Fig 2: Loss of Sox2 rescues forestomach/esophageal transformation induced by Cdx2 deletion.(A) Whole-mount images and subsequent histological analyses using H&E staining of control and Sox2;Cdx2 doubly deleted guts at E18.5 (n ≥ 3 each). Cre-LoxP recombination was induced at E8.5. Scale bars, 100 μm. (B) Immunofluorescence images show CDX2, SOX2, TP63, PCNA, and SOX9 staining in control, Sox2 KO, Cdx2 KO, and Sox2;Cdx2 DKO embryos. Scale bars, 100 μm. (C) Comparison of E16.5 common-, forestomach-, and small intestine–enriched ATAC-seq peaks in publicly available Cdx2 KO ATAC-seq data. Ectopic forestomach-specific peaks in Cdx2 KO embryos are associated with SOX2 binding (SOX2 ChIP-seq). Cdx2KO ATAC-seq was performed in E16 intestine tissue by Banerjee et al. and retrieved from GSM3181688. SOX2 ChIP-Seq was performed in E16.5 forestomach epithelium.

Fig 3: DiOs are phenotypically similar to primary intestinal organoids (IOs)(A) Bright-field images comparing dissection iPSC-derived intestinal organoids to patient rectal biopsy-derived organoids under matched culture conditions in 1:1 OGM/LWRN medium.Culture day refers to time elapsed after splitting prior cell material. All images were cropped using Nikon software after imaging with a four objective.(B) Single cell RNA sequencing data of DiO and primary IO cells using genotype-matched sources homozygous for CFTR G542X, R1162X, and W1282X variants, cultured for 7 days in 1:1 OGM/LWRN medium to maintain stem/progenitor populations.Lineage scores categorized by intestinal cell type (Intestinal stem cell: ISC, transit amplifying: TA, secretory progenitor: SecProgenitor, goblet, Enteroendocrine: EEC).(C) Dot plots of the top genes upregulated and down regulated by cluster in both DiO and primary IO populations for day 7 of culture.(D) Feature plots of both DiO and primary IO cells for the genes LGR5, OLFM4, CFTR, CDX2, and MUC2, in both DiO and primary IO populations for day 7 of culture.(E) Confocal images showing immunofluorescence detection of known intestinal stem/progenitor proteins identified through single cell RNA sequencing. Both DiO and primary IO sources were cultured for two days post passaging prior to FFPE block preparation, sectioning, and antibody staining. Green (Alexa FluorTM 488 nm) indicates either LGR5 or MUC2, red marks OLFM4 (Alexa FluorTM 594 nm), blue indicates CDX2 (Alexa FluorTM 647 nm). All images were collected using a 20x objective, then converted into maximum intensity projections from Z-stacks.