The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.

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Aims: The influence of enteric glia on the regulation of intestinal functions is unknown. Our aim was to determine the phenotype of enteric neurones in a model of glia alterations and the putative changes in intestinal motility and permeability. Methods: Transgenic mice expressing haemagglutinin (HA) in glia were used. Glia disruption was induced by injection of activated HA specific CD8+ T cells. Control mice consisted of non-transgenic littermates injected with activated HA specific CD8+ T cells. Immunohistochemical staining for choline acetyltransferase (ChAT), substance P (SP), vasoactive intestinal peptide (VIP), and nitric oxide synthase (NOS) was performed on jejunal submucosal plexus (SMP) and myenteric plexus (MP). Neurally induced jejunal muscle activity was characterised in vitro. Gastrointestinal transit and paracellular permeability were measured using fluorescein isothiocyanate-dextran markers. Results: CD3 positive T cells infiltrates were observed in the MP of transgenic mice. In the SMP, the proportions of VIP and SP positive neurones decreased in transgenic mice compared with control mice. ChAT remained unchanged. In the MP, the proportions of ChAT and NOS positive neurones increased and decreased, respectively, in transgenic mice. In contrast, VIP and SP remained unchanged. Neurally mediated jejunal relaxation was lower in transgenic mice than in controls. This relaxation was reduced by N G -nitro-L-arginine methyl ester in control mice but not in transgenic mice. Gastrointestinal transit was delayed and intestinal permeability increased in transgenic mice compared with control mice. Conclusion: Glia disruption induces changes in the neurochemical coding of enteric neurones, which may partly be responsible for dysfunctions in intestinal motility and permeability.

Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer1. Cell entry of HCV2 and other pathogens3-5 is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver-chimeric mouse model6 we show that a monoclonal antibody specific for TJ protein claudin-17 eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection via host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy.

Although recent studies have shown that enteric neurons control intestinal barrier function, the role of enteric glial cells (EGCs) in this control remains unknown. Therefore, our goal was to characterize the role of EGCs in the control of intestinal epithelial cell proliferation using an in vivo transgenic and an in vitro coculture model. Assessment of intestinal epithelial cell proliferation after ablation of EGCs in transgenic mice demonstrated a significant increase in crypt cell hyperplasia. Furthermore, mucosal glial network (assessed by immunohistochemical detection of S-100beta) is altered in colon adenocarcinoma compared with control tissue. In an in vitro coculture model of subconfluent Caco-2 cells seeded onto Transwell filters with EGCs, Caco-2 cell density and [3H]thymidine incorporation were significantly lower than in control (Caco-2 cultured alone). Flow cytometry analysis showed that EGCs had no effect on Caco-2 cell viability. EGCs induced a significant increase in Caco-2 cell surface area without any sign of cellular hypertrophy. These effects by EGCs were also seen in various transformed or nontransformed intestinal epithelial cell lines. Furthermore, TGF-beta1 mRNA was expressed, and TGF-beta1 was secreted by EGCs. Exogenously added TGF-beta1 reproduced partly the EGC-mediated effects on cell density and surface area. In addition, EGC effects on Caco-2 cell density were significantly reduced by a neutralizing TGF-beta antibody. In conclusion, EGCs have profound antiproliferative effects on intestinal epithelial cells. Functional alterations in EGCs may therefore modify intestinal barrier functions and be involved in pathologies such as cancer or inflammatory bowel diseases.

Gut 2003;52:84-90 Background: Morphological and functional changes in the enteric nervous system (ENS) have been reported in inflammatory bowel diseases but it is still uncertain whether neurochemical coding of myenteric neurones is altered in ulcerative colitis (UC). Aims: In this study we investigated transmitter co-localisation in myenteric neurones of normal colon and the colon of patients with UC. Methods: Choline acetyltransferase (ChAT), neurone specific enolase (NSE), vasoactive intestinal peptide (VIP), and substance P (SP) were detected by immunohistochemical methods in whole mounts of colonic myenteric plexus of UC patients (n=10) and controls (n=8). Results: The proportion of ChAT positive and VIP positive neurones relative to the NSE population did not differ in inflamed (33.3% and 9.3%, respectively) and non-inflamed segments (33.6% and 9.7%) of UC colon compared with controls (35.0% and 6.9%). The proportion of SP positive neurones was significantly larger in both inflamed (15.5%) and non-inflamed (20.3%) segments than in controls (5.9%). Analysis of changes in subpopulations showed that 26.9% of neurones were only ChAT positive in controls but that the proportion was significantly smaller in inflamed (18.8%) and non-inflamed (15.8%) areas of UC. The proportions of neurones containing ChAT and SP were significantly higher in inflamed (11.8%) and non-inflamed (13.9%) areas than in controls (5.0%). Conclusion: Remodelling of myenteric neurones in UC involves a shift from mainly cholinergic to more SP positive innervation. This effect may constitute part of the neuronal basis for the motility disturbances observed in UC.

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Enteric glial cells (EGCs) are essential in the control of gastrointestinal functions. Although lesions of EGCs are associated with neuronal degeneration in animal models, their direct neuroprotective role remains unknown. Therefore, the aims of this study were to demonstrate the direct neuroprotective effects of EGCs and to identify putative glial mediators involved. First, viral targeted ablation of EGCs in primary cultures of enteric nervous system increased neuronal death both under basal conditions and in the presence of oxidative stress (dopamine, hydrogen peroxide). Second, direct or indirect coculture experiments of EGC lines with primary cultures of enteric nervous system or neuroblastoma cell lines (SH-SY5Y) prevented neurotoxic effects induced by oxidative stress (increased membrane permeability, release of neuronal specific enolase, caspase-3 immunoreactivity, changes in [Ca(2+)](i) response). Finally, combining pharmacological inhibition and mRNA silencing methods, we demonstrated that neuroprotective effects of EGCs were mediated in part by reduced glutathione but not by oxidized glutathione or by S-nitrosoglutathione. Our study identified the neuroprotective effects of EGCs via their release of reduced glutathione, extending their critical role in physiological contexts and in enteric neuropathies.-Abdo, H., Derkinderen, P., Gomes, P., Chevalier, J., Aubert, P., Masson, D., Galmiche, J.-P., Vanden Berghe, P., Neunlist, M., Lardeux, B. Enteric glial cells protect neurons from oxidative stress in part via reduced glutathione.