Search

The goal of this study was to determine if transplantation of enteric neural stem cells (ENSCs) can rescue the enteric nervous system, restore gut motility, reduce colonic inflammation, and improve survival in the Ednrb-KO mouse model of Hirschsprung disease (HSCR). ENSCs were isolated from mouse intestine, expanded to form neurospheres, and microinjected into the colons of recipient Ednrb-KO mice. Transplanted ENSCs were identified in recipient colons as cell clusters in neo-ganglia. Immunohistochemical evaluation demonstrated extensive cell migration away from the sites of cell delivery and across the muscle layers. Electrical field stimulation and optogenetics showed significantly enhanced contractile activity of aganglionic colonic smooth muscle following ENSC transplantation and confirmed functional neuromuscular integration of the transplanted ENSC-derived neurons. ENSC injection also partially restored the colonic migrating motor complex. Histological examination revealed a significant reduction in inflammation in ENSC-transplanted aganglionic recipient colon compared with that of sham-operated mice. Interestingly, mice that received cell transplant also had prolonged survival compared with controls. This study demonstrates that ENSC transplantation can improve outcomes in HSCR by restoring gut motility and reducing the severity of Hirschsprung-associated enterocolitis, the leading cause of death in human HSCR.

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.

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.

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.

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.

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.

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.

The enteric nervous system (ENS) is an extensive network of neurons and glia within the wall of the gastrointestinal (GI) tract that regulates many essential GI functions. Consequently, disorders of the ENS due to developmental defects, inflammation, infection, or age-associated neurodegeneration lead to serious neurointestinal diseases. Despite the prevalence and severity of these diseases, effective treatments are lacking as they fail to directly address the underlying pathology. Neuronal stem cell therapy represents a promising approach to treating diseases of the ENS by replacing the absent or injured neurons, and an autologous source of stem cells would be optimal by obviating the need for immunosuppression. We utilized the swine model to address key questions concerning cell isolation, delivery, engraftment, and fate in a large animal relevant to human therapy. We successfully isolated neural stem cells from a segment of small intestine resected from 1-month-old swine. Enteric neuronal stem cells (ENSCs) were expanded as neurospheres that grew optimally in low-oxygen (5%) culture conditions. Enteric neuronal stem cells were labeled by lentiviral green fluorescent protein (GFP) transduction, then transplanted into the same swine from which they had been harvested. Endoscopic ultrasound was then utilized to deliver the ENSCs (10,000-30,000 neurospheres per animal) into the rectal wall. At 10 and 28 days following injection, autologously derived ENSCs were found to have engrafted within rectal wall, with neuroglial differentiation and no evidence of ectopic spreading. These findings strongly support the feasibility of autologous cell isolation and delivery using a clinically useful and minimally invasive technique, bringing us closer to first-in-human ENSC therapy for neurointestinal diseases.

Cell therapy offers the potential to replace the missing enteric nervous system (ENS) in patients with Hirschsprung disease (HSCR) and to restore gut function. The Schwann cell (SC) lineage has been shown to generate enteric neurons pre- and post-natally. Here, we aimed to isolate SCs from the aganglionic segment of HSCR and to determine their potential to restore motility in the aganglionic colon. Proteolipid protein 1 (PLP1) expressing SCs were isolated from the extrinsic nerve fibers present in the aganglionic segment of postnatal mice and patients with HSCR. Following 7-10 days of in vitro expansion, HSCR-derived SCs were transplanted into the aganglionic mouse colon ex vivo and in vivo. Successful engraftment and neuronal differentiation were confirmed immunohistochemically and calcium activity of transplanted cells was demonstrated by live cell imaging. Organ bath studies revealed the restoration of motor function in the recipient aganglionic smooth muscle. These results show that SCs isolated from the aganglionic segment of HSCR mouse can generate functional neurons within the aganglionic gut environment and restore the neuromuscular activity of recipient mouse colon. We conclude that HSCR-derived SCs represent a potential autologous source of neural progenitor cells for regenerative therapy in HSCR.

Hirschsprung disease is most often characterized by aganglionosis limited to the distal colon and rectum, and mice lacking the Endothelin receptor type B (Ednrb) faithfully recapitulate this phenotype. However, despite the presence of enteric ganglia in the small intestine, both human patients and Ednrb-/- mice suffer from dysmotility and altered gastrointestinal function, thus raising the possibility of enteric nervous system (ENS) abnormalities proximal to the aganglionic region. We undertook the present study to determine whether abnormalities with the ENS in ganglionated regions may account for abnormal gastrointestinal function. We performed single-cell RNA sequencing on ENS cells from the small intestine of Ednrb-/- mice and compared the results to a published single-cell dataset. Our results identified a missing population of neurons marked by the enzyme Gad2, which catalyzes the production of γ-Aminobutyric acid (GABA), in the small intestine of Ednrb-/- animals. This result was confirmed by immunostaining enteric ganglia from Ednrb-/- mice and their wild-type littermates. These data show for the first time that ganglionated regions of the Hirschsprung gut lack a neuronal subpopulation, which may explain the persistent gastrointestinal dysfunction after surgical correction of Hirschsprung disease.

With a steadily aging population there is an increasing prevalence of neurological disorders. Given the lack of effective treatment strategies and a limited ability for the central nervous system (CNS) to regenerate endogenously, there is a critical need to better understand exogenous strategies for nervous system repair. Stem cell therapy offers a promising approach to promote the repair of neurologic tissue and function, however studies to date have been limited by various factors including challenges in harvesting donor cells from the CNS, ethical concerns regarding use of embryonic or fetal tissue, tumorigenic potential of induced pluripotent stem cells, and immune-mediated rejection of non-autologous cell sources. Here we review and propose two alternative sources of autologous cells derived from the peripheral nervous system (PNS) for CNS repair: enteric neuronal stem cells (ENSCs) and neural crest-derived Schwann cells found in subcutaneous adipose tissue (termed SAT-NSCs). ENSCs can be successfully isolated from the postnatal enteric nervous system, propagated in vitro, and transplanted successfully into models of CNS injury via both direct intracerebral injection and systemic tail vein injection. Similarly, SAT-NSCs can be readily isolated from both human and mouse adipose tissue and, although not yet utilized in models of CNS injury, have successfully been transplanted and restored function in models of colonic aganglionosis and gastroparesis. These unique sources of PNS-derived autologous cells offer an exciting option for stem cell therapies for the CNS as they have proven neurogenic potential and eliminate concerns around tumorigenic risk, ethical considerations, and immune-mediated rejection.