Fig 2: Rebuilding the Repairing Epithelium In Vitro Based on Defined Components(A) Murine small intestinal epithelial cells cultured in the presence of EGF, Noggin, and R-spondin1 (ENR) or with the addition of Wnt3a (+Wnt3a) in either Matrigel or collagen type I. Scale bars, 100 µm.(B) Heatmap of differentially expressed probe sets between culture conditions using MG/ENR+Wnt3a (ENRW, green) and COL/ENRW (red) (n = 6; fold change > 1.5; FDR < 0.1). Examples of differentially expressed genes are indicated.(C) Detection of F-actin with Phalloidin (gray) counterstained with DAPI (blue) and YAP (green) counterstained with E-cadherin (red) and DAPI (blue) in Matrigel and collagen type I cultures from the small intestine. Insets show enlarged view of the indicated regions. Scale bars, 50 µm in the top panels and 100 µm in the bottom panels.(D) Heatmap of Z score-transformed relative expression levels as determined by qPCR for selected YAP/TAZ target genes and markers of the fetal and adult states in cultures of small intestinal epithelial cells grown in either Matrigel (MG) or collagen type I (Col) in the presence of EGF/Noggin/Rspondin (ENR) with or without Wnt3a (W).(E) Detection of Sca1 (green, left) and Anxa1 (green, right) in collagen type I cultures from the small intestine. Inset shows enlarged view of the region indicated. Images are counterstained with DAPI (blue) and image for Anxa1 is additionally counterstained with E-cadherin (red). Scale bars, 50 µm.(F) GSEA showing enrichment of the collagen culture gene signature in the repairing epithelium.(G) GSEA showing enrichment of YAP and fetal gene signatures in ENRW-Collagen relative to ENRW-Matrigel cultures.(H) Mouse and human colonic organoids propagated in collagen type I. For human colonic crypts, Prostaglandin E2 (PGE2) is supplemented. Scale bar, 100 µm.See also Figure S4.

Fig 3: The Repairing Epithelium Adopts a Fetal-like Expression Profile(A) Sca1 (green) expression in organoids from adult animals (Adult) and in fetal organoids (Fetal) derived from the proximal small intestine. Insets show the enlarged view of the indicated region. Images are counterstained with DAPI (blue). Scale bar, 50 µm.(B) Heatmap of differentially expressed probe sets showing genes upregulated more than 2-fold comparing adult organoids (green) and fetal organoids (red) (n = 3; fold change > 2; FDR < 0.05). Multiple members of Ly6 and Annexin gene families are upregulated in the fetal cells.(C) Sca1 (green) expression in fetal colon at embryonic day (E)16.5 counterstained with EpCAM (red) and DAPI (blue). Scale bars, 50 µm.(D) GSEA showing enrichment of the fetal intestinal gene signature in the repairing epithelium.(E) Anxa1 (green) expression in adult organoids (Adult) and fetal organoids (Fetal) derived from the proximal small intestine. Images are counterstained with E-cadherin (red) and DAPI (blue). Scale bar, 100 µm.(F) Anxa1 (green) expression in fetal colon at E16.5, colon in homeostasis and in the repair phase. Images are counterstained with E-cadherin (red) and DAPI (blue). Scale bars, 50 µm for fetal colon and 100 µm for adult colon.(G) ANXA1 (green) in colonic biopsy from non-inflamed and inflamed regions obtained from a patient with ulcerative colitis. Images are counterstained with E-cadherin (red) and DAPI (blue). Scale bar, 100 µm.(H and I) GSEAs showing enrichment of the transcriptional signatures for the mouse repairing epithelium (H, mRepair) and mouse fetal state (I, mFetal) in samples isolated from patients with active colitis (hColitis) compared to control individuals (hNormal).See also Figure S2 and Table S2.

Fig 4: Injury-Induced Cellular Reprogramming Is Reversible(A and B) Serial sections of engrafted patches from Matrigel cultures analyzed for (A) tdTomato (red) and Sca1 (green) and (B) YAP (green) and E-cadherin (red) 1 day after transplantation (day 12). Scale bar, 100 µm.(C) Diagram of the transplantation strategy using cells from the conditional YAP/TAZ cdKO cells and control animals. Indicated are the administration of DSS (day 0–5), time points for transplantation (days 8 and 11), administration of 4-hydroxy tamoxifen (days 12 and 13), as well as the final analysis (day 16).(D) Whole-mount analysis of the colon for control (tdTomato+/red) and Villin CreER YAP/TAZ cDKO cells (eGFP+/green) before (day 12) and after (day 16) tamoxifen administration. Arrows illustrate areas of GFP+ cells.(E) Quantification of the ratio of the area covered by GFP- versus RFP-expressing cells before and after tamoxifen treatment. Each dot represents independent animals, and data are presented as the mean ± SEM (p = 0.029 based on a Mann-Whitney exact one-sided test).(F) Section of area with engrafted RFP- (red) and GFP- (green) expressing cells. The engraftments are shown at higher magnification in F’ and F”. Scale bar, 100 µm.(G) Small intestinal epithelial cells cultured for 3 passages (P3) in collagen type I with ENRW grow as organoids, when replated in Matrigel (replating passage 3) in the presence of ENR. Inset shows secretory cells in an organoid. Scale bar, 100 µm.(H) Heatmap of Z score-transformed relative expression levels as determined by qPCR for selected various markers expressed by fetal and adult epithelia grown in ENR in MG (Matrigel), then transferred into collagen and cultured in the presence of ENRW (Collagen), and lastly replated in Matrigel and cultured in ENR (Matrigel replating).(I) Engrafted patches (red) from tdTomato-expressing cells cultured for 3 passages in either collagen type I or Matrigel. Scale bars, 2 mm.(J) Model depicting how environmental changes control cellular identity during tissue repair via YAP/TAZ signaling.See also Figure S6.

Fig 5: Repairing Intestinal Epithelium Has a Cellular and Molecular Profile Distinct from the Normal Epithelium(A) Macroscopic images of the distal part of the colon in an untreated mouse (left) and a mouse 2 weeks post-administration of DSS (right). The demarcated area indicates regions undergoing active re-epithelialization. Scale bar, 2 mm.(B) H&E staining of homeostatic tissue and tissue in the repair phase at 2 weeks following DSS administration. Scale bar, 100 µm.(C) The mucosal/submucosal thickness at homeostasis and repair phase (2 weeks). Shown are mean distances ± SEM (n = 4 animals; p = 0.007 based on two-sided Student’s t test).(D) Sca1 (green) expression in colonic epithelium in homeostatic and repair phase. Sections are counterstained with DAPI (blue). The demarcated line indicates the epithelial structure. Scale bar, 100 µm.(E) Flow cytometric analysis of cells from the colonic epithelium during homeostasis and in repair phase (2 weeks post-administration of DSS). Diagrams show representative plots for Sca1-PECy5 in live CD45-veCD31-veEpcam+ve cell population.(F) Quantification of the percentages of Sca1high cells in homeostasis and repair phase. Diagram shows average ± SEM (n = 3; p = 4 × 10-5 based on two-sided Student’s t test).(G) qRT-PCR analyses in Epcam+Sca1high cells sorted via FACS from the repairing epithelium and in homeostatic epithelial cells (Epcam+). Bars represent average levels ± SEM (n = 3; Ly6a, p = 1.3 × 10-4; Reg3b, p = 9.7 × 10-3; Reg3g, p = 8.5 × 10-3 based on two-sided Student’s t test).(H) Heatmap analysis of differentially expressed probe sets (fold change > 2.0; FDR < 0.05), comparing the expression profile of epithelial cells isolated from homeostatic tissue and Sca1high cells from the repairing epithelium.(I) Detection of Lrig1 (green) in homeostasis, early and late repair phase counterstained for E-cadherin (red) with DAPI (blue). Scale bar, 50 µm.(J) GSEA of repairing and homeostatic epithelium using Lgr5 intestinal stem cell gene signature.(K) Organoid formation for Epcam+ cells isolated from normal homeostasis, as well as Epcam+ Sca1low and Sca1high cells during tissue repair showing representative images of formed organoids. Bars represent average number of organoids formed ± SEM (n = 3 animals).See also Figure S1 and Table S1.