Figure 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 S2H (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.