Fig 2: p40 supplementation in early stage has a long-term effect on the increase in Tgfb gene expression in mice. (A) For testing the short-term effects of p40 in neonate and adult mice, WT mice were supplemented with p40 from postnatal day 2 to day 14 or from week 6 to week 8. Mice were killed at the end of treatment. (B) For testing the long-term effects of p40 supplementation in neonatal stage and in adulthood, WT mice were supplemented with p40 from postnatal day 2 to day 21 (neo-p40) and from week 6 to week 8 (adult-p40). Mice supplemented with hydrogels without p40 were used as controls. Mice were killed 4 weeks after the end of p40 supplementation. Each symbol represents one mouse in A and B. (C) For testing the sustained effects of p40 on Tgfb gene expression in mouse enteroids, enteroids from WT mice were treated with p40 (100 ng/mL) and passaged as indicated. RNA was isolated from colonic tissues and enteroids for RT-PCR analysis of Tgfb1 gene expression. The Tgfb1 mRNA expression levels in the control groups were set as 1. The mRNA expression levels in treated groups were compared with the control group. (C) Data are quantified from 3 independent cultures.

Fig 3: p40 stimulates TGFß production in IECs. (A and B) Enteroids and colonoids from WT mice were cultured in IntestiCult organoid growth medium in the presence and absence of p40 (100 ng/mL). (A) Representative images of colonoids at the indicated days after culture are shown. Images were taken using a light microscope at a magnification of 10×. (C and D) YAMC cells were treated with p40 at 10 ng/mL for the indicated times. RNA was isolated from (B) enteroids and colonoids cultured for 9 days, and (C) YAMC cells for RT-PCR analysis of the expression levels of Tgfb mRNA. The Tgfb mRNA expression levels in the control groups were set as 1. The mRNA expression levels in treated groups were compared with the control group. (D) Supernatants from YAMC cell culture with and without p40 treatment were collected for assessing the amount of TGFß release using enzyme-linked immunosorbent assay. The TGFß concentration is presented as picograms per 106 cells. (B–D) Data are quantified from 3 independent experiments. (B) For each experiment, data represent the average of at least 5 enteroids or colonoids in each group. *P < .05 compared with the control group. #P < .05 compared with the 2-hour p40 treatment group.

Fig 4: Transplantation of TGFB1-treated organoids enhances engraftment into DSS-treated mice.(A) Gene signatures of regenerative epithelium, fetal spheroids, revival stem cells and YAP signaling (Ayyaz et al., 2019; Gregorieff et al., 2015; Mustata et al., 2013; Wang et al., 2019; Yui et al., 2018) are each elevated post-TGFB1 treatment, as assayed by GSEA in non-irradiated conditions (n=3 independent organoid cultures, Kolmogorov-Smirnov test, P < 0.001). (B) Heatmaps display that RNA-seq expression levels of fetal/regenerative genes are highly expressed upon TGFB1 treatment compared to the vehicle controls (n=3 independent organoid cultures). Schematic of experimental design for bulk RNA-seq for panels A-B is depicted in Figure S7A. (C) For non-IR organoids, to deplete Tgfbr2, the primary organoids were treated with 1 µM tamoxifen for 12 hours on Day 3, followed with TGFB1 (2 ng/ml) treatment on Day 6. Organoids were collected 24 hours after TGFB1 treatment. Transcript levels are relative to WT; statistical comparisons were performed using one-way ANOVA followed by Dunnett’s post at P < 0.001***, P < 0.01** or P < 0.05* (n=3 independent organoid cultures). (D) GSEA reveals that genes upregulated or downregulated upon TGFB1 treatment strongly correlate with transcriptional changes in Ki67-RFP cells from the intestine of mice with irradiation vs. non-irradiation, respectively, as described in Figure 1 (Kolmogorov-Smirnov test, P < 0.001, n=3 independent organoid cultures). (E) Experimental design for organoid transplantation assay, to determine the ability of TGFB1 to prime organoids in culture prior to transplantation for engrafting into damaged colonic tissue. Transgenic organoid lines were used to later help visualize transplants. Organoids were treated with either vehicle or with TGFB1 to induce regenerative properties. To induce epithelial damage in the mouse intestine, 3.5% DSS was prepared in drinking water and fed to NOD mice for 7 days. After a period of water recovery, the treated and control organoids were mixed 1:1 and used for enema-based transplant. Either vehicle-treated organoids with RFP were mixed with TGFB1-treated organoids with GFP; or vehicle-treated organoids with GFP were mixed with TGFB1-treated organoids with RFP. Organoid mixtures were transferred into DSS-treated mice on Day 10. Colon tissues were collected on Day 19, and cryosections were prepared for checking GFP or RFP under fluorescence microscope. (F) Representative images of organoids used for transplantation. (G-I) Representative images and quantification of transplant efficiency upon TGFB1 pre-treatment. (G) Fluorescent micrographs demonstrating transgenic organoid grafts into mice. (H) The size of grafts observed. (I) The average area of organoid grafts per mouse. Color indicates whether transplanted organoids derived from red or green fluorescent lines. Symbol type represents a single mouse used in the competition assay (n=21 grafts from 5 mice, Student’s t-test at P < 0.001*** or P < 0.05*).

Fig 5: TGFB1-treated mesenchyme promotes fetal-like conversion of intestinal organoids.(A) UMAP indicates Pdgfra-positive mesenchymal cells express Tgfbr2. Pdgfra positive mesenchymal cells were subset from the scRNA-seq data set featured in Figure 2 (GSE165318). (B-C) TGFB1-induces aggregation of Pdgfra-positive mesenchymal cells in a dose- and time-dependent manner (n=3 independent experiments, passaged mesenchyme). (D-F) TGFB1-treated mesenchyme induces fetal-like gene signatures in intestinal organoids upon co-culture. (D) Schematic of experimental design of co-culture. Passaged intestinal mesenchyme cells were pre-treated with vehicle, TGFB1 or TGFBR inhibitors for 3 days, and then co-cultured as overlaid matrigel bubbles containing primary organoids at day 3 post isolation. After 2 days of co-culture, organoids were collected in their matrix bubbles for qRT-PCR (n=6 independent organoid cultures with 2 different cell densities of mesenchyme). TGFB1 was removed during co-culture and only used for pre-treatment. TGFBR inhibitors were either kept (E) or removed (Figure S5E) in co-culture. (F) Mesenchyme cells pre-treated with vehicle, TGFB1 or TGFBR inhibitors for 3 days were also collected for qRT-PCR (n=3 independent mesenchyme cultures). (G) Presence of TGFBR inhibitors suppresses fetal-like conversion of intestinal organoids co-cultured with mesenchyme isolated from mice 3 days post-irradiation (n=6 independent organoid cultures with 2 different cell lines of mesenchyme). All the qRT-PCR data are presented as mean ± SEM. Transcript levels relative to vehicle control, and statistical comparisons were performed using one-way ANOVA followed by Dunnett’s post at P < 0.001***, P < 0.01** or P < 0.05*. (H-J) Tgfbr2 knockout via UBC-CreERT2 restricts regeneration after irradiation. 5-week old mice were treated with tamoxifen to inactivate Tgfbr2 in the whole body. Intestine was collected 3 days post-IR and scored for regenerative foci using OLFM4 immunostaining (n=4-5 biological replicates, duodenum, Student’s t-test at P < 0.001***). (H) Schematic of experimental design. (I) Representative images. (J) Quantification.