Fig 2: Identification of dysplastic stem cells in Meta4.a–e Clonal sphere forming efficiency from Meta4 subpopulations. a Meta4 organoids were dissociated to single cells, stained for CD44, CD133, CD166, and FACS isolated. Following doublet-discrimination and dead cell exclusion, CD133/CD166 positive cells (gray gate) were applied (red arrow) to a CD44 positive gate to distinguish between double-positive CD44neg/CD133+/CD166+ (orange gate) and triple-positive CD44+/CD133+/CD166+cells (green gate). Double-positive and Triple-positive cells were collected by FACS and applied to CRAs. b Immediately after plating, single cells were quantified and their physical address on the CRA was determined. c, d After 8 days of culture, spheres were quantified and their physical address on the CRA was determined. Upper-panels are low magnification of each CRA containing 2025 wells. Red-boxes indicate the regions in the higher magnification images depicted in the lower panels. e Spheres-Forming Efficiency (SFE) of clonal events was determined by quantifying the number of spheres that developed from all single cell events at t = 0. There were 619 Double-positive clonal events identified in the CRAs, and 28.9% of those generated spheres (orange chart). There were 509 Triple-positive clonal events identified in the CRAs, and 6.3% of those generated spheres (green chart). The data represent binary outcomes. f Quantitation of immunofluorescence staining of Ki67-positive cells in either Double-positive (DP) or Triple-positive (TP) cell zone in Mist1-Kras mouse stomach corpus at 4 months after tamoxifen injection. Data are presented as mean values with standard deviation (n = 12). P-values were calculated using paired two-tailed t-test. g Immunostaining for CSC markers, CD44 (basolateral membrane), CD133 (apical membrane) and CD166 (basolateral membrane), metaplasia and gastric cancer markers, Sox9 and CD44v9, and a proliferation marker, Ki67 in Mist1-Kras mice at 4 months after tamoxifen injection. Yellow arrow indicates Double-positive (DP) cells and white arrow indicates Triple-positive (TP) cells. Dotted boxes indicate enlarged area. Scale bars indicate 100 µm. Source data are provided as a Source Data file.

Fig 3: Lineage tracing and scRNA-seq by Gif+ chief cells identify injury-responsive chief cells(A) Diagram of the Gif-Cre-nTnG allele used in this study.(B) Experimental scheme of the L635 treatment study in Gif-Cre-nTnG mice. Dox was administered for 1 week and off for 2 weeks to label chief cells with GFP. The mice were then left untreated or treated with 3 doses of L635.(C and D) Paraffin sections of Gif-Cre-nTnG mouse stomachs untreated or treated with 3 doses of L635 were immunolabeled with antibodies against (C) GFP (green), Mist1 (chief cells, red), GSII-lectin (neck cells, blue), and P120 (epithelial cell membrane, white) or (D) GFP (green), p57 (red), and Ki67 (proliferating cells, white). GFP+Mist1+ cells indicate chief cells and GFP+GSII+ cells indicate injury-responsive chief cells. Arrowheads indicate GFP+p57+ double-positive cells (yellow) and GFP+Ki67+p57-proliferating injury-responsive chief cells (red), respectively. Nuclei were counterstained with hoechst (blue) in (D). Yellow boxes indicate enlarged areas. Orange-dotted boxes indicate enlarged areas shown in Figure S3C. Scale bars, 100 µm.(E) UMAP plots of scRNA-seq from Gif lineage cells (GFP+TdTom-) of Gif-Cre-nTnG mice in both conditions (Total, left), uninjured (Control, middle), and 2 days after DMP-777-induced corpus injury (Injury, right). Gifhigh and Giflow populations are marked by brown-dotted lines.(F) UMAP plots for control (light blue) and injury (red) cells.(G) UMAP plots of Gif expression in control (left) and injury (right).(H) Dot plot for expression of marker genes of each cell type in Gifhigh and Giflow cell populations.(I) Dot plot for expression of marker genes of proliferation or cell cycle in Gifhigh and Giflow cell populations.(J) Projection of Gifhigh and Giflow cells on the UMAP plot for Pgc+ scRNA-seq. The cells in control and injury are denoted as light blue and red, respectively. Brown-dotted circles indicate the injury-responsive chief cell population.(K) UMAP plots of p57 and Ki67 expressions in control (left) and injury (right).See also Figure S3.

Fig 4: Defective regeneration of colonic epithelium in Irf2?IEC mice. (A) H&E (left) and PAS staining (middle) of distal colon tissue from Irf2fl/fl and Irf2?IEC mice (n = 3). Proliferating cells were detected by Ki67 staining (right). Scale bars: 100 µm. (B,C) Graphs show changes in body weight (BW) and mortality over time between Irf2fl/fl and Irf2?IEC mice that were given 2% DSS (w/v) in drinking water for 7 days followed by 11 days of plain water (n = 8 per group). (D) Low-magnification and (E) high-magnification images show histologic analysis of the distal colon in Irf2fl/fl and Irf2?IEC mice treated with 2% DSS in drinking water for 5 days followed by 6 days of plain water (n = 3). Regenerating crypts were detected by Ki67 staining. Scale bars: (D) 200 µm and (E) 100 µm. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t test.

Fig 5: Additional NRF2 activation in Keap1-cKO::Nrf2SAmice promotes elimination of NRF2-activated cells. (A) Immunohistochemical staining for NQO1. NQO1-positive cells were distributed segmentally in the esophageal epithelium of both Keap1-cKO::Nrf2SA mice and Keap1-cKO mice. Scale bars: 400 µm. (B) Quantification of esophageal epithelial length. The epithelial length was greatest in Keap1-cKO::Nrf2SA mice (n = 9–12 mice per group). (C) Quantification of NQO1-positive cells in the esophageal epithelium. The number of NQO1-positive cells decreased earlier in Keap1-cKO::Nrf2SA mouse esophagus than in Keap1-cKO mouse esophagus (n = 8–10 mice per group). (D and E) Immunohistochemical staining for Ki67. A greater increase in number of Ki67-positive cells was observed in the Keap1-cKO::Nrf2SA mouse esophagus than in the Keap1-cKO mouse esophagus. Scale bars: 50 µm. (F and G) Immunohistochemical staining for ?H2A.X. Greater increase in number of ?H2A.X-positive cells was observed in Keap1-cKO::Nrf2SA mouse esophagus than in Keap1-cKO mouse esophagus. Scale bars: 50 µm. Data are presented as means ± standard deviations. *P < .05, **P < .01, and ***P < .001 according to Welch t test.