Skip to main content
Fig. 5 | BMC Biology

Fig. 5

From: Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types

Fig. 5

Effect of dedifferentiation of ENT organoids on expression of biomarker immunofluorescence and histological staining. From ISC precursors, ENT organoids were cultured for 3 days with C59 + valproic acid to full differentiation as indicated by the peak expression of biomarkers. To force dedifferentiation, the Wnt inhibitor C59 was removed from the medium, and ENT organoids were then exposed for 36 h to 6 μM CHIR (dENT). a ENT and dENT organoids were stained with H&E as well as PAS. The morphology of and cell dimensions within dENT organoids were similar to those of ENT organoids. However, the immunofluorescence of the biomarkers indicated there was a significant loss of ENT-marker sucrase isomaltase (SI) staining in dENT organoids as well as an increase in secretion of stem cell marker OLFM4. b The expression of TJ mRNA in dENT organoids. Cldn2 increased with dedifferentiation (n = 4 for ENTs, n = 3 for dENTs and ISCs). Cldn7 decreased slightly with dedifferentiation (n = 4), while Ocln levels did not change (n = 5 for ENTs, n = 3 for dENTs, and n = 4 for ISCs). Tjp1 (ZO-1) tended to increase (P = 0.09) with dedifferentiation (n = 5 for ENTs, n = 4 for dENTs, and n = 3 for ISCs). Pa,b,c ≤ 0.05. c Immunofluorescence staining of CLDN2, OCLN, and ZO-1 in dedifferentiated cells. Nuclei are stained blue. Organoids were stained with the TJ protein CLDN2 (CL2; red), OCLN (green), and ZO-1 (green). Representative organoids are shown at 60× magnification. Bars are 25 μm. d Functional changes due to dedifferentiation. 4 kDa dextran permeability decreased in dENT organoids, Pa,b,c ≤ 0.05 (n = 4 for ENTs, 3 for dENTs, and 5 for ISCs). Supporting data sets are deposited in the figshare repository [50]. Cldn claudin, dENT dedifferentiated enterocyte, ENT enterocyte, FITC fluorescein isothiocyanate, H&E hematoxylin and eosin stain, ISC intestinal stem cell, L lumen, Ocln occludin, OLFM4 olfactomedin-4, PAS periodic acid-Schiff stain, SI, sucrase isomaltase, TJ tight junction, Tjp or ZO-1 tight junction protein

Back to article page