Fig 2: Optimization of GI tissue-derived ECM hydrogels for organoid culture as an alternative to Matrigel.a Brightfield images of gastric organoids grown in SEM hydrogels and Matrigel (MAT) at day 5 (scale bar = 200 µm, independent experiments = 3). b Quantification of gastric organoid formation efficiency in SEM hydrogels compared to in MAT (N = 6, independent experiments = 3). c qPCR analysis to compare mRNA expression in gastric organoids grown in each hydrogel (SEM 7 mg ml-1 versus MAT, *p = 0.0174 for Pgc, **p = 0.0051 for Atp4a, ***p < 0.0001 for Atp4b; SEM 5 mg ml-1 versus MAT, **p = 0.0029; N = 4, independent experiments = 3). d Comparison of mRNA expression in gastric organoids grown in 5 mg ml-1 SEM hydrogel and MAT (N = 4, independent experiments = 3). e Immunofluorescent staining for stemness markers (SOX9 and KI67), differentiation markers (MUC5AC, CHGA, and HK), a tight junction marker (ZO1), and a cell–cell adhesion/interaction marker (ECAD) in gastric organoids grown in 5 mg ml-1 SEM hydrogel and MAT (scale bars = 50 µm, independent experiments = 3). f Fluorescent staining with acridine orange for gastric organoids grown in 5 mg ml-1 SEM hydrogel and MAT (scale bars = 100 µm), and quantification of fluorescence (600–650 nm)/fluorescence (500–550 nm) from organoids in each hydrogel (N = 12 for SEM and N = 14 for MAT, independent experiments = 2). The color scale indicates the relative number of pixels displayed in the area. g Brightfield images of intestinal organoids grown in IEM hydrogels and MAT at day 6 (scale bar = 200 µm, independent experiments = 3). h Quantification of intestinal organoid formation efficiency in IEM hydrogels compared to in MAT (N = 4, independent experiments = 3). i qPCR analysis to compare mRNA expression of intestinal organoids within each hydrogel (IEM 2 mg ml-1 versus MAT, ***p < 0.0001 for Lgr5, ***p = 0.0009 for Muc2; IEM 3 mg ml-1 versus MAT, ***p = 0.0003 for Lgr5, **p = 0.0032 for Muc2; IEM 4 mg ml-1 versus MAT, *p = 0.0455 for Muc2; N = 4, independent experiments = 3). j Comparison of mRNA expression of intestinal organoids grown in 2 mg ml-1 IEM hydrogel and MAT (IEM versus MAT, ***p < 0.0001 for Lgr5, ***p < 0.0001 for Axin2, **p = 0.0035 for Muc2; N = 4, independent experiments = 3). k Immunofluorescent staining for a stemness marker, differentiation markers (MUC2, LYZ, CHGA, and VILLIN), a tight junction marker, and a cell–cell adhesion/interaction marker in intestinal organoids grown in 2 mg ml-1 IEM hydrogel and MAT (scale bars = 50 µm, independent experiments = 3). l Brightfield images of intestinal organoids grown in each hydrogel after forskolin treatment (scale bar = 100 µm), and m the area of forskolin-treated organoids normalized to the organoid area prior to forskolin treatment in each group (N = 4, independent experiments = 3). The data in b–d, f, h–j, and m are presented as mean ± S.D. Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test (c, i) and unpaired, two-sided student’s t-test (d, j).

Fig 3: ACE2 and TMPRSS2 are expressed in the gastrointestinal tract and in PSC-HIOs. (A) Paraffin-embedded sections of indicated gastrointestinal tissues and embedded Caco-2 cells were stained for ACE2 and TMPRSS2 protein. Strong luminal expression is found in epithelia of duodenum, colon, and gallbladder, with weaker expression in the stomach and the mucosal lining of the esophagus. Representative images are shown. Scale bars = 20 µm. (B) Duodenum biopsy was stained for ACE2 and TMPRSS2 using primary antibodies and fluorophore-conjugated secondary antibodies and imaged by fluorescence microscopy. Scale bar = 50 µM. (C) ACE2 and TMPRSS2 expression from panel A was graded according to signal intensity (relative to the positive control, Caco-2 cells). (D) Microscopy of PSC-HIOs. Scale bar = 50 µM. (E) PSC-HIOs were stained for ACE2 (left panels) and TMPRSS2 (right panels). Nuclei are stained with DAPI in blue. (F–H) PSC-HIOs were costained for ACE2 and (F) enteroendocrine cell marker CHGA, (G) Paneth cell marker LYZ, or (H) goblet cell marker MUC2 showing coexpression with CHGA and LYZ but not with MUC2. Nuclei are stained with DAPI in blue. Arrows indicate co-expression. Left panels show representative images, right panels magnifications.

Fig 4: Epithelial cell subclusters in SLs.A, UMAP representation of dimensionally reduced data following graph-based clustering with marker-based cell type assignments (left) and proportion of epithelial cell types in NC and SLs (right). B, Dot plot depicting expression levels of canonical colonocyte marker genes together with the percentage of cells expressing the marker. C, UMAP plot showing Seurat clusters of epithelial cells and association of Seurat clusters with defined epithelial cell types. D, Expression of GUCA2A and GUCA2B in epithelia of different groups. E, UMAP plot showing the sample origin of epithelial cells. F, Volcano plot depicting the differentially expressed genes between Epi-SL cluster and normal colonocytes (BEST4+ colonocytes, mature colonocytes, and goblet cells). G, Expression of LYZ, RNF43, and MYC in the epithelia of different groups.

Fig 5: Paneth cell apoptosis and exfoliation shown by anti-lysozyme Ab IHC and development of ileal pathology in the post-weaning DKO mice. Panel A shows the normal appearance of Paneth cells stained by anti-Lyz1 IHC in a non-DKO (GPx1-/-GPx2+/-) mouse (DAB substrate; hematoxylin counterstained). Panel B shows a putative apoptotic Paneth cell judging by shriveled Lys1+ appearance in the DKO crypt (pointed by a white arrow). Apoptotic Paneth cells, in situ, are also demonstrated in Supplementary Fig. 3 as double-stained Paneth cells with sequential TUNEL IHC (dark grey to black color stain) and anti-Lyz1 Ab IHC (red/brown stain). Panel C shows Lyz+ exfoliated cells (green arrows) in DKO crypt. Panel D shows that Paneth cell number begins to decline at 26 days and nearly depleted by 32-day-old. From 27 days on, the Paneth cell number is significantly lower in DKO compared to the control mice (*, one-way ANOVA test). The progression of the apoptosis pathology (Panel E), crypt exfoliation (Panel F), counts of MPO+ cells in the ileal submucosa (Panel G), appearance of ileum crypt abscesses (Panel H) and increase of Nox1 mRNA levels (Panel I) were analyzed from the ileum of 24- to 35-day-old DKO mice. Two to four mice were analyzed for each day of DKO mice and 35-day-old non-DKO controls. All panels are shown as mean±SD. Panel D, E, F and H were analyzed by histology. The numbers of non-DKO and DKO mice analyzed were 7, 8 of 24-day-old; 2, 7 of 25-day-old; 4, 12 of 26-day-old; 6 each of 27-day-old; 8 each of 28-day-old; 5, 7 of 29-day-old; 5 each of 30-day-old; 3, 5 of 31-day-old; and 14, 13 of 35-day-old, respectively. MPO IHC was done on 4 samples each and Nox1 qPCR on 2 to 4 samples at each time point. For Panel D the comparison is between the non-DKO and DKO at each age using t-test. In Panel E–H analysis is done by one-way ANOVA test using 24- to 35-day-old sets as reference sequentially against the remaining sets using Dunnett's multiple-correction test. Panel I was analyzed by pair-wise t-test.