CXCR4 and CXCL12 signaling regulates the development of extrinsic innervation to the colorectum.

The gastrointestinal tract is innervated by an intrinsic neuronal network, known as the enteric nervous system (ENS), and by extrinsic axons arising from peripheral ganglia. The nerve of Remak (NoR) is an avian-specific sacral neural crest-derived ganglionated structure that extends from the cloaca to the proximal midgut and, similar to the pelvic plexus, provides extrinsic innervation to the distal intestine. The molecular mechanisms controlling extrinsic nerve fiber growth into the gut is unknown. In vertebrates, CXCR4, a cell-surface receptor for the CXCL12 chemokine, regulates migration of neural crest cells and axon pathfinding. We have employed chimeric tissue recombinations and organ culture assays to study the role of CXCR4 and CXCL12 molecules in the development of colorectal innervation. CXCR4 is specifically expressed in nerve fibers arising from the NoR and pelvic plexus, while CXCL12 is localized to the hindgut mesenchyme and enteric ganglia. Overexpression of CXCL12 results in significantly enhanced axonal projections to the gut from the NoR, while CXCR4 inhibition disrupts nerve fiber extension, supporting a previously unreported role for CXCR4 and CXCL12 signaling in extrinsic innervation of the colorectum.

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.

RET overactivation leads to concurrent Hirschsprung disease and intestinal ganglioneuromas.

Appropriately balanced RET signaling is of crucial importance during embryonic neural crest cell migration, proliferation and differentiation. RET deficiency, for example, leads to intestinal aganglionosis (Hirschsprung disease), whereas overactive RET can lead to multiple endocrine neoplasia (MEN) syndromes. Some RET mutations are associated with both intestinal aganglionosis and MEN-associated tumors. This seemingly paradoxical occurrence has led to speculation of a 'Janus mutation' in RET that causes overactivation or impairment of RET activity depending on the cellular context. Using an intestinal catenary culture system to test the effects of GDNF-mediated RET activation, we demonstrate the concurrent development of distal colonic aganglionosis and intestinal ganglioneuromas. Interestingly, the tumors induced by GDNF stimulation contain enteric neuronal progenitors capable of reconstituting an enteric nervous system when transplanted into a normal developmental environment. These results suggest that a Janus mutation may not be required to explain co-existing Hirschsprung disease and MEN-associated tumors, but rather that RET overstimulation alone is enough to cause both phenotypes. The results also suggest that reprogramming tumor cells toward non-pathological fates may represent a possible therapeutic avenue for MEN-associated neoplasms.

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.

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.

Collagen 18 and agrin are secreted by neural crest cells to remodel their microenvironment and regulate their migration during enteric nervous system development.

The enteric nervous system (ENS) arises from neural crest cells that migrate, proliferate, and differentiate into enteric neurons and glia within the intestinal wall. Many extracellular matrix (ECM) components are present in the embryonic gut, but their role in regulating ENS development is largely unknown. Here, we identify heparan sulfate proteoglycan proteins, including collagen XVIII (Col18) and agrin, as important regulators of enteric neural crest-derived cell (ENCDC) development. In developing avian hindgut, Col18 is expressed at the ENCDC wavefront, while agrin expression occurs later. Both proteins are normally present around enteric ganglia, but are absent in aganglionic gut. Using chick-mouse intestinal chimeras and enteric neurospheres, we show that vagal- and sacral-derived ENCDCs from both species secrete Col18 and agrin. Whereas glia express Col18 and agrin, enteric neurons only express the latter. Functional studies demonstrate that Col18 is permissive whereas agrin is strongly inhibitory to ENCDC migration, consistent with the timing of their expression during ENS development. We conclude that ENCDCs govern their own migration by actively remodeling their microenvironment through secretion of ECM proteins.

Sonic hedgehog controls enteric nervous system development by patterning the extracellular matrix.

The enteric nervous system (ENS) develops from neural crest cells that migrate along the intestine, differentiate into neurons and glia, and pattern into two plexuses within the gut wall. Inductive interactions between epithelium and mesenchyme regulate gut development, but the influence of these interactions on ENS development is unknown. Epithelial-mesenchymal recombinations were constructed using avian hindgut mesenchyme and non-intestinal epithelium from the bursa of Fabricius. These recombinations led to abnormally large and ectopically positioned ganglia. We hypothesized that sonic hedgehog (Shh), a secreted intestinal epithelial protein not expressed in the bursa, mediates this effect. Inhibition of Shh signaling, by addition of cyclopamine or a function-blocking antibody, resulted in large, ectopic ganglia adjacent to the epithelium. Shh overexpression, achieved in ovo using Shh-encoding retrovirus and in organ culture using recombinant protein, led to intestinal aganglionosis. Shh strongly induced the expression of versican and collagen type IX, whereas cyclopamine reduced expression of these chondroitin sulfate proteoglycans that are known to be inhibitory to neural crest cell migration. Shh also inhibited enteric neural crest-derived cell (ENCC) proliferation, promoted neuronal differentiation, and reduced expression of Gdnf, a key regulator of ENS formation. Ptc1 and Ptc2 were not expressed by ENCCs, and migration of isolated ENCCs was not inhibited by Shh protein. These results suggest that epithelial-derived Shh acts indirectly on the developing ENS by regulating the composition of the intestinal microenvironment.

Sleep condition and cognitive decline in Japanese community-dwelling older people: Data from a 4-year longitudinal study.

This study examined whether sleep duration and excessive daytime sleepiness (EDS) are related to cognitive decline among community-dwelling older adults with intact cognition at baseline, using 4-year longitudinal data. A total of 3,151 community-dwelling older individuals aged ≥65 years were studied. They were assessed for cognitive function, including memory, attention, executive function and processing speed. Cognitive impairment was defined based on a score >1.5 standard deviations below the age- and education-specific mean. Cognitive decline was defined in one or more cognitive tests at follow-up. Self-reported sleep duration (short, ≤6.0 hr; medium, 6.1-8.9 hr; long, ≥9.0 hr) and EDS at first-wave examination were assessed and logistic regression analyses were used to examine the associations of sleep duration and EDS with cognitive status at second-wave examination. The incidence of cognitive decline differed significantly among the sleep-duration groups (short, 15.9%; medium, 11.9%; long, 20.1%; p = 0.001). The prevalence of having EDS was 13.1%, which was associated with a higher rate of cognitive decline than having no EDS (18.9% vs. 12.5%, p = 0.004). Long sleep duration compared with medium sleep duration (OR, 1.50; 95% CI, 1.05-2.13) and EDS (1.43; 1.01-2.03) independently impacted the incidence of cognitive decline. The results were similar after multiple imputations (long, 1.68, 1.12-2.52; EDS, 1.55, 1.05-2.29). In conclusion, our study revealed that both long sleep duration and EDS were independent risk factors associated with cognitive decline after 4 years among older adults.

Effects of Driving Skill Training on Safe Driving in Older Adults with Mild Cognitive Impairment.

BACKGROUND: Driving cessation is strongly associated with adverse health outcomes in the older adults. Although there were numerous documentations of driving rehabilitation in disabled adults, the effects of interventions on safe driving were not clear in older adults with cognitive impairment who had low driving skills. OBJECTIVE: This randomized controlled trial was designed to determine whether a safe driving skill program consisting of classroom and on-road training could enhance driving performance of older drivers with cognitive impairment in Japan. METHODS: A total of 160 community-living older drivers participated in the randomized controlled trial with blinded endpoint assessment. Participants randomized to intervention underwent 10 1-h classroom sessions and 10 1-h on-road sessions focused on common problem areas of older drivers. Controls received 1 classroom education. On-road driving performance was assessed by certified driving school instructors in a driving school. The participants carried out dynamic vision and cognitive performance tests. RESULTS: One hundred and forty-six (intervention group, n = 71) subjects completed the 3-month follow-up. Mean adherence to classroom-based vision training and driving simulator training and on-road training programs, including the 71 participants, was 99.0 ± 6.4 and 99.0 ± 7.2%, respectively. Regarding the safe driving skill score, there were group × time interactions (p < 0.01) indicating benefits of the intervention over time. Although there were no significant group × time interactions in cognitive tests, dynamic vision showed group × time interactions (p < 0.01). CONCLUSION: The driving skill program significantly improved safe driving performance in older adults with cognitive impairment who were at a potentially high risk of a car accident.

Dianionic Phase-Transfer Catalyst for Asymmetric Fluoro-cyclization.

Inspired by the dicationic nature of the electrophilic fluorinating reagent, Selectfluor (1), we rationally designed a series of dicarboxylic acid precatalysts (2), which, when deprotonated, act as anionic phase-transfer catalysts for asymmetric fluorination of alkenes. Among them, 2a having the shortest linker moiety efficiently catalyzed unprecedented 6-endo-fluoro-cyclization of various allylic amides, affording fluorinated dihydrooxazine compounds with high enantioselectivity (up to 99% ee). In addition to cyclic substrates, acyclic trisubstituted alkenes underwent the reaction with good diastereoselectivity, whereas low diastereoselectivity was observed for linear disubstituted alkenes. Results suggest that the reaction proceeds via a fluoro-carbocation intermediate.

Intraganglionic macrophages: a new population of cells in the enteric ganglia.

The enteric nervous system shares embryological, morphological, neurochemical, and functional features with the central nervous system. In addition to neurons and glia, the CNS includes a third component, microglia, which are functionally and immunophenotypically similar to macrophages, but a similar cell type has not previously been identified in enteric ganglia. In this study we identify a population of macrophages in the enteric ganglia, intermingling with the neurons and glia. These intraganglionic macrophages (IMs) are highly ramified and express the hematopoietic marker CD45, major histocompatibility complex (MHC) class II antigen, and chB6, a marker specific for B cells and microglia in avians. These IMs do not express antigens typically associated with T cells or dendritic cells. The CD45+ /ChB6+ /MHCII+ signature supports a hematopoietic origin and this was confirmed using intestinal chimeras in GFP-transgenic chick embryos. The presence of green fluorescent protein positive (GFP+) /CD45+ cells in the intestinal graft ENS confirms that IMs residing within enteric ganglia have a hematopoietic origin. IMs are also found in the ganglia of CSF1RGFP chicken and CX3CR1GFP mice. Based on the expression pattern and location of IMs in avians and rodents, we conclude that they represent a novel non-neural crest-derived microglia-like cell population within the enteric ganglia.

Impact of fear of falling and fall history on disability incidence among older adults: Prospective cohort study.

OBJECTIVE: Fear of falling (FOF) is a major health problem for older adults, present not just in fallers, but also nonfallers. This study examined the impact of FOF and fall history on disability incidence among community-dwelling older adults from a prospective cohort study. METHODS: A total of 5104 older adults living in community settings participated in baseline assessment and were followed up for about 4 years (median 52 mo, range 49-55 mo). At baseline, participants were assessed the presence of FOF and their fall history, and divided into 4 groups: Fall (-) FOF (-), Fall (+) FOF (-), Fall (-) FOF (+), and Fall (+) FOF (+). Disability incidence was defined as national long-term care insurance certification for personal support or care. RESULTS: During the follow-up period, 429 participants (9.9%) were newly certified as having a disability and needing personal support for long-term care insurance. Fall (-) FOF (+) group and Fall (+) FOF (+) group showed a significantly higher risk of disability incidence than Fall (-) FOF (-) group even after adjusting for covariates (Fall (-) FOF (+): hazard ratio 1.28, 95% confidence interval, 1.01-1.62, Fall (+) FOF (+): hazard ratio 1.44, 95% confidence interval, 1.05-1.98). CONCLUSIONS: Fear of falling could be a simple and useful predictor of disability incidence in community-dwelling older adults. Identifying and decreasing fall risk factors may prevent fall-related injuries, but excessive FOF may be associated with increased risk of disability incidence.

Associations of Near-Miss Traffic Incidents with Attention and Executive Function among Older Japanese Drivers.

BACKGROUND: Attention and executive function may play an important role in ensuring safe driving as they involve paying attention to complex information and making an instantaneous judgment during driving. We hypothesized that poor performance in attention and executive function may increase the risk of near-miss incidents among older drivers. OBJECTIVE: The aim of this study was to examine associations of current experience of near-miss traffic incidents with attention and executive function among older Japanese drivers. METHODS: The study included 3,421 general older drivers (mean age: 71.7 ± 4.9 years; 56.3% men) with a valid driver's license who were currently driving at least once per week and who had participated in a community-based cohort study between February 2015 and August 2016. The participants were asked about their experiences of near-miss traffic incidents in 10 situations that had almost happened during driving in the previous year. RESULTS: Of the 3,421 older drivers, 1,840 (53.8%) had experienced near-miss incidents during driving in the previous year at least once. Male sex (OR 1.46, 95% CI 1.27-1.69) and high driving frequency (OR 1.11, 95% CI 1.07-1.15) were significantly associated with the current experience of near-miss traffic incidents when the overall data were analyzed. In young-old drivers aged 65-74 years, poor performance in attention as assessed by the Trail Making Test-part A (OR 1.45, 95% CI 1.05-2.00) was significantly associated with near-miss traffic incidents. CONCLUSION: Male sex, high driving frequency, and poor performance in attention (in young-old drivers) were associated with near-miss traffic incidents. Improvement in attention may play a role in decreasing the risk of traffic accidents among older drivers.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies.

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.

Postnatal human enteric neuronal progenitors can migrate, differentiate, and proliferate in embryonic and postnatal aganglionic gut environments.

BACKGROUND: Enteric neural stem/progenitor cells (ENSCs) offer an innovative approach to treating Hirschsprung disease (HSCR) and other enteric neuropathies. However, postnatal-derived human ENSCs have not been thoroughly characterized and their behavior in the embryonic and postnatal intestinal environment is unknown. METHODS: ENSCs were isolated from the intestines of 25 patients undergoing bowel resection, including 7 children with HSCR. Neuronal differentiation and proliferation of ENSCs from submucosal and myenteric plexuses from patients with and without HSCR were characterized. ENSC migration and differentiation were studied following transplantation into embryonic chick neural crest, embryonic chick hindgut, and postnatal mouse aganglionic colon. RESULTS: The proliferative and neurogenic potential of ENSCs from HSCR intestine is equivalent to that of non-HSCR controls. Similarly, no difference was observed between myenteric- and submucosal-derived ENSCs. Postnatal ENSCs transplanted to embryonic neural crest pathways and to aneural hindgut migrate normally and differentiate into appropriate neural crest-derived cell types. ENSCs in postnatal mouse aganglionic colon differentiate into neurons and glia both ex vivo and in vivo. CONCLUSIONS: ENSCs isolated from the postnatal intestine of patients with and without HSCR can behave like embryonic neural crest-derived cells. These results support the feasibility of cell-based therapy for future treatment of neurointestinal disease.

Subjective Memory Complaints are Associated with Incident Dementia in Cognitively Intact Older People, but Not in Those with Cognitive Impairment: A 24-Month Prospective Cohort Study.

OBJECTIVE: Although subjective memory complaints (SMCs) are considered a risk factor for incident dementia in older people, the effect might differ based on cognitive function. The aim of the present study was to investigate whether the effect of SMCs on the incidence of dementia in older people differed based on cognitive function. DESIGN: A 24-month follow-up cohort study. SETTING: Japanese community. PARTICIPANTS: Prospective, longitudinal data for incident dementia were collected for 3,672 participants (mean age: 71.7 years; 46.5% men) for up to 24 months. MEASUREMENTS: Baseline measurements included covariates for incident dementia, SMCs, and cognitive function. Associations between SMCs, cognitive impairment, and incident dementia were examined using Cox proportional hazards models. RESULTS: Incidences of dementia in the cognitively intact without SMC, cognitively intact with SMC, cognitive impairment without SMC, and cognitive impairment with SMC groups were 0.3%, 1.8%, 3.4%, and 4.8%, respectively. In the cognitively intact participants, SMCs were associated with a significantly higher risk of dementia (hazard ratio [HR]: 4.95, 95% confidence interval [CI]: 1.52-16.11, p = 0.008). Incident dementia with cognitive impairment was not significantly different based on SMC presence (p = 0.527). Participants with cognitive impairment in multiple domains had a significantly higher risk of incident dementia (HR: 2.07, 95% CI: 1.01-4.24, p = 0.046) CONCLUSION: SMCs were related with dementia in cognitively intact older people, but not in those with cognitive impairment.Multiple domains of cognitive impairment were associated with a higher risk of incident dementia.

Prospective associations between sedentary behaviour and incident depressive symptoms in older people: a 15-month longitudinal cohort study.

OBJECTIVE: This study aimed to investigate whether sitting time, as a form of sedentary behaviour, is related to incident depressive symptoms in older people. METHODS: This study included 3503 participants (mean age 71.7 years, 50.1% female) from the 'Obu Study of Health Promotion for the Elderly' cohort study. At baseline and then 15 months later, the participants reported their status of depressive symptoms using the 15-item Geriatric Depression Scale. During the baseline assessment, the participants were also asked about their sedentary behaviour on weekdays over the past 7 days and, from there, categorized into three groups (<240, 240-480, ≥480 min/day). Demographic data and the other health behaviours were also assessed at the baseline. RESULTS: Cross-sectional analysis revealed that 437 participants (12.0%) had depressive symptoms. In a prospective analysis, the logistic regression model revealed that the odds ratio for depressive symptom incidence was higher in participants who, at baseline, spent 480 min or more per day sitting (1.636; 95% confidence interval [CI] 1.015 to 2.636, p = 0.043), and in those who spent 240-480 min (1.605; 95% CI 1.085 to 2.375, p = 0.018) in comparison with those who spent less than 240 min. CONCLUSIONS: Sedentary behaviour significantly affects the risk of incident depressive symptoms. Further research is needed to develop an intervention strategy to manage depressive symptoms, as the second most common cause of burden of disease among older adults. Copyright © 2016 John Wiley & Sons, Ltd.

Engraftment of enteric neural progenitor cells into the injured adult brain.

BACKGROUND: A major area of unmet need is the development of strategies to restore neuronal network systems and to recover brain function in patients with neurological disease. The use of cell-based therapies remains an attractive approach, but its application has been challenging due to the lack of suitable cell sources, ethical concerns, and immune-mediated tissue rejection. We propose an innovative approach that utilizes gut-derived neural tissue for cell-based therapies following focal or diffuse central nervous system injury. RESULTS: Enteric neuronal stem and progenitor cells, able to differentiate into neuronal and glial lineages, were isolated from the postnatal enteric nervous system and propagated in vitro. Gut-derived neural progenitors, genetically engineered to express fluorescent proteins, were transplanted into the injured brain of adult mice. Using different models of brain injury in combination with either local or systemic cell delivery, we show that transplanted enteric neuronal progenitor cells survive, proliferate, and differentiate into neuronal and glial lineages in vivo. Moreover, transplanted cells migrate extensively along neuronal pathways and appear to modulate the local microenvironment to stimulate endogenous neurogenesis. CONCLUSIONS: Our findings suggest that enteric nervous system derived cells represent a potential source for tissue regeneration in the central nervous system. Further studies are needed to validate these findings and to explore whether autologous gut-derived cell transplantation into the injured brain can result in functional neurologic recovery.

Optimizing neurogenic potential of enteric neurospheres for treatment of neurointestinal diseases.

BACKGROUND: Enteric neurospheres derived from postnatal intestine represent a promising avenue for cell replacement therapy to treat Hirschsprung disease and other neurointestinal diseases. We describe a simple method to improve the neuronal yield of spontaneously formed gut-derived neurospheres. MATERIALS AND METHODS: Enteric neurospheres were formed from the small and large intestines of mouse and human subjects. Neurosphere size, neural crest cell content, cell migration, neuronal differentiation, and neuronal proliferation in culture were analyzed. The effect of supplemental neurotrophic factors, including glial cell line-derived neurotrophic factor (GDNF) and endothelin-3, was also assessed. RESULTS: Mouse small intestine-derived neurospheres contained significantly more P75-expressing neural crest-derived cells (49.9 ± 15.3% versus 21.6 ± 11.9%, P < 0.05) and gave rise to significantly more Tuj1-expressing neurons than colon-derived neurospheres (69.9 ± 8.6% versus 46.2 ± 15.6%, P < 0.05). A similar pattern was seen in neurospheres isolated from human small and large intestine (32.6 ± 17.5% versus 10.2 ± 8.2% neural crest cells, P < 0.05; 29.7 ± 16.4% versus 16.0 ± 13.5% enteric neurons, P < 0.05). The addition of GDNF to the culture media further improved the neurogenic potential of small intestinal neurospheres (75.9 ± 4.0% versus 67.8 ± 5.8%, P < 0.05) whereas endothelin-3 had no effect. CONCLUSIONS: Enteric neurospheres formed from small intestine and supplemented with GDNF yield an enriched population of neural crest-derived progenitor cells and give rise to a high density of enteric neurons.