Fig 1: 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.