Fig. 4: Mesenchymal stem cell-derived extracellular vesicle (EV39)-induced enterocyte maturation in human preterm intestinal epithelial cells via cystic fibrosis transmembrane conductance regulator (CFTR).
From: Functional maturation of preterm intestinal epithelium through CFTR activation
a Schematic illustration of the experimental procedure showing preterm intestinal epithelial cells treated with EV39 (2 × 109 EVs/mL) and a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor (CFTR inhibitor 172, 10 μM) for seven days. Relative mRNA expression of BEST4+ enterocyte marker genes (b), adult enterocyte marker gene (c), and functional enterocyte markers: digestive enzyme genes (d), fatty acid transporter genes (e), inorganic solute transporter genes (f), amino acid transporter genes (g), and cell junction genes (h) in preterm intestinal epithelial cells treated with EV39 (2 × 109 EVs/mL), with or without CFTR inhibitor (CFTRinh, 10 μM) for seven days. mRNA levels of each gene are normalized to HPRT1 and RPLP0 levels. NT, non-treated group (n = 3, 3 wells per group, EV39 vs. NT, or EV39 + CFTR inhibitor). i Relative mRNA expression of adult enterocyte marker gene (TM4SF20, THSD4, SLC46A1, LCT, ABTB2) in preterm intestinal epithelial cells treated with forskolin (10 μM) for seven days. mRNA levels of each gene are normalized to HPRT1 and RPLP0 levels. NT, non-treated group (n = 3, 3 wells per group, NT vs. forskolin). j Relative mRNA expression of adult enterocyte marker gene (TM4SF20, THSD4, SLC46A1, LCT, ABTB2) in preterm intestinal epithelial cells treated with VX-770 (CFTR potentiator, 3 μM), with or without VX-445 (CFTR corrector, 3 μM) for seven days. 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). k (left panel) Representative immunofluorescence images showing EdU-stained nuclei of proliferating cells (red) and CFTR expression (green) in preterm intestinal epithelial cells treated with EV39 (2 × 109 EVs/mL) for seven days. All nuclei were counterstained with Hoechst. (right panel) Quantification of total CFTR-positive cells and the number of EdU-positive versus EdU-negative cells among CFTR-positive cells in the indicated experimental groups. White-filled arrowheads indicate EdU-labeled CFTR-positive cells, and unfilled arrowheads indicate EdU-negative CFTR-positive cells. NT, non-treated group (n = 10, comprising 10 organoids from 2 wells per group, NT vs. EV39). Scale bar = 20 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 by one-way ANOVA with Dunnett’s post hoc test (b–h, j) and unpaired two-tailed Student’s ttests (i, k). See also Supplementary Fig. 4, 5.
