Wheat gliadin induces severe intestinal symptoms and small-bowel mucosal damage in coeliac disease patients. At present, the only effective treatment for the disease is a strict life-long gluten-free diet. In this study we investigated whether probiotics Lactobacillus fermentum or Bifidobacterium lactis can inhibit the toxic effects of gliadin in intestinal cell culture conditions. The ability of live probiotics to inhibit peptic-tryptic digested gliadin-induced damage to human colon cells Caco-2 was evaluated by measuring epithelial permeability by transepithelial resistance, actin cytoskeleton arrangements by the extent of membrane ruffling and expression of tight junctional protein ZO-1. B. lactis inhibited the gliadin-induced increase dose-dependently in epithelial permeability, higher concentrations completely abolishing the gliadin-induced decrease in transepithelial resistance. The same bacterial strain also inhibited the formation of membrane ruffles in Caco-2 cells induced by gliadin administration. Furthermore, it also protected the tight junctions of Caco-2 cells against the effects of gliadin, as evinced by the pattern of ZO-1 expression. We conclude thus that live B. lactis bacteria can counteract directly the harmful effects exerted by coeliac-toxic gliadin and would clearly warrant further studies of its potential as a novel dietary supplement in the treatment of coeliac disease.

Intestinal barrier dysfunction and translocation of endotoxins are involved in the pathogenesis of alcoholic liver disease. Exposure to ethanol and its metabolite, acetaldehyde at relatively high concentrations have been shown to disrupt intestinal epithelial tight junctions in the conventional two dimensional cell culture models. The present study investigated quantitatively and qualitatively the effects of ethanol at concentrations detected in the blood after moderate ethanol consumption, of its metabolite acetaldehyde and of the combination of both compounds on intestinal barrier function in a three-dimensional cell culture model. Caco-2 cells were grown in a basement membrane matrix (Matrigel™) to induce spheroid formation and were then exposed to the compounds at the basolateral side. Morphological differentiation of the spheroids was assessed by immunocytochemistry and transmission electron microscopy. The barrier function was assessed by the flux of FITC-labeled dextran from the basal side into the spheroids' luminal compartment using confocal microscopy. Caco-2 cells grown on Matrigel assembled into fully differentiated and polarized spheroids with a central lumen, closely resembling enterocytes in vivo and provide an excellent model to study epithelial barrier functionality. Exposure to ethanol (10–40 mM) or acetaldehyde (25–200 µM) for 3 h, dose-dependently and additively increased the paracellular permeability and induced redistribution of ZO-1 and occludin without affecting cell viability or tight junction-encoding gene expression. Furthermore, ethanol and acetaldehyde induced lysine residue and microtubules hyperacetylation. These results indicate that ethanol at concentrations found in the blood after moderate drinking and acetaldehyde, alone and in combination, can increase the intestinal epithelial permeability. The data also point to the involvement of protein hyperacetylation in ethanol- and acetaldehyde-induced loss of tight junctions integrity.

The production of functional retinal pigment epithelium (RPE) cells from human embryonic (hESCs) and human induced pluripotent stem cells (hiPSCs) in defined and xeno-free conditions is highly desirable, especially for their use in cell therapy for retinal diseases. In addition, differentiated RPE cells provide an individualized disease model and drug discovery tool. In this study, we report the differentiation of functional RPE-like cells from several hESC lines and one hiPSC line in culture conditions, enabling easy translation to clinical quality cell production under Good Manufacturing Practice regulations. Pluripotent stem cells were cultured on human fibroblast feeder cells in serum-free medium. The differentiation toward RPE was induced by removing basic fibroblast growth factor and feeder cells from the serum-free conditions. RPE differentiation was also achieved using xeno-free and defined culture conditions. The RPE cell morphology and pigmentation of the cells were analyzed and the expression of genes and proteins characteristic for RPE cells was evaluated. In vitro functionality of the cells was analyzed using ELISA measurements for pigment epithelium derived factor (PEDF) secretion and phagocytosis of photoreceptor outer segments (POS). The integrity of the generated RPE layers was analyzed using transepithelial electric resistance measurements. We generated putative RPE cells with typical pigmented cobblestone-like morphology. The expression of RPE-specific markers was confirmed at the gene and protein level. The differentiated cells were able to phagocytose POS and secrete PEDF characteristic of native RPE cells. In addition, cultured cells formed a polarized epithelium with high integrity and exhibited excellent transepithelial electric resistance values, indicating well established, tight junctions. Moreover, we introduced an improved method to generate functional putative RPE cells without xeno-components under defined conditions. We have developed a progressive differentiation protocol for the production of functional RPE-like cells from hESCs and hiPSCs. Our results demonstrate that putative hESC-RPE and hiPSC-RPE express genes and proteins characteristic for RPE cells, as well as being able to phagocytose POS and secrete PEDF. Furthermore, our results show that RPE-like cells can be differentiated in xeno-free and defined culture conditions, which is mandatory for Good Manufacturing Practice-production of these cells for clinical use.

Extracellular matrix (ECM) interactions play a vital role in cell morphology, migration, proliferation, and differentiation of cells. We investigated the role of ECM proteins on the structure and function of human embryonic stem cell–derived retinal pigment epithelial (hESC-RPE) cells during their differentiation and maturation from hESCs into RPE cells in adherent differentiation cultures on several human ECM proteins found in native human Bruch's membrane, namely, collagen I, collagen IV, laminin, fibronectin, and vitronectin, as well as on commercial substrates of xeno-free CELLstart™ and Matrigel™. Cell pigmentation, expression of RPE-specific proteins, fine structure, as well as the production of basal lamina by hESC-RPE on different protein coatings were evaluated after 140 days of differentiation. The integrity of hESC-RPE epithelium and barrier properties on different coatings were investigated by measuring transepithelial resistance. All coatings supported the differentiation of hESC-RPE cells as demonstrated by early onset of cell pigmentation and further maturation to RPE monolayers after enrichment. Mature RPE phenotype was verified by RPE-specific gene and protein expression, correct epithelial polarization, and phagocytic activity. Significant differences were found in the degree of RPE cell pigmentation and tightness of epithelial barrier between different coatings. Further, the thickness of self-assembled basal lamina and secretion of the key ECM proteins found in the basement membrane of the native RPE varied between hESC-RPE cultured on compared protein coatings. In conclusion, this study shows that the cell culture substrate has a major effect on the structure and basal lamina production during the differentiation and maturation of hESC-RPE potentially influencing the success of cell integrations and survival after cell transplantation.

Retinal pigment epithelial (RPE) cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP), the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC) and RPE derived from the hESC (hESC-RPE). Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE -derived diseases, drug testing and targeted drug therapy.

Purpose.: Aquaporins (AQPs), a family of transmembrane water channel proteins, are essential for allowing passive water transport through retinal pigmented epithelial (RPE) cells. Even though human native RPE cells and immortalized human RPEs have been shown to express AQPs, the expression of AQPs during the differentiation in stem cell–derived RPE remains to be elucidated. Methods.: In human embryonic (hESCs) and induced pluripotent stem cells (hiPSCs)–derived RPE cells, the expression of several AQPs was determined by quantitative real-time PCR and the localization of AQP1 was assessed with confocal microscopy. The functionality of AQP water channels was determined by cell volume assay in hESC-derived RPE cells. Results.: AQP1, AQP3, AQP4, AQP5, AQP6, AQP7, AQP10, AQP11, and AQP12 were expressed in hESC- and hiPSC-derived RPE cells. Furthermore, the expression of AQP1 and AQP11 genes were significantly upregulated during the maturation of both hESC and iPSC into RPE. Confocal microscopy shows the expression of AQP1 at the apical plasma membrane of polarized cobblestone hESC- and hiPSC-derived RPE cells. Lastly, aquaporin inhibitors significantly reduced AQP functionality in hESC-RPE cells. Conclusions.: hESC-RPE and hiPSC-RPE cells express several AQP genes, which are functional in mature hESC-derived RPE cells. The localization of AQP1 on the apical plasma membrane in mature RPE cells derived from both hESC and hiPSC suggests its functionality. These data propose that hESC- and hiPSC-derived RPE cells, grown and differentiated under serum-free conditions, resemble their native counterpart in the human eye.