultured in an expansion medium composed of advanced DMEM/F12 supplemented with penicillin–streptomycin, 10 mM HEPES, GlutaMAX, N2 (Life Technologies), B27 (Life Technologies), 1 mM N -acetylcysteine (Sigma-Aldrich), R-spondin1 (conditioned medium from 293T-HA-RspoI-Fc cells, 10% final volume), 100 ng ml −1 NOGGIN (Peprotech), 10 nM human gastrin I (Sigma-Aldrich), 500 nM A83-01 (Tocris), WNT3A (conditioned medium from WNT-producing L-cell line, 50% final volume), 50 ng ml −1 mouse recombinant epidermal growth factor (EGF; Peprotech), 100 ng ml −1 human insulin-like growth factor-1 (IGF-1; BioLegend), and 50 ng ml −1 human recombinant fibroblast growth factor-basic (FGF-2; Peprotech) .

Techniques: Staining, Cell Culture, Control, Recombinant, Expressing, Irradiation, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Schematic outline of transgenic mice that conditionally express a constitutively active RANK mutant in the ROSA26 locus using gene targeting, termed LSL-caRANK mice. Crossing this line with Villin1Cre mice removes the stop cassette and thereby drives specific expression of the RANK transgene in the intestinal epithelium; these mice are termed caRANK vil-Tg . LSL, LoxP-STOP-LoxP; NeoR, neomycin resistance cassette; eGFP, enhanced green fluorescence protein. b , Representative images of eGFP expression in intestinal epithelial cells of caRANK vil-Tg mice and control mice, using cryo-sections. Phalloidin and DAPI are shown to visualize F-actin and nuclei, respectively. Scale bars, 50 μm. c , Small intestinal villi length, volume and surface areas of 3-week-old control (n = 6 mice, n = 126 villi analysed) and caRANK vil -Tg mice (n = 7/145). Each data point represents the average length, volume, and surface area of villi per mouse. The original data is the same as Fig. . d,e , Reduced cell death of small intestinal epithelial cells in caRANK vil-Tg mice. Representative images (left in d ) and quantification (right in d , e ) of cleaved caspase-3 (CLC3) immunostaining (arrows) in the small intestine are shown for three wks old control mice (n = 7 mice, n = 93 regions analysed) and caRANK vil -Tg mice (n = 8/126). Each data point represents the number of CLC3 positive cells in villi per 2 mm of the intestine ( d ) and the average number per mouse ( e ). Scale bars, 100 μm. f,g , Representative images (left in f ) and quantification of phosphor-Histone H3 (pHH3) immunostaining in the crypts of the upper small intestines from control and caRANK vil -Tg mice (right in f , g ). 3 wks old control mice (n = 3 mice, n = 254 crypts analysed) and caRANK vil -Tg mice (n = 4/395); 4 wks old control (n = 6/379) and caRANK vil -Tg mice (n = 4/351); and 5–8 wks old control (n = 4/556) and caRANK vil -Tg littermates (n = 4/487). Each data point represents the number of pHH3 positive cells per individual crypts ( f ) and average number per mouse ( g ). Scale bars, 20 μm. h , Kaplan–Meier survival curve of control ( n = 31) and caRANK vil -Tg ( n = 37) littermates. i , Numbers of OLFM4 + cells/ crypt in the indicated mice. Each data point represents the average length, volume, and surface area of villi per mouse in the indicated mice described in Fig. . j , Small intestinal villi length, volume and surface areas the indicated mice. Each data point represents the averaged length, volume, and surface area of villi per mouse. The original data is the same as Fig. . k,l , Numbers of villi (left) and crypts (right) per 2 mm were quantified in histological H&E sections of the upper small intestine from control (n = 4 mice, n = 48 regions) and caRANK vil -Tg (n = 4/46) mice at 3 and control (n = 5 mice, n = 63 regions) and caRANK vil -Tg (n = 5/56) mice at 3 and 5–8 wks of age. Each data point represents the number of Numbers of villi (left) and crypts (right) per 2 mm of the intestine ( k ) and the average number per mouse ( l ). m , Representative images of mouse jejunal organoids from 2 weeks old control caRANK vil -Tg mice, cultured in E low NR (50 pg/ml EGF) or ENR (50 ng/ml EGF) media for 3 days after the first passage. Scale bars, 100 μm. n , Representative images (left) of EdU + proliferating cells in control small intestinal organoids (n = 7) and caRANK vil -Tg mice-derived organoids (n = 6). Scale bars, 50 μm. Right; ratios of EdU labelled proliferating cells to all cells presents in each bud. The analysed bud number is n = 27 (control) and n = 24 ( caRANK vil -Tg ). o , Ratios of organoid numbers after prolonged culture of control- and caRANK vil -Tg mice-derived jejunal organoids. Numbers of organoids were counted at each passage. The ratio of organoid numbers in the caRANK vil -Tg mice-derived jejunal organoids was normalized to untreated control jejunal organoids. Data were combined from two independent experiments. n = 6 (control), n = 6 ( caRANK vil -Tg ). p , Representative images of control and caRANK vil -Tg mice-derived small intestinal organoids cultured with/without recombinant human RANKL (rhRANKL; 100, 500 ng/ml) for 4 days. Scale bars, 100 μm. q , Percentages of surviving control and caRANK vil -Tg jejunal organoids after irradiation, as compared to non-irradiated organoids of the same genotypes. Organoids were cultured in ENR medium and irradiated with 4 Gy, followed by subsequent culture for 7 days. Data are from two independent experiments. n = 5 (control), n = 5 ( caRANK vil -Tg ). AVG, average. Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant. Two-tailed Mann–Whitney U -test ( c,e,g,i,j,l,o,q); Kaplan–Meier survival curve with a log-rank test ( h ); Two-tailed Student’s t-test ( d,f,k,n ).

Article Snippet: The medium was replaced every 48–72 h. Once organoids were established, they were further cultured in an expansion medium composed of advanced DMEM/F12 supplemented with penicillin–streptomycin, 10 mM HEPES, GlutaMAX, N2 (Life Technologies), B27 (Life Technologies), 1 mM N -acetylcysteine (Sigma-Aldrich), R-spondin1 (conditioned medium from 293T-HA-RspoI-Fc cells, 10% final volume), 100 ng ml −1 NOGGIN (Peprotech), 10 nM human gastrin I (Sigma-Aldrich), 500 nM A83-01 (Tocris), WNT3A (conditioned medium from WNT-producing L-cell line, 50% final volume), 50 ng ml −1 mouse recombinant epidermal growth factor (EGF; Peprotech), 100 ng ml −1 human insulin-like growth factor-1 (IGF-1; BioLegend), and 50 ng ml −1 human recombinant fibroblast growth factor-basic (FGF-2; Peprotech) .

Techniques: Transgenic Assay, Mutagenesis, Expressing, Fluorescence, Control, Immunostaining, Cell Culture, Derivative Assay, Recombinant, Irradiation, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a,b , Representative immunostaining to detect RANKL expressing cells (arrows) in small intestinal cross-sections (Left in a ). Sections were also stained for EpCAM to detect intestinal epithelial cells and PDGFRα to visualize subepithelial cells in villi. Scale bars, 25 μm. Quantification of RANKL positive cells per villus and 200 μm of crypt area from nulliparous Rankl WT (n = 5 mice, n = 47 villi, n = 26 regions analysed) and Rankl ΔTwist2 (n = 3/9/23) mice and lactating (L5) Rankl WT (n = 4/15/20) and Rankl ΔTwist2 (n = 3/11/12) mice (right in a , b ). Each data point represents the measurements of numbers of RANKL + cells per villi or 2 mm of crypt area ( a ) and the average number per mouse ( b ). c , Quantitative RT–PCR analyses to compare expression levels of Rankl in mesenchymal cultures derived from lamina propria. Data represent the relative expression of Rankl in recombinant mouse Prolactin (rmProlactin)-stimulated mesenchymal cells to control (no rmProlactin) mesenchymal cells (set at 1). rmProlactin stimulation was for 48 h. n = 3 (control), n = 3 (rmProlactin). d , Left panels, 3D reconstruction of small intestinal tissue from Twist2 -Cre-tdTomato mice using confocal microscopy. Scale bars, 300 μm. Right panels, gating strategy to determine tdTomato-expression in mesenchymal cells from Twist2 -Cre-tdTomato mice using FACS. e,f , Representative 3D reconstructions of small intestine from age-matched nulliparous Rankl WT and Rankl ΔTwist2 and lactating (L5) Rankl WT and Rankl ΔTwist2 dams. Grid spacing is 200 μm (upper in e ). Villous length, volume and surface areas in nulliparous Rankl WT (n = 4 mice, n = 49 villi analysed); nulliparous Rankl ΔTwist2 (n = 4/63); L5 Rankl WT (n = 3/53); Rankl ΔTwist2 (n = 5/82) mice(lower in e , f ). Each data point represents the measurements of length, volume and surface area of individual villi ( e ) and the average value per mouse. g,h , Lengths, volumes and surface areas of villi were measured in age matched L5 Rankl WT (n = 3 mice, n = 53 villi analysed), L5 Rankl ΔTwist2 - Het (n = 4/50) and L5 Rankl ΔTwist2 (n = 5/82) littermates, using confocal microscopy and 3D tissue reconstructions. Each data point represents the measurements of length, volume and surface area of individual villi ( g ) and the average value per mouse( h ). i,j , Small intestinal images from age-matched nulliparous and lactating (L5) Rankl WT and Rankl ΔCd4 littermates. Left: Representative H&E stained intestinal sections. Scale bars, 100 μm (left in i ). Quantification of villus length in nulliparous Rankl WT (n = 5 mice, n = 89 villi analysed) and Rankl ΔCd4 (n = 5/135) mice and L5 Rankl WT (n = 3/106) and Rankl ΔCd4 (n = 7/403) mice (right in i , j ). Each data point represents the measurements of length of individual villi ( i ) and the average value per mouse( j ). k,l , Quantification of villus length in age-matched L5 Rankl WT (n = 3 mice, n = 149 villi analysed) and Rankl ΔRorc (n = 2/148) mice, quantified from H&E cross sections. Each data point represents the measurements of length, volume and surface area of individual villi ( k ) and the average value per mouse( l ). Data are mean ± s.e.m. *P < 0.05; ns, not significant. One-way analysis of variance (ANOVA) with Tukey’s post hoc test ( a,e,g,i ); Two-tailed Paired t-test ( c ); Two-tailed Mann–Whitney U -test ( b,f,h,j ); Two-tailed Student’s t-test ( k ).

Article Snippet: The medium was replaced every 48–72 h. Once organoids were established, they were further cultured in an expansion medium composed of advanced DMEM/F12 supplemented with penicillin–streptomycin, 10 mM HEPES, GlutaMAX, N2 (Life Technologies), B27 (Life Technologies), 1 mM N -acetylcysteine (Sigma-Aldrich), R-spondin1 (conditioned medium from 293T-HA-RspoI-Fc cells, 10% final volume), 100 ng ml −1 NOGGIN (Peprotech), 10 nM human gastrin I (Sigma-Aldrich), 500 nM A83-01 (Tocris), WNT3A (conditioned medium from WNT-producing L-cell line, 50% final volume), 50 ng ml −1 mouse recombinant epidermal growth factor (EGF; Peprotech), 100 ng ml −1 human insulin-like growth factor-1 (IGF-1; BioLegend), and 50 ng ml −1 human recombinant fibroblast growth factor-basic (FGF-2; Peprotech) .

Techniques: Immunostaining, Expressing, Staining, Quantitative RT-PCR, Derivative Assay, Recombinant, Control, Confocal Microscopy, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Uniform manifold approximation and projection (UMAP) of 2,265 human intestinal epithelial cells. Data were taken from Fujii et al. . 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 .

Article Snippet: The medium was replaced every 48–72 h. Once organoids were established, they were further cultured in an expansion medium composed of advanced DMEM/F12 supplemented with penicillin–streptomycin, 10 mM HEPES, GlutaMAX, N2 (Life Technologies), B27 (Life Technologies), 1 mM N -acetylcysteine (Sigma-Aldrich), R-spondin1 (conditioned medium from 293T-HA-RspoI-Fc cells, 10% final volume), 100 ng ml −1 NOGGIN (Peprotech), 10 nM human gastrin I (Sigma-Aldrich), 500 nM A83-01 (Tocris), WNT3A (conditioned medium from WNT-producing L-cell line, 50% final volume), 50 ng ml −1 mouse recombinant epidermal growth factor (EGF; Peprotech), 100 ng ml −1 human insulin-like growth factor-1 (IGF-1; BioLegend), and 50 ng ml −1 human recombinant fibroblast growth factor-basic (FGF-2; Peprotech) .

Techniques: Expressing, Quantitative RT-PCR, Control, Cell Culture, Recombinant, MTT Assay, Immunostaining, Solvent, Staining, Two Tailed Test

Journal: Nature

Article Title: RANK drives structured intestinal epithelial expansion during pregnancy

doi: 10.1038/s41586-024-08284-1

Figure Lengend Snippet: a , Human duodenal organoids were cultured without (control) and with recombinant human RANKL (rhRANKL; 500 ng ml −1 ) for 2 days. Left, representative images. Scale bars, 100 μm. Right, quantification of organoid sizes. n = 83 (control) and n = 83 (rhRANKL). Data were combined from three independent experiments. b , Organoids were cultured with or without rhRANKL (500 ng ml −1 ) for 2 days and then irradiated and cultured for additional 7 days. Data show the number of surviving organoids combined from three independent experiments. n = 12 (control) and n = 12 (rhRANKL). c , Cell cycle analyses of RANK-stimulated human duodenal organoids. Left, representative fluorescence-activated cell sorting (FACS) plots of the EdU cell cycle analysis assessed at 24 h and 72 h of rhRANKL stimulation. Right, quantification of S phase and subG1 + G1 entry. Data were combined from two independent experiments. n = 12 for each condition. d , The ratio of the numbers of wild-type (WT) and BMPR1A -knockout ( BMPR1A -KO) human small intestinal organoids in the absence or presence of rhRANKL (500 ng ml −1 ). The ratio of organoid numbers in the WT + RANKL group was normalized to untreated WT organoids; and the ratio of organoid numbers in the BMPR1A- KO + RANKL organoids was normalized to the untreated BMPR1A- KO organoids. n = 3 for each condition. e , Cell cycle analyses of RANK-stimulated duodenal organoids in the absence (control) and presence of the BMPi LDN193189 (1.6 μM). Left, representative FACS plots depicting EdU cell cycle analysis after 72 h rhRANKL stimulation with or without BMPi. Right, quantification of S phase and sbG1 + G1 entry. n = 11 (control), n = 12 (control + RANKL), n = 12 (BMPi) and n = 12 (BMPi + RANKL). Data were combined from two independent experiments. f , Representative images of OLFM4 + stem cells in WT and BMPR1A -KO human small intestinal organoids with or without rhRANKL (500 ng ml −1 ). Scale bars, 50 μm. Dots represent individual datapoints. Data are mean ± s.e.m. Statistical analysis was performed using two-tailed Student’s t -tests ( a – c ) and one-way ANOVA with Tukey’s post hoc test ( d and e ). Further details on statistics and reproducibility are provided in the .

Article Snippet: The medium was replaced every 48–72 h. Once organoids were established, they were further cultured in an expansion medium composed of advanced DMEM/F12 supplemented with penicillin–streptomycin, 10 mM HEPES, GlutaMAX, N2 (Life Technologies), B27 (Life Technologies), 1 mM N -acetylcysteine (Sigma-Aldrich), R-spondin1 (conditioned medium from 293T-HA-RspoI-Fc cells, 10% final volume), 100 ng ml −1 NOGGIN (Peprotech), 10 nM human gastrin I (Sigma-Aldrich), 500 nM A83-01 (Tocris), WNT3A (conditioned medium from WNT-producing L-cell line, 50% final volume), 50 ng ml −1 mouse recombinant epidermal growth factor (EGF; Peprotech), 100 ng ml −1 human insulin-like growth factor-1 (IGF-1; BioLegend), and 50 ng ml −1 human recombinant fibroblast growth factor-basic (FGF-2; Peprotech) .

Techniques: Cell Culture, Control, Recombinant, Irradiation, Fluorescence, FACS, Cell Cycle Assay, Knock-Out, Two Tailed Test