Fig 1: Generation and characterization of human M cell organoids.(a,b) Representative bright-filed images of cultured M cell organoids versus normal intestinal organoids (a). The number of buds per organoid is quantified in (b). n = 20 organoids for each group. Each dot represents one organoid. Three independent experiments are performed on two donors with similar results. Data are presented as mean values +/− SEM. P-values are derived from two-tailed t-test. Scale bars, 100 µm. (c,d) Representative confocal images showing the EdU+ proliferating cells in cultured M cell organoids versus normal intestinal organoids (c). The number of EdU+ cells per organoid is quantified in (d). n = 20 organoids for each group. Each dot represents one organoid. Three independent experiments are performed on two donors with similar results. Data are presented as mean values +/− SEM. P-values are derived from two-tailed t-test. Scale bars, 100 µm. (e,f) Representative flow cytometry analysis (e) and quantification of DAPI− live cells or GP2+ M cells (f) in cultured M cell organoids (Ctrl) or adding LTB during M cell differentiation. n = 3 independent wells on one donor. Each dot represents one well. Data are presented as mean values +/− SEM. P-values are derived from two-tailed t-test. (g) Illustration of the knock-in reporter organoids containing a P2A-tdTomato cassette inserted at the C-terminus, before the stop codon, of the SPIB gene. (h) Representative confocal image of SPIB-P2A-tdTomato reporter organoids cultured in M cell medium. M cells are marked by tdTomato fluorescence (red). Three independent experiments are performed on one donor with similar results. Scale bar, 50 µm. (i) qPCR analysis of the expression levels of a set of M cell markers in FACS-sorted SPIB− and SPIB+GP2+ cells. n = 2 technical replicates are shown. Data are presented as mean values. Results are representative of two independent experiments on one donor. (j) Quality control of scRNA-seq dataset derived from human M cell organoids. Numbers of features (top) and counts (bottom) per cell are shown across different cell types. n = 375 single cells. (k) Heatmap of cell type-enriched genes in scRNA-seq dataset of human M cell organoids. Each column represents a single cell and each row represents one marker gene. The colors, ranging from purple to yellow, indicate low to high relative gene expression levels. (l) scRNA-seq analysis of primary human intestinal cells. Cell clusters are visualized in UMAP plots and colored by different cell types. n = 15,543 single cells. Annotations of M cells based on a previous study (left) and in this study (right) are shown, respectively. (m) Dot plot showing the expression levels of a set of cell type-specific markers across primary intestinal cell types. n = 15,543 single cells. Dot color relates to normalized mean expression values and dot size to fraction of expressing cells. (n) UMAP plot showing the primary M cells colored by region code. Annotations of M cells are based on a previous study. n = 301 single cells. (o) IHC staining of TFF2 antibody on human duodenum tissue sections. Positive staining signals are detected in Brunner’s gland cells. Images are derived from the Human Protein Atlas (proteinatlas.org). Similar results are observed in tissues from two donors. Scale bars, 100 µm. (p) Representative transmission electron microscopy images of M cell organoids. M cells are identified by having fewer apical microvilli compared to the neighboring enterocytes. M cell organoids from two donors are tested with similar results (see also Fig. 1h). Scale bars, 5 µm in low-magnification image and 2 µm in high-magnification images. (q) Schematic of air-liquid interface (ALI) system for M cell differentiation in monolayer culture. Images were created in BioRender. van Es, J. (2025) https://BioRender.com/xh7haut. (r) Representative images showing the ALI monolayer culture containing GP2+ M cells. Three independent experiments are performed on two donors with similar results. Scale bar, 50 µm. (s) Representative flow cytometry analysis of GP2+ M cells in ALI monolayer culture. Three independent experiments are performed on two donors with similar results. (t) Representative confocal images showing the GP2+ M cells with fewer apical microvilli. M cells are identified by IF staining of GP2 antibody (green). Microvilli structures are detected by staining of F-actin using Phalloidin (red/white). Two GP2− cells also exhibit fewer microvilli (arrows). Three independent experiments are performed on two donors with similar results. Scale bar, 20 µm. Source Data