Fig. 5: Identification of TGFβ1 and FGF2 as mediators of enterocyte maturation within extracellular vesicle EV39.

a Venn diagram of LC-MS/MS-based proteomic analysis of MSCs-derived extracellular vesicles (EV39 and EV42). Among the abundant or unique proteins in EV39, 16 ligand candidates, including fibroblast growth factor II (FGF2) and transforming growth factor I (TGFβ1), were identified based on the CellChat v2 database. b Bar plot of relative mRNA expression of TGFβ signaling pathway-associated genes in preterm intestinal epithelial cells treated with EV39 (2 × 10⁹ EVs/mL for seven days) in the presence of a TGFβ1 receptor inhibitor (A83-01, 500 nM, n = 3, 3 wells per group, NT vs. EV39). c Relative mRNA expression of adult enterocyte marker genes (TM4SF20, THSD4, SLC46A1, LCT, ABTB2) in preterm intestinal epithelial cells. The cells were treated with EV39 (2 × 10⁹ EVs/mL), TGFβ1 (0.1 ng/mL), FGF2 (10 ng/mL), or co-treatment of TGFβ1 and FGF2 for seven days, in the absence of A83-01. mRNA levels of each gene are normalized to HPRT1 and RPLP0 levels. NT, non-treated group (n = 3, 3 wells per group, NT vs. other groups). d Summary of gene expression patterns for EV39-induced enterocyte markers, including the effects of TGFβ1 or co-treatment of TGFβ1 and FGF2. e Relative mRNA expression of adult enterocyte marker genes and functional enterocyte markers: digestive enzyme genes, fatty acid transporter genes, amino acid transporter genes, inorganic solute transporter genes, and cell junction genes in the preterm intestinal epithelial cells co-treated with TGFβ1 (0.1 ng/mL) and FGF2 (10 ng/mL), with or without CFTR inhibitor (CFTRinh, 10 μM) for seven days. mRNA levels were normalized to HPRT1 and RPLP0. NT, non-treated group (n = 3, 3 wells per group, TGFβ1 + FGF2 vs. NT, or TGFβ1 + FGF2 + CFTR inhibitor). f (left panel) Representative immunofluorescence images showing the EdU-stained nuclei of proliferating cells (red) in the preterm intestinal epithelial cells co-treated with TGFβ1 (0.1 ng/mL) and FGF2 (10 ng/mL), with or without CFTR inhibitor (CFTRinh, 10 μM) for seven days. All nuclei were counterstained with Hoechst. (right panel) Quantification of EdU-positive cells in the indicated experimental groups. NT, non-treated group (n = 10, comprising 10 organoids from 2 wells per group, TGFβ1 + FGF2 vs. NT, or TGFβ1 + FGF2 + CFTR inhibitor). Scale bar = 100 µm. Quantitative data are expressed as the mean ± standard error of the mean (S.E.M). *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined using unpaired two-tailed Student’s t tests (b) and one-way ANOVA with Dunnett’s post hoc test (c,e,f). See also Supplementary Fig. 6.