Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts’ views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.

Mechanisms mediating adult enteric neurogenesis are largely unknown. Using inflammation-associated neurogenesis models and a transgenic approach, we aimed to understand the cell-source for new neurons in infectious and inflammatory colitis. Dextran sodium sulfate (DSS) and Citrobacter rodentium colitis (CC) was induced in adult mice and colonic neurons were quantified. Sox2GFP and PLP1GFP mice confirmed the cell-type specificity of these markers. Sox2CreER:YFP and PLP1creER:tdT mice were used to determine the fate of these cells after colitis. Sox2 expression was investigated in colonic neurons of human patients with Clostridium difficile or ulcerative colitis. Both DSS and CC led to increased colonic neurons. Following colitis in adult Sox2CreER:YFP mice, YFP initially expressed predominantly by glia becomes expressed by neurons following colitis, without observable DNA replication. Similarly in PLP1CreER:tdT mice, PLP1 cells that co-express S100b but not RET also give rise to neurons following colitis. In human colitis, Sox2-expressing neurons increase from 1–2% to an average 14% in colitis. The new neurons predominantly express calretinin, thus appear to be excitatory. These results suggest that colitis promotes rapid enteric neurogenesis in adult mice and humans through differentiation of Sox2- and PLP1-expressing cells, which represent enteric glia and/or neural progenitors. Further defining neurogenesis will improve understanding and treatment of injury-associated intestinal motility/sensory disorders.

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhea among children, human immunodeficiency virus-infected patients, and travelers to developing regions of the world. The pathogenesis of EAEC strains involves the production of biofilm. In this study, we determined the association between presence of putative EAEC virulence genes and biofilm formation in 57 EAEC isolates (as defined by HEp-2 adherence) from travelers with diarrhea and in 18 EAEC isolates from travelers without diarrhea. Twelve nondiarrheagenic E. coli isolates from healthy travelers were used as controls. Biofilm formation was measured by using a microtiter plate assay with the crystal violet staining method, and the presence of the putative EAEC virulence genes aap, aatA, aggR, astA, irp2, pet, set1A, and shf was determined by PCR. EAEC isolates were more likely to produce biofilm than nondiarrheagenic E. coli isolates (P ‫؍‬ 0.027), and the production of biofilm was associated with the virulence genes aggR, set1A, aatA, and irp2, which were found in 16 (40%), 17 (43%), 10 (25%), and 27 (68%) of the biofilm producers versus only 4 (11%), 6 (6%), 2 (6%), and 15 (43%) in non-biofilm producers (P ‫؍‬ 0.008 for aggR, P ‫؍‬ 0.0004 for set1A, P ‫؍‬ 0.029 for aatA, and P ‫؍‬ 0.04 for irp2). Although the proportion of EAEC isolates producing biofilm in patients with diarrhea (51%) was similar to that in patients without diarrhea (61%), biofilm production was related to the carriage of aggR (P ‫؍‬ 0.015), set1A (P ‫؍‬ 0.001), and aatA (P ‫؍‬ 0.025). Since aggR is a master regulator of EAEC, the presence of aap (P ‫؍‬ 0.004), astA (P ‫؍‬ 0.001), irp2 (P ‫؍‬ 0.0006), pet (P ‫؍‬ 0.002), and set1A (P ‫؍‬ 0.014) in an aggR versus an aggR-lacking background was investigated and was also found to be associated with biofilm production. This study suggests that biofilm formation is a common phenomenon among EAEC isolates derived from travelers with or without diarrhea and that multiple genes associated with biofilm formation are regulated by aggR.

These results show that colitis promotes enteric neurogenesis in the adult colon through a serotonin-dependent mechanism that drives glial cells to transdifferentiate into neurons.

Little is known about postnatal enteric nervous system (ENS) development, but some reports suggest that the postnatal bowel may contain neural stem cells. Therefore, we created an in vitro model of desegregation using an enzymatic and mechanical tissue technique. This approach yielded a group of cells from the small intestine of lactating and adult mice, which ex vivo attach to the culture dish; actively proliferate; and express nestin, vimentin, and the pro‐neural transcription factors neurogenin‐2 (ngn‐2), Sox‐10, and Mash‐1. In the conditions grown, double immunostains suggest that they differentiate into various cell types, particularly neurons, smooth muscle, and glia including 04 protein–positive cells. They also express the neurotrophic‐protein tyrosine kinase (Trk) receptors TrkA, TrkB, and TrkC; the low‐affinity neurotrophin receptor p75NTR; and the glial‐derived neurotrophic factor receptors (GFR)α‐1, GFRα‐2, and GFRα‐3. The neurons expressed several sensory and motor neurotransmitters present in the central and enteric nervous systems, including calcitonin gene‐related peptide, neuropeptideY, peptideYY, substance P, vasoactive intestinal polypeptide, and galanin; along with glia, these neurons formed elaborate intercellular connections. They also express c‐KIT, CD34, CD20, and CD45RO, suggesting they either have a hematogenous origin or may differentiate toward hematogenous lines. These findings suggest that these cells may be enteric neural stem cells (ENSCs); may normally be present in the small intestine; and may have the capacity to proliferate and differentiate into neurons, glia, and smooth muscle. Further identification and purification of intestinal ENSCs will provide a means to study the regulation of their differentiation and should give insight into the mechanisms involved in development and remodeling of the ENS. The possible therapeutic application of postnatal stem cells such as ENSCs needs to be evaluated, including their use for transplantation in the central nervous system.

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Background The intestinal microbiota plays an important role in regulating gastrointestinal (GI) physiology in part through interactions with the enteric nervous system (ENS). Alterations in the gut microbiome frequently occur together with disturbances in enteric neural control in pathophysiological conditions. However, the mechanisms by which the microbiota regulates GI function and the structure of the ENS are incompletely understood. Using a mouse model of antibiotic (Abx)-induced bacterial depletion, we sought to determine the molecular mechanisms of microbial regulation of intestinal function and the integrity of the ENS. Spontaneous reconstitution of the Abx-depleted microbiota was used to assess the plasticity of structure and function of the GI tract and ENS. Microbiota-dependent molecular mechanisms of ENS neuronal survival and neurogenesis were also assessed. Results Adult male and female Abx-treated mice exhibited alterations in GI structure and function, including a longer small intestine, slower transit time, increased carbachol-stimulated ion secretion, and increased intestinal permeability. These alterations were accompanied by the loss of enteric neurons in the ileum and proximal colon in both submucosal and myenteric plexuses. A reduction in the number of enteric glia was only observed in the ileal myenteric plexus. Recovery of the microbiota restored intestinal function and stimulated enteric neurogenesis leading to increases in the number of enteric glia and neurons. Lipopolysaccharide (LPS) supplementation enhanced neuronal survival alongside bacterial depletion, but had no effect on neuronal recovery once the Abx-induced neuronal loss was established. In contrast, short-chain fatty acids (SCFA) were able to restore neuronal numbers after Abx-induced neuronal loss, demonstrating that SCFA stimulate enteric neurogenesis in vivo. Conclusions Our results demonstrate a role for the gut microbiota in regulating the structure and function of the GI tract in a sex-independent manner. Moreover, the microbiota is essential for the maintenance of ENS integrity, by regulating enteric neuronal survival and promoting neurogenesis. Molecular determinants of the microbiota, LPS and SCFA, regulate enteric neuronal survival, while SCFA also stimulates neurogenesis. Our data reveal new insights into the role of the gut microbiota that could lead to therapeutic developments for the treatment of enteric neuropathies.

Anogenital distance (AGD) at birth is regarded as a useful measurement that reflects the prenatal androgenic status in rodents. However, the impact of xenoantiandrogens on human development is largely unknown. The aim of this study was to evaluate the potential antiandrogenic impact of prenatal DDT metabolites (p,p′-DDE and p,p′-DDT) exposure on infant AGD, using a non-age–dependent anal position index (API). As part of an ongoing perinatal cohort study on the effects of organochlorine pesticides in children’s neurodevelopment, we conducted a cross-sectional study in 71 infants (37 males and 34 females). Maternal serum levels of DDT metabolites (p,p′-DDE and p,p′-DDT) before and during each trimester of pregnancy were determined by electron capture gas–liquid chromatography. During postnatal home visits at 3, 6, and 12 or 18 months of age, the children’s weight and API were evaluated. Multiple lineal regression models were used to estimate the potential endocrine disruptor activity of prenatal p,p′-DDE exposure. Boys had significantly higher API values than girls (0.6 versus 0.5; P < 0.001). Only among boys, a doubling increase of maternal p,p′-DDE serum levels during the first trimester of pregnancy, were associated with a significant reduction of API (β = −0.02; P = 0.02). No effect of p,p′-DDT on AGD was observed. Evidence of the effect of prenatal p,p′-DDE on external genital differentiation is scarce and not consistent in the literature. Further studies are needed to confirm a hormonal disruptive effect on the development of external genitalia, due not only to p,p′-DDE but also due to other antiandrogenic persistent compounds.