Fig 2: CD73+cells mediate epithelial renewal in human esophageal epithelium. (A–D) PDOs or EPC2-hTERT organoids were harvested on day 11 (1° organoids) and dissociated for FACS. The cells were sorted based on CD73 and CD104 expression and resuspended in Matrigel for organoid establishment and growth. OFR and organoid size (mean area) of 2° organoids were (B and C) quantified on a Celigo image cytometer and (D) harvested on day 11 and again dissociated for flow cytometry. (A) Representative phase-contrast images of organoids formed by corresponding cell populations. (B) OFR and (C) mean size were compared among organoids from the 3 populations. (D) The scheme of this experiment and the representative flow cytometry plots of 2° organoids are shown. The percentage of CD73+ cells in EPC2-hTERT were compared. The data are shown as means ± SD (n = 3). (E–H) EPC2-hTERT cells were treated for 48 hours with CD73 siRNA or nonsilencing (scramble) siRNA in 2D culture, followed by seeding into organoid cultures. (E) CD73 silencing efficiency was evaluated by flow cytometry. (F) Representative phase-contrast images of organoids formed by corresponding cells. (G) OFR and (H) mean size of organoids from EPC2-hTERT cells transfected with CD73 or scramble siRNA. The data are shown as means ± SD (n = 10). Two-tailed Student t test or analysis of variance were performed for single or multiple comparisons, respectively. *P < .05, **P < .01, ***P < .001, and ****P < .0001.

Fig 3: CD73+CD104+cells represent a less-differentiated and slow-cycling cell population within the basal zone. (A and B) EPC2-hTERT organoids were harvested on day 11 and dissociated for FACS. (A) The cells in EPC2-hTERT organoids were sorted based on CD73 and CD104 expression. (B) CD73, CD104, IVL, and FLG relative gene expression in CD73+CD104+ and CD73+CD104- populations was validated by qRT-PCR. (C) EPC2-hTERT cells in 2-dimensional culture (organoid day 0) and EPC2-hTERT organoid (day 7 and day 11) were harvested for flow cytometry and the representative flow cytometry plots for CD73 and CD104 expression are shown. (D) Representative IHC images for CD73 staining in EPC2-hTERT organoids harvested on day 5, day 7, day 9, and day 11. Scale bars: 100 µm. (E–G) EPC2-hTERT organoids were harvested on day 11 after incubation in culture medium with EdU for 2 hours, and dissociated for flow cytometry. (E) A representative FACS dot plot of cells from EPC2-hTERT organoids subjected to EdU, with the frequencies of each population according to CD73 and CD104 expression, is shown. (F) Representative histograms of EdU staining in each cell population. The percentage of EdU+ cells in each population is shown. (G) Percentages of EdU+ cells in each population. All data are shown as means ± SD (n = 3). A 2-tailed Student t test or analysis of variance were performed for comparing 2 or multiple comparisons, respectively. *P < .05, **P < .01, ***P < .001, and ****P < .0001.

Fig 4: The therapeutic potential of STAT6 inhibition in EoE. (A) IHC staining for phosphorylated STAT6 was performed, with representative images shown. Scale bars: 100 µm. (B) The positive rate of phosphorylated STAT6 in esophageal epithelium are compared. The data are shown as means ± SD (n = 5). (C) Relative expression of CD73 gene in EPC2-hTERT organoids treated with STAT6 inhibitor (AS151749, 400 nmol/L), IL4 (10 ng/mL), or IL13 (10 ng/mL). (D) The frequencies of CD73+CD104+ cells in organoids treated with AS151749 or dimethyl sulfoxide (DMSO) under the presence of Th2 cytokines. STAT6 silencing efficiency was evaluated by (E) qRT-PCR and (F) Western blot. (G) CD73 gene expression in EPC2-hTERT organoids treated with STAT6 siRNA or nonsilencing (scramble) siRNA with or without IL13. The data are shown as means ± SD (n = 3). Analysis of variance was performed with multiple comparisons. **P < .01, ****P < .0001.

Fig 5: Single-cell RNA sequencing analysis of PDOs from 4 non-EoE patients. PDOs from 4 non-EoE patients were treated with IL13 (10 ng/mL) or vehicle (phosphate-buffered saline) from day 7 to day 11, and harvested and dissociated for single-cell RNA sequencing analysis on day 11. (A) Seurat’s Uniform Manifold Approximation and Projection (UMAP) visualization of 4 classified cell groups, basal-1, basal-2, suprabasal, and superficial, within PDOs from non-EoE controls grown with or without IL13 (10 ng/mL). (B) Average normalized gene expression values of MKI67, CD73, basal markers (TP63, NGFR), and differentiated markers (IVL, KRT13, FLG) in each cell cluster. Circle size reflects the percentage of cells with a nonzero expression level for the indicated genes. Color intensity reflects the average expression level across all cells within each cluster. (C) Expression z-scores for the top 8 up-regulated genes in each cluster. (D) The proportions of each cell cluster were compared in PDOs (n = 4) with IL13 or vehicle. (E–G) The normalized expression of (E) IVL and FLG, (F) SOX2, and (G) CD73 in nontreated (NT) PDOs was compared with that in PDOs treated with IL13 for each cluster. ****P < .0001, Wilcoxon signed-rank test. (H) GO analysis of differentially expressed gene profiles of CD73+CD104+ cells, compared with CD73-CD104+ cells in NT PDOs.