The glandular stomach is composed of two regenerative compartments termed corpus and antrum, and our understanding of the transcriptional networks that maintain these tissues is incomplete. Here we show that cell types with equivalent functional roles in the corpus and antrum share similar transcriptional states including the poorly characterized stem cells of the isthmus region. To further study the isthmus, we developed a monolayer two-dimensional (2D) culture system that is continually maintained by Wnt-responsive isthmus-like cells capable of differentiating into several gastric cell types. Importantly, 2D cultures can be converted into conventional three-dimensional organoids, modelling the plasticity of gastric epithelial cells in vivo. Finally, we utilized the 2D culture system to show that Sox2 is both necessary and sufficient to generate enterochromaffin cells. Together, our data provide important insights into gastric homeostasis, establish a tractable culture system to capture isthmus cells and uncover a role for Sox2 in enterochromaffin cells.
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Bulk and single-cell RNA sequencing data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE217473. Previously published single-cell RNA sequencing data of the primary mouse intestine that were re-analysed for this study are available under accession code GSE92332. Previously published bulk RNA sequencing data of mouse intestinal EC cells that were re-analysed for this study are available under accession code GSE98794. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.
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We thank all members of the Hochedlinger lab for feedback and critical reading of the manuscript, and A. Coffey and N. Bourdon for help with animal husbandry. We are further grateful to the Single Cell Core at Harvard Medical School for performing sample preparation and to M. Hadley and the rest of the HSCI flow cytometry core for assistance. We are grateful to S. Wang and C. C. Morton at the Brigham and Women’s Hospital cytogenetics laboratory for karyotyping. This work was supported by the NIH (5R01DK096034), MGH and the Gerald and Darlene Jordan Chair in Regenerative Medicine.
The authors declare no competing interests.
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Extended Data Fig. 1 Molecular comparison of epithelial cell types in gastrointestinal tissues.
(a) Known markers of facultative and/or constitutive stem cells in the glandular stomach. (b) Joint UMAP embedding of corpus and antrum tuft and EE cells and classification of cell types based on Conos clustering and intracluster gene expression. (c) Multidimensional scaling (MDS) plot comparing the aggregate expression profiles of each of the defined cell types in the corpus and antrum. (d) Normalized heatmaps showing the top 10 DEGs expressed in pit, gland base, EE, and tuft cells isolated from corpus or antrum using 100 randomly selected cells from the pool. Genes highlighted in red or blue indicate genes that are differentially expressed in multiple cell types (red in corpus, blue in antrum). (e) Normalized heatmap showing selected genes that are differentially expressed in the tissue/cell types present in the 6,261 corpus cells sequenced and the 4,844 antrum cells sequenced. (f) Joint UMAP embedding of primary corpus and antrum epithelial cells (this study) with previously published primary intestinal epithelial cells34. Middle panel indicates intestinal clusters identified in relation to embedded gastric epithelial cells. Bottom panel highlights corpus/antrum clusters (this study) in relation to embedded intestinal epithelial cells. The cells from the complementary dataset are shown in black. (g) Normalized heatmap showing the top 10 shared or most differentially expressed genes between intestinal and stomach EE, Paneth/Chief, and tuft cells using 100 randomly selected cells from the respective pools of cells.
Extended Data Fig. 2 Identification of gastric stem cells based on gene expression/cell cycle status.
(a) UMAP embedding of primary corpus cells showing overlap of Stmn1 and Mki67 expression. (b) UMAP embedding of antrum epithelial cells showing regional enrichment of Sox2 and Lgr5. (c) RNA expression (CPM) of tuft markers relative to Lgr5 and Epcam expression in the primary corpus, n = 2 biologically independent samples. (d) Cell cycle analysis on primary antrum and corpus epithelia using transcripts associated with S and G2M and scored based on their expression levels in each cell.
Extended Data Fig. 3 Wnt signaling, TGFβ inhibition, and feeders are required to maintain 2D culture.
(a) Representative phase contrast image of p5 corpus and antrum 2D culture using recombinant Wnt3a and Rspo1 in lieu of conditioned media, scale bars = 50 μm. (b) Representative immunofluorescence images of corpus and antrum 2D culture stained with Epcam following the iterative removal of Wnt signaling from the culture, scale bars = 50 μm. (c) Quantification of corpus 2D culture following iterative removal of Wnt agonists from the culture, staining for Epcam, and measuring colony size. 5 total fields quantified for each replicate, n = 3 biologically independent experiments. *** = p > 0.001 using students 2-sided t-test. (d) 2D culture in the presence or absence of recombinant Jagged1 and HGF were stained for Epcam and the number of Epcampos cells, as well as colony size per field, were quantified. 8 total fields quantified, n = 3 biologically independent experiments. *** = p > 0.001 using students 2-sided t-test. (e) A8301 was included or removed from 2D media conditions and proliferation was measured in epithelial cells by Epcam and Mki67 immunofluorescence staining. Representative colony from each condition is shown. (f) 2D culture was moved onto various extracellular matrices and the ability of the culture to expand was monitored. Representative phase contrast images are shown, scale bars, 50 μm. (g) 2D complete media preconditioned on feeders for 72 hours was used to expand 2D culture without the use of feeders. Representative phase contrast images are shown for each passage, scale bars, 50 μm. (h) 2D culture was serially passaged at a 1:2 ratio in the absence of feeders. Representative phase contrast images are shown for each passage, scale bars, 50 μm.
Extended Data Fig. 4 Characterization of mouse 2D culture.
(a) Entropy score on 2D culture embedded as shown in Fig. 3g. (b) Joint UMAP embedding of combined corpus and antrum 2D cultures with primary corpus and antrum cells showing expression levels of isthmus markers, Mki67 and Birc5, base/neck markers, Gkn3 and Aqp5, and pit markers Tff1 and Klf4. (c) MA plot comparing corpus 2D-cultured cells with antrum 2D-cultured cells using bulk RNA-sequencing counts. Genes in red were identified as being significantly differentially expressed based on a cutoff of FC > 2 and FDR < 0.05 (n = 2, biologically independent samples). Genes highlighted in green have been shown to discriminate corpus from antrum in vivo. (d) Joint UMAP embedding of primary corpus or antrum cells showing expression levels corpus-specific DEG Esrrg and antrum-specific DEG Nkx6-3. (e) Representative expression analysis of Gif in mouse antrum 2D culture by immunofluorescence, scale bar, 100 μm.
Extended Data Fig. 5 Characterization of human 2D culture.
(a) Section of human stomach that was used to isolate and expand glandular stomach cells using 2D media. Yellow box indicates portion of antrum that was taken, and blue box indicates section of corpus that was taken to derive 2D lines. (b) Representative phase contrast image of passage 6 human epithelial cells expanded in 2D culture conditions, scale bars, 100 μm. (c) Representative expression and distribution of GKN1 and MUC5AC protein in 2D culture by immunofluorescence, scale bars, 50 μm. (d) Single cell RNA sequencing on p6 human antrum and corpus 2D culture, embedded using UMAP, and clustered using Conos (Fig. 4c). Normalized heatmap showing top 20 upregulated DEGs using a representative number of cells within each cluster is shown. (e) Joint UMAP embedding of human 2D culture showing expression levels of the base/neck markers LGR5, AQP5, INSC, NFATC4, and SLC9A4.
Extended Data Fig. 6 Comparison of 2D-cultured cells derived from human and mouse(a) Strategy used to polarize human 2D culture.
(b) Representative immunofluorescence staining of the parietal cell marker GIF in human antrum cultured using ALI, scale bar, 25 μm. (c) Correlation plot using the cumulative expression value for each gene that can be compared between species. Cumulative log expression of mouse genes are plotted on the Y-axis and the cumulative log expression of human genes are plotted on the X-axis. R was calculated using a two-sided Pearson correlation test. (d) Single cell expression of Clu in mouse primary gastric epithelial cells embedded using UMAP. (e,f,g) Human and mouse cells cultured in 2D culture conditions were jointly embedded using UMAP and clustered using Conos (Fig. 4k). Normalized heatmap showing the top 10 most shared or most differentially expressed genes between human and mouse for each cluster identified using 100 randomly selected cells from each group.
Extended Data Fig. 7
Lgr5pos cells are not required for the maintenance of 2D culture(a) Representative FACS gating of wildtype 2D and organoid cultures used to quantify Lgr5pos cells in Fig. 5a. (b) Joint t-SNE embedding of isogenic 2D and organoid cells showing expression of the isthmus marker Birc5, the pit marker Tff1, and the base/neck marker Aqp5. (c) Quantification of proliferation by FACS CellTrace dye detection following ablation of Lgr5pos cells in 2D culture (top) (n = 3 independent experiments, representative quantification shown). Representative phase contrast images of Lgr5DTR-GFP 2D cultures + /- DT continually for 10 passages, scale bars, 50 μm (n = 3 independent experiments). (d) Lgr5DTR-GFP organoids were treated with DT for 5 passages or passaged using standard conditions. Number of visible organoids were counted manually and recorded before splitting 1:3. Horizontal lines indicate mean number of organoids for each passage, n = 8 independent experiments. Representative FACS plots and representative images of organoids quantified at p5 are shown below and on the right of the graph, respectively.
Extended Data Fig. 8 Organoid culture and 2D culture systems can be interconverted.
(a) Gene expression of pit and gland base markers in corpus-derived organoids and corpus 2D cells converted to organoids. Expression is shown relative to 2D cultures and normalized to Gapdh (right). Data are presented as mean values + /- SD, n = 3 biologically independent experiments. (b) Gene expression of isthmus and gland base markers in antrum-derived organoids converted to 2D-cultured cells. Expression is shown relative to organoid cultures and normalized to Gapdh (right). Data are presented as mean values + /- SD, n = 3 biologically independent experiments. (c) Gene expression of pit and gland base markers in corpus-derived organoids converted to 2D-cultured cells. Expression is shown relative to organoid cultures and normalized to Gapdh (right). Data are presented as mean values + /- SD, n = 3 biologically independent experiments. (d) Representative phase contrast image showing the expansion of Lgr5neg organoids, n = 8 independent experiments. (e) Size of organoids derived from Lgr5pos and Lgr5neg 2D cells shown in Fig. 6c was measured by calculating the area of each organoid (mean is shown as the horizontal bars, n = 6 independent experiments using 3 biologically-independent samples, n.s. = not significant using students 2-sided t-test).
Extended Data Fig. 9 Relationship between Sox2 and EC cell identity.
(a) Representative expression of endogenous Sox2 protein in the antrum of mouse stomach by immunofluorescence (left). Colocalization of Sox2 and TdTomato in the antrum of Sox2TdTomato reporter mice by immunofluorescence (right). Higher magnification of the isthmus region is shown on the right of the image. Closed arrowheads point to cells with lower levels of endogenous Sox2 staining in the nucleus that is colocalized with cytoplasmic TdTomato signal. Open arrowheads point to cells with high levels of nuclear Sox2 expression and high cytoplasmic TdTomato signal, scale bars, 50 μm, n = 5 animals examined. (b) Colocalization of Sox2 and TdTomato in the corpus of Sox2TdTomato reporter mice by immunofluorescence. Higher magnification of the base and isthmus region are shown on the right. Closed arrowheads point to nuclear Sox2 staining identified in both the isthmus and base of corpus glands, scale bars, 50 μm, n = 5 animals examined. (c) RNA sequencing of primary Sox2neg and Sox2pos cells taken from the corpus. RPKM values of gland base/chief markers Lgr5, Pgc, Gif, and Tnfrsf19 are plotted data are presented as mean values, n = 3 biologically independent samples. (d) Sox2 expression levels in sorted Sox2neg, Sox2pos, and Sox2high cells by RNA sequencing data are presented as mean values, n = 3 biologically independent samples. (e) RNA sequencing of primary Sox2pos and Sox2high cells taken from the antrum. RPKM values of isthmus markers Hmgb2, Mki67, and Set are plotted data are presented as mean values, n = 3 biologically independent samples. (f) Single-cell gene expression of ChgA and/or ChgB in primary antrum EE cells embedded using UMAP embedding. EC cell population is circled. (g) MDS plot comparing Sox2high and Sox2low cells from the glandular stomach to ChgApos and ChgAneg cells in the intestine. (h) Representative image showing colocalization of SOX2 and 5−HT expression in human antrum by immunofluorescence. Higher magnification of outlined area is shown below. Closed arrowheads point to cells that are double positive for both nuclear SOX2 and cytoplasmic 5-HT, scale bars, 100 μm, n = 3 biologically independent experiments. (i) Percentage of 5-HT positive cells relative to total epithelial nuclei present in the field in the stomachs shown in Fig. 7j. Data are presented as mean values + /- SD, n = 3 biologically independent samples for each group, 5 fields quantified per replicate.
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Huebner, A.J., Gorelov, R.A., Deviatiiarov, R. et al. Dissection of gastric homeostasis in vivo facilitates permanent capture of isthmus-like stem cells in vitro. Nat Cell Biol 25, 390–403 (2023). https://doi.org/10.1038/s41556-022-01079-4