Figure 6.

IL-22 treatment recapitulates the emergence of IA cellular states in human colonic organoids

(A) UMAP plots of control and IL-22-treated organoid datasets colored by condition (left) and cell type annotation (right).

(B and C) UMAP plots showing enrichment for STAT3 transcriptional regulon (C) and the shared IA signature (C) in control and IL-22-treated organoids.

(D) UMAP plots with a heatmap of normalized expression REG1A in control and IL-22-treated organoids.

(E and F) Violin plots showing normalized enrichment score of the indicated signatures and expression levels of the indicated lineage-specific genes (CA2 and ZG16) in the cell type clusters identified in organoids treated with IL-22.

(G) Representative flow cytometry analysis of REG1A-mNeon human organoid reporter line (clone#3) following IL-22 treatment for 3 days. Simplified schematic of the reporter is shown above.

(H) Quantification of REG1A-expressing cells in human colonic organoids treated with IL-22. Bar plots showing percentage of mNeon-positive cells (mean ± SD) in the REG1A-mNeon reporter line (clone#3; left) and percentage of high REG1A cells (normalized expression in the third quantile) in the scRNA-seq dataset (right). Each dot represents an independent experiment. ^∗∗ by paired Student’s t test.

(I) Representative images of REG1A-mNeon reporter line (clone#3) treated with IL-22 and/or tofacitinib for 3 days. Images show merged phase contrast and GFP channels. The secreted REG1A-mNeon fusion protein accumulates in the lumen of the organoids. Scale bars: 200 μm.

(J) RT-qPCR analysis showing relative expression levels of the indicated genes in the fluorescence-activated cell sorting (FACS)-purified cell populations from the REG1A-mNeon reporter line (clone#3). Fold change relative to mNeon− cells ±SEM. Each dot represents an independent experiment. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; two-way ANOVA with Tukey’s multiple comparison test.

(K) Organoid-forming assay of purified mNeon-negative and mNeon-positive cells from IL-22-treated organoids (clone#3). mNeon-negative cells from untreated organoids were used as control. Left shows representative images of organoids formed from each purified cell fraction. Scale bar: 300 μm. Right shows quantification of formation efficiency and organoid size, as described in (2I). Efficiency plot shows mean ± SEM. Each dot represents an independent experiment. Size is shown as Tukey plot in log2 scale. The number of organoids analyzed within one representative experiment is shown below the bars. ^∗∗ for p < 0.01 by one-way ANOVA.