A distinct transcriptome characterizes neural crest-derived cells at the migratory wavefront during enteric nervous system development.

Enteric nervous system development relies on intestinal colonization by enteric neural crest-derived cells (ENCDCs). This is driven by a population of highly migratory and proliferative ENCDCs at the wavefront, but the molecular characteristics of these cells are unknown. ENCDCs from the wavefront and the trailing region were isolated and subjected to RNA-seq. Wavefront-ENCDCs were transcriptionally distinct from trailing ENCDCs, and temporal modelling confirmed their relative immaturity. This population of ENCDCs exhibited altered expression of ECM and cytoskeletal genes, consistent with a migratory phenotype. Unlike trailing ENCDCs, the wavefront lacked expression of genes related to neuronal or glial maturation. As wavefront ENCDC genes were associated with migration and developmental immaturity, the genes that remain expressed in later progenitor populations may be particularly pertinent to understanding the maintenance of ENCDC progenitor characteristics. Dusp6 expression was specifically upregulated at the wavefront. Inhibiting DUSP6 activity prevented wavefront colonization of the hindgut, and inhibited the migratory ability of post-colonized ENCDCs from midgut and postnatal neurospheres. These effects were reversed by simultaneous inhibition of ERK signaling, indicating that DUSP6-mediated ERK inhibition is required for ENCDC migration in mouse and chick.

Avian ceca are indispensable for hindgut enteric nervous system development.

The enteric nervous system (ENS), which is derived from enteric neural crest cells (ENCCs), represents the neuronal innervation of the intestine. Compromised ENCC migration can lead to Hirschsprung disease, which is characterized by an aganglionic distal bowel. During the craniocaudal migration of ENCCs along the gut, we find that their proliferation is greatest as the ENCC wavefront passes through the ceca, a pair of pouches at the midgut-hindgut junction in avian intestine. Removal of the ceca leads to hindgut aganglionosis, suggesting that they are required for ENS development. Comparative transcriptome profiling of the cecal buds compared with the interceca region shows that the non-canonical Wnt signaling pathway is preferentially expressed within the ceca. Specifically, WNT11 is highly expressed, as confirmed by RNA in situ hybridization, leading us to hypothesize that cecal expression of WNT11 is important for ENCC colonization of the hindgut. Organ cultures using embryonic day 6 avian intestine show that WNT11 inhibits enteric neuronal differentiation. These results reveal an essential role for the ceca during hindgut ENS formation and highlight an important function for non-canonical Wnt signaling in regulating ENCC differentiation.

Bone Marrow Stem Cells Derived from Nerves Have Neurogenic Properties and Potential Utility for Regenerative Therapy.

Neurons and glia of the peripheral nervous system are derived from progenitor cell populations, originating from embryonic neural crest. The neural crest and vasculature are intimately associated during embryonic development and in the mature central nervous system, in which they form a neurovascular unit comprised of neurons, glia, pericytes, and vascular endothelial cells that play important roles in health and disease. Our group and others have previously reported that postnatal populations of stem cells originating from glia or Schwann cells possess neural stem cell qualities, including rapid proliferation and differentiation into mature glia and neurons. Bone marrow receives sensory and sympathetic innervation from the peripheral nervous system and is known to contain myelinating and unmyelinating Schwann cells. Herein, we describe a population of neural crest-derived Schwann cells residing in a neurovascular niche of bone marrow in association with nerve fibers. These Schwann cells can be isolated and expanded. They demonstrate plasticity in vitro, generating neural stem cells that exhibit neurogenic potential and form neural networks within the enteric nervous system in vivo following transplantation to the intestine. These cells represent a novel source of autologous neural stem cells for the treatment of neurointestinal disorders.

Essential Role of BMP4 Signaling in the Avian Ceca in Colorectal Enteric Nervous System Development.

The enteric nervous system (ENS) is principally derived from vagal neural crest cells that migrate caudally along the entire length of the gastrointestinal tract, giving rise to neurons and glial cells in two ganglionated plexuses. Incomplete migration of enteric neural crest-derived cells (ENCDC) leads to Hirschsprung disease, a congenital disorder characterized by the absence of enteric ganglia along variable lengths of the colorectum. Our previous work strongly supported the essential role of the avian ceca, present at the junction of the midgut and hindgut, in hindgut ENS development, since ablation of the cecal buds led to incomplete ENCDC colonization of the hindgut. In situ hybridization shows bone morphogenetic protein-4 (BMP4) is highly expressed in the cecal mesenchyme, leading us to hypothesize that cecal BMP4 is required for hindgut ENS development. To test this, we modulated BMP4 activity using embryonic intestinal organ culture techniques and retroviral infection. We show that overexpression or inhibition of BMP4 in the ceca disrupts hindgut ENS development, with GDNF playing an important regulatory role. Our results suggest that these two important signaling pathways are required for normal ENCDC migration and enteric ganglion formation in the developing hindgut ENS.

Neuroinflammation as an etiological trigger for depression comorbid with inflammatory bowel disease.

Patients with inflammatory bowel disease (IBD) suffer from depression at higher rates than the general population. An etiological trigger of depressive symptoms is theorised to be inflammation within the central nervous system. It is believed that heightened intestinal inflammation and dysfunction of the enteric nervous system (ENS) contribute to impaired intestinal permeability, which facilitates the translocation of intestinal enterotoxins into the blood circulation. Consequently, these may compromise the immunological and physiological functioning of distant non-intestinal tissues such as the brain. In vivo models of colitis provide evidence of increased blood-brain barrier permeability and enhanced central nervous system (CNS) immune activity triggered by intestinal enterotoxins and blood-borne inflammatory mediators. Understanding the immunological, physiological, and structural changes associated with IBD and neuroinflammation may aid in the development of more tailored and suitable pharmaceutical treatment for IBD-associated depression.

Mesenchymal stem cell treatment for enteric neuropathy in the Winnie mouse model of spontaneous chronic colitis.

Inflammatory bowel disease (IBD) is a chronic gut inflammation with periods of acute flares and remission. Beneficial effects of a single dose of mesenchymal stem cell (MSC)-based treatment have been demonstrated in acute models of colitis. No studies investigated therapeutic effects of MSCs for the attenuation of enteric neuropathy in a chronic model of colitis. The short and long-term effects of MSC treatment in modulating inflammation and damage to the enteric nervous system (ENS) were studied in the Winnie mouse model of spontaneous chronic colitis highly representative of human IBD. Winnie mice received a single dose of either 1 × 106 human bone marrow-derived MSCs or 100µL PBS by intracolonic enema. C57BL/6 mice received 100µL PBS. Colon tissues were collected at 3 and 60 days post MSC administration to evaluate the short-term and long-term effects of MSCs on inflammation and enteric neuropathy by histological and immunohistochemical analyses. In a separate set of experiments, multiple treatments with 4 × 106 and 2 × 106 MSCs were performed and tissue collected at 3 days post treatment. Chronic intestinal inflammation in Winnie mice was associated with persistent diarrhea, perianal bleeding, morphological changes, and immune cell infiltration in the colon. Significant changes to the ENS, including impairment of cholinergic, noradrenergic and sensory innervation, and myenteric neuronal loss were prominent in Winnie mice. Treatment with a single dose of bone marrow-derived MSCs was ineffective in attenuating chronic inflammation and enteric neuropathy in Winnie.

Enteric mesenchymal cells support the growth of postnatal enteric neural stem cells.

Interplay between embryonic enteric neural stem cells (ENSCs) and enteric mesenchymal cells (EMCs) in the embryonic gut is essential for normal development of the enteric nervous system. Disruption of these interactions underlies the pathogenesis of intestinal aganglionosis in Hirschsprung disease (HSCR). ENSC therapy has been proposed as a possible treatment for HSCR, but whether the survival and development of postnatal-derived ENSCs similarly rely on signals from the mesenchymal environment is unknown and has important implications for developing protocols to expand ENSCs for cell transplantation therapy. Enteric neural crest-derived cells (ENCDCs) and EMCs were cultured from the small intestine of Wnt1-Rosa26-tdTomato mice. EMCs promoted the expansion of ENCDCs 9.5-fold by inducing ENSC properties, including expression of Nes, Sox10, Sox2, and Ngfr. EMCs enhanced the neurosphere-forming ability of ENCDCs, and this persisted after withdrawal of the EMCs. These effects were mediated by paracrine factors and several ligands known to support neural stem cells were identified in EMCs. Using the optimized expansion procedures, neurospheres were generated from small intestine of the Ednrb-/- mouse model of HSCR. These ENSCs had similar proliferative and migratory capacity to Ednrb+/+ ENSCs, albeit neurospheres contained fewer neurons. ENSCs derived from Ednrb-/- mice generated functional neurons with similar calcium responses to Ednrb+/+ ENSCs and survived after transplantation into the aganglionic colon of Ednrb-/- recipients. EMCs act as supporting cells to ENSCs postnatally via an array of synergistically acting paracrine signaling factors. These properties can be leveraged to expand autologous ENSCs from patients with HSCR mutations for therapeutic application.

Molecular Targets to Alleviate Enteric Neuropathy and Gastrointestinal Dysfunction.

Enteric neuropathy underlies long-term gastrointestinal (GI) dysfunction associated with several pathological conditions. Our previous studies have demonstrated that structural and functional changes in the enteric nervous system (ENS) result in persistent alterations of intestinal functions long after the acute insult. These changes lead to aberrant immune response and chronic dysregulation of the epithelial barrier. Damage to the ENS is prognostic of disease progression and plays an important role in the recurrence of clinical manifestations. This suggests that the ENS is a viable therapeutic target to alleviate chronic intestinal dysfunction. Our recent studies in preclinical animal models have progressed into the development of novel therapeutic strategies for the treatment of enteric neuropathy in various chronic GI disorders. We have tested the anti-inflammatory and neuroprotective efficacy of novel compounds targeting specific molecular pathways. Ex vivo studies in human tissues freshly collected after resection surgeries provide an understanding of the molecular mechanisms involved in enteric neuropathy. In vivo treatments in animal models provide data on the efficacy and the mechanisms of actions of the novel compounds and their combinations with clinically used therapies. These novel findings provide avenues for the development of safe, cost-effective, and highly efficacious treatments of GI disorders.

Optogenetic Activation of Cholinergic Enteric Neurons Reduces Inflammation in Experimental Colitis.

BACKGROUND & AIMS: Intestinal inflammation is associated with loss of enteric cholinergic neurons. Given the systemic anti-inflammatory role of cholinergic innervation, we hypothesized that enteric cholinergic neurons similarly possess anti-inflammatory properties and may represent a novel target to treat inflammatory bowel disease. METHODS: Mice were fed 2.5% dextran sodium sulfate (DSS) for 7 days to induce colitis. Cholinergic enteric neurons, which express choline acetyltransferase (ChAT), were focally ablated in the midcolon of ChAT::Cre;R26-iDTR mice by local injection of diphtheria toxin before colitis induction. Activation of enteric cholinergic neurons was achieved using ChAT::Cre;R26-ChR2 mice, in which ChAT+ neurons express channelrhodopsin-2, with daily blue light stimulation delivered via an intracolonic probe during the 7 days of DSS treatment. Colitis severity, ENS structure, and smooth muscle contractility were assessed by histology, immunohistochemistry, quantitative polymerase chain reaction, organ bath, and electromyography. In vitro studies assessed the anti-inflammatory role of enteric cholinergic neurons on cultured muscularis macrophages. RESULTS: Ablation of ChAT+ neurons in DSS-treated mice exacerbated colitis, as measured by weight loss, colon shortening, histologic inflammation, and CD45+ cell infiltration, and led to colonic dysmotility. Conversely, optogenetic activation of enteric cholinergic neurons improved colitis, preserved smooth muscle contractility, protected against loss of cholinergic neurons, and reduced proinflammatory cytokine production. Both acetylcholine and optogenetic cholinergic neuron activation in vitro reduced proinflammatory cytokine expression in lipopolysaccharide-stimulated muscularis macrophages. CONCLUSIONS: These findings show that enteric cholinergic neurons have an anti-inflammatory role in the colon and should be explored as a potential inflammatory bowel disease treatment.

Bone marrow-derived mesenchymal stem cells mitigate chronic colitis and enteric neuropathy via anti-inflammatory and anti-oxidative mechanisms.

Current treatments for inflammatory bowel disease (IBD) are often inadequate due to limited efficacy and toxicity, leading to surgical resection in refractory cases. IBD's broad and complex pathogenesis involving the immune system, enteric nervous system, microbiome, and oxidative stress requires more effective therapeutic strategies. In this study, we investigated the therapeutic potential of bone marrow-derived mesenchymal stem cell (BM-MSC) treatments in spontaneous chronic colitis using the Winnie mouse model which closely replicates the presentation and inflammatory profile of ulcerative colitis. The 14-day BM-MSC treatment regimen reduced the severity of colitis, leading to the attenuation of diarrheal symptoms and recovery in body mass. Morphological and histological abnormalities in the colon were also alleviated. Transcriptomic analysis demonstrated that BM-MSC treatment led to alterations in gene expression profiles primarily downregulating genes related to inflammation, including pro-inflammatory cytokines, chemokines and other biomarkers of inflammation. Further evaluation of immune cell populations using immunohistochemistry revealed a reduction in leukocyte infiltration upon BM-MSC treatment. Notably, enteric neuronal gene signatures were the most impacted by BM-MSC treatment, which correlated with the restoration of neuronal density in the myenteric ganglia. Moreover, BM-MSCs exhibited neuroprotective effects against oxidative stress-induced neuronal loss through antioxidant mechanisms, including the reduction of mitochondrial-derived superoxide and attenuation of oxidative stress-induced HMGB1 translocation, potentially relying on MSC-derived SOD1. These findings suggest that BM-MSCs hold promise as a therapeutic intervention to mitigate chronic colitis by exerting anti-inflammatory effects and protecting the enteric nervous system from oxidative stress-induced damage.

Agrin Inhibition in Enteric Neural Stem Cells Enhances Their Migration Following Colonic Transplantation.

Regenerative cell therapy to replenish the missing neurons and glia in the aganglionic segment of Hirschsprung disease represents a promising treatment option. However, the success of cell therapies for this condition are hindered by poor migration of the transplanted cells. This limitation is in part due to a markedly less permissive extracellular environment in the postnatal gut than that of the embryo. Coordinated interactions between enteric neural crest-derived cells (ENCDCs) and their local environment drive migration along the embryonic gut during development of the enteric nervous system. Modifying transplanted cells, or the postnatal extracellular environment, to better recapitulate embryonic ENCDC migration could be leveraged to improve the engraftment and coverage of stem cell transplants. We compared the transcriptomes of ENCDCs from the embryonic intestine to that of postnatal-derived neurospheres and identified 89 extracellular matrix (ECM)-associated genes that are differentially expressed. Agrin, a heparin sulfate proteoglycan with a known inhibitory effect on ENCDC migration, was highly over-expressed by postnatal-derived neurospheres. Using a function-blocking antibody and a shRNA-expressing lentivirus, we show that inhibiting agrin promotes ENCDC migration in vitro and following cell transplantation ex vivo and in vivo. This enhanced migration is associated with an increased proportion of GFAP + cells, whose migration is especially enhanced.

Autologous cell transplantation for treatment of colorectal aganglionosis in mice.

Neurointestinal diseases cause significant morbidity and effective treatments are lacking. This study aimes to test the feasibility of transplanting autologous enteric neural stem cells (ENSCs) to rescue the enteric nervous system (ENS) in a model of colonic aganglionosis. ENSCs are isolated from a segment of small intestine from Wnt1::Cre;R26iDTR mice in which focal colonic aganglionosis is simultaneously created by diphtheria toxin injection. Autologous ENSCs are isolated, expanded, labeled with lentiviral-GFP, and transplanted into the aganglionic segment in vivo. ENSCs differentiate into neurons and glia, cluster to form neo-ganglia, and restore colonic contractile activity as shown by electrical field stimulation and optogenetics. Using a non-lethal model of colonic aganglionosis, our results demonstrate the potential of autologous ENSC therapy to improve functional outcomes in neurointestinal disease, laying the groundwork for clinical application of this regenerative cell-based approach.

The Oxidative Stress and Nervous Distress Connection in Gastrointestinal Disorders.

Oxidative stress is increasingly recognized as a central player in a range of gastrointestinal (GI) disorders, as well as complications stemming from therapeutic interventions. This article presents an overview of the mechanisms of oxidative stress in GI conditions and highlights a link between oxidative insult and disruption to the enteric nervous system (ENS), which controls GI functions. The dysfunction of the ENS is characteristic of a spectrum of disorders, including neurointestinal diseases and conditions such as inflammatory bowel disease (IBD), diabetic gastroparesis, and chemotherapy-induced GI side effects. Neurons in the ENS, while essential for normal gut function, appear particularly vulnerable to oxidative damage. Mechanistically, oxidative stress in enteric neurons can result from intrinsic nitrosative injury, mitochondrial dysfunction, or inflammation-related pathways. Although antioxidant-based therapies have shown limited efficacy, recognizing the multifaceted role of oxidative stress in GI diseases offers a promising avenue for future interventions. This comprehensive review summarizes the literature to date implicating oxidative stress as a critical player in the pathophysiology of GI disorders, with a focus on its role in ENS injury and dysfunction, and highlights opportunities for the development of targeted therapeutics for these diseases.

Oxaliplatin-Induced Damage to the Gastric Innervation: Role in Nausea and Vomiting.

Nausea and vomiting are common gastrointestinal side effects of oxaliplatin chemotherapy used for the treatment of colorectal cancer. However, the mechanism underlying oxaliplatin-induced nausea and vomiting is unknown. The stomach is involved in the emetic reflex but no study investigated the effects of oxaliplatin treatment on the stomach. In this study, the in vivo effects of oxaliplatin treatment on eating behaviour, stomach content, intrinsic gastric neuronal population, extrinsic innervation to the stomach, levels of mucosal serotonin (5-hydroxytryptamine, 5-HT), and parasympathetic vagal efferent nerve activity were analysed. Chronic systemic oxaliplatin treatment in mice resulted in pica, indicated by increased kaolin consumption and a reduction in body weight. Oxaliplatin treatment significantly increased the stomach weight and content. The total number of myenteric and nitric oxide synthase-immunoreactive neurons as well as the density of sympathetic, parasympathetic, and sensory fibres in the stomach were decreased significantly with oxaliplatin treatment. Oxaliplatin treatment significantly increased the levels in mucosal 5-HT and the number of enterochromaffin-like cells. Chronic oxaliplatin treatment also caused a significant increase in the vagal efferent nerve activity. The findings of this study indicate that oxaliplatin exposure has adverse effects on multiple components of gastric innervation, which could be responsible for pica and gastric dysmotility.

APE1/Ref-1 as a Therapeutic Target for Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is characterized by chronic relapsing inflammation of the gastrointestinal tract. The prevalence of IBD is increasing with approximately 4.9 million cases reported worldwide. Current therapies are limited due to the severity of side effects and long-term toxicity, therefore, the development of novel IBD treatments is necessitated. Recent findings support apurinic/apyrimidinic endonuclease 1/reduction-oxidation factor 1 (APE1/Ref-1) as a target in many pathological conditions, including inflammatory diseases, where APE1/Ref-1 regulation of crucial transcription factors impacts significant pathways. Thus, a potential target for a novel IBD therapy is the redox activity of the multifunctional protein APE1/Ref-1. This review elaborates on the status of conventional IBD treatments, the role of an APE1/Ref-1 in intestinal inflammation, and the potential of a small molecule inhibitor of APE1/Ref-1 redox activity to modulate inflammation, oxidative stress response, and enteric neuronal damage in IBD.

Differential Gene Expression of Checkpoint Markers and Cancer Markers in Mouse Models of Spontaneous Chronic Colitis.

The presence of checkpoint markers in cancer cells aids in immune escape. The identification of checkpoint markers and early cancer markers is of utmost importance to gain clarity regarding the relationship between colitis and progressive inflammation leading to cancer. Herein, the gene expression levels of checkpoint makers, cancer-related pathways, and cancer genes in colon tissues of mouse models of chronic colitis (Winnie and Winnie-Prolapse mice) using next-generation sequencing are determined. Winnie mice are a result of a Muc2 missense mutation. The identification of such genes and their subsequent expression and role at the protein level would enable novel markers for the early diagnosis of cancer in IBD patients. The differentially expressed genes in the colonic transcriptome were analysed based on the Kyoto Encyclopedia of Genes and Genomes pathway. The expression of several oncogenes is associated with the severity of IBD, with Winnie-Prolapse mice expressing a large number of key genes associated with development of cancer. This research presents a number of new targets to evaluate for the development of biomarkers and therapeutics.

Oxidative Stress and Neural Dysfunction in Gastrointestinal Diseases: Can Stem Cells Offer a Solution?

Oxidative stress is involved in many gastrointestinal (GI) disorders as either the primary pathogenesis (radiation, chemotherapy, toxicity, ischemia-reperfusion) or a secondary driving force of disease progression (inflammation and diabetes). The GI tract is innervated intrinsically by the enteric nervous system (ENS) with a diverse role in maintaining gut homeostasis and GI motility. Complications in the physiological functioning of the ENS results in GI dysfunction that can result in debilitating sequelae from dysmotility greatly impacting quality of life and leading to potentially fatal complications. Therapeutics to remedy either oxidative stress or enteric neuronal dysfunction are severely limited, resulting in a critical gap in clinical care for GI disease and neurointestinal complications. Stem cell therapies have shown great promise in the treatment of several gut disorders via mechanisms including cell regeneration, anti-inflammatory activity, providing trophic support, and emerging evidence of antioxidant and neuroprotective functions. The potential of mesenchymal stem cell (MSC) therapies and recent evidence of their antioxidant and neuroprotective activity in several GI conditions are discussed. Finally, future therapeutic aspects of stem cell-based tools for combatting oxidative stress and enteric neuropathies in GI disease are considered.

Single-cell multiome sequencing clarifies enteric glial diversity and identifies an intraganglionic population poised for neurogenesis.

The enteric nervous system (ENS) consists of glial cells (EGCs) and neurons derived from neural crest precursors. EGCs retain capacity for large-scale neurogenesis in culture, and in vivo lineage tracing has identified neurons derived from glial cells in response to inflammation. We thus hypothesize that EGCs possess a chromatin structure poised for neurogenesis. We use single-cell multiome sequencing to simultaneously assess transcription and chromatin accessibility in EGCs undergoing spontaneous neurogenesis in culture, as well as small intestine myenteric plexus EGCs. Cultured EGCs maintain open chromatin at genomic loci accessible in neurons, and neurogenesis from EGCs involves dynamic chromatin rearrangements with a net decrease in accessible chromatin. A subset of in vivo EGCs, highly enriched within the myenteric ganglia and that persist into adulthood, have a gene expression program and chromatin state consistent with neurogenic potential. These results clarify the mechanisms underlying EGC potential for neuronal fate transition.

Site-specific pathophysiology in a neonatal mouse model of gastroparesis.

BACKGROUND: Early-life events impact maturation of the gut microbiome, enteric nervous system, and gastrointestinal motility. We examined three regions of gastric tissue to determine how maternal separation and gut microbes influence the structure and motor function of specific regions of the neonatal mouse stomach. METHODS: Germ-free and conventionally housed C57BL/6J mouse pups underwent timed maternal separation (TmSep) or nursed uninterrupted (controls) until 14 days of life. We assessed gastric emptying by quantifying the progression of gavaged fluorescein isothiocyanate (FITC)-dextran. With isolated rings of forestomach, corpus, and antrum, we measured tone and contractility by force transduction, gastric wall thickness by light microscopy, and myenteric plexus neurochemistry by whole-mount immunostaining. KEY RESULTS: Regional gastric sampling revealed site-specific differences in contractile patterns and myenteric plexus structure. In neonatal mice, TmSep prolonged gastric emptying. In the forestomach, TmSep increased contractile responses to carbachol, decreased muscularis externa and mucosa thickness, and increased the relative proportion of myenteric plexus nNOS+ neurons. Germ-free conditions did not appreciably alter the structure or function of the neonatal mouse stomach and did not impact the changes caused by TmSep. CONCLUSIONS AND INFERENCES: A regional sampling approach facilitates site-specific investigations of murine gastric motor physiology and histology to identify site-specific alterations that may impact gastrointestinal function. Delayed gastric emptying in TmSep is associated with a thinner muscle wall, exaggerated cholinergic contractile responses, and increased proportions of inhibitory myenteric plexus nNOS+ neurons in the forestomach. Gut microbes do not profoundly affect the development of the neonatal mouse stomach or the gastric pathophysiology that results from TmSep.

Transplanted ENSCs form functional connections with intestinal smooth muscle and restore colonic motility in nNOS-deficient mice.

BACKGROUND: Enteric neuropathies, which result from abnormalities of the enteric nervous system, are associated with significant morbidity and high health-care costs, but current treatments are unsatisfactory. Cell-based therapy offers an innovative approach to replace the absent or abnormal enteric neurons and thereby restore gut function. METHODS: Enteric neuronal stem cells (ENSCs) were isolated from the gastrointestinal tract of Wnt1-Cre;R26tdTomato mice and generated neurospheres (NS). NS transplants were performed via injection into the mid-colon mesenchyme of nNOS-/- mouse, a model of colonic dysmotility, using either 1 (n = 12) or 3 (n = 12) injections (30 NS per injection) targeted longitudinally 1-2 mm apart. Functional outcomes were assessed up to 6 weeks later using electromyography (EMG), electrical field stimulation (EFS), optogenetics, and by measuring colorectal motility. RESULTS: Transplanted ENSCs formed nitrergic neurons in the nNOS-/- recipient colon. Multiple injections of ENSCs resulted in a significantly larger area of coverage compared to single injection alone and were associated with a marked improvement in colonic function, demonstrated by (1) increased colonic muscle activity by EMG recording, (2) faster rectal bead expulsion, and (3) increased fecal pellet output in vivo. Organ bath studies revealed direct neuromuscular communication by optogenetic stimulation of channelrhodopsin-expressing ENSCs and restoration of smooth muscle relaxation in response to EFS. CONCLUSIONS: These results demonstrate that transplanted ENSCs can form effective neuromuscular connections and improve colonic motor function in a model of colonic dysmotility, and additionally reveal that multiple sites of cell delivery led to an improved response, paving the way for optimized clinical trial design.