Objective Hirschsprung disease (HSCR) is a rare congenital intestinal condition that, despite corrective surgery, can lead to recurrent hospitalisations and reduced quality of life throughout childhood. There is limited population-level evidence on the causes of these admissions or associated educational needs. Design/methods Using linked health and education data from the Education and Child Health Insights from Linked Data database, we created a cohort of all children born in England between 2002 and 2020. We compared admission rates, mortality, surgical procedures and causes of admission for children with and without HSCR at ages 0-4, 5-9 and 10-14 years. We also assessed the prevalence of recorded Special Educational Needs and Disability (SEND) by Year 1 of primary school (age 6). Results Among 11 261 227 children, 3227 (0.03%) had HSCR. By age 4, 95.0% of children with HSCR had been readmitted, compared with 40.2% without. Common admission reasons included constipation, gastroenteritis, intestinal infection, abdominal pain and nausea or vomiting. 81.4% of children with HSCR had two or more surgical procedures between ages 0 and 4, compared with 2.9% in children without HSCR. Mortality by age 4 was 3.2% for HSCR versus 0.5% for non-HSCR children. By age 6, 44.0% of children with HSCR had recorded SEND compared with 17.9% of those without. Conclusion Children with HSCR experience substantially higher hospital readmission rates, more surgeries and greater mortality up to age 14 than their peers. They are also more likely to require educational support, independent of comorbidities such as Down syndrome. Improvements in surgical and long-term care, including novel or complementary approaches, alongside enhanced educational and psychosocial support, are needed to improve outcomes and quality of life across childhood and adolescence.

Background and aims Intestinal motility relies on inputs from multiple cells within a complex neuromuscular syncytium located in the gut wall. While it is known that motility is dependent on the development of motor patterns which govern coordinated contractile activity within the gut wall, knowledge regarding the onset of coordinated motor activity in humans is still lacking. This study assessed the emergence of coordinated motor patterns, and the molecular mechanisms underpinning developing motility, in the human fetal gut. Methods Human fetal gut samples (obtained via the MRC-Wellcome Trust Human Developmental Biology Resource-UK) were characterised by live imaging, spatiotemporal mapping, immunohistochemistry and RNAseq. Results Human small intestinal samples displayed the presence of key cell types including enteric neurons, interstitial cells of Cajal, platelet-derived growth factor receptor alpha positive (PDGFRα+) cells and smooth muscle at post conception week (PCW) 12. Between PCW12 and PCW16, functional assessment revealed a marked increase in the velocity (p= 0.0341) of propagating contractions. Subsequently, between PCW16 and PCW20 the number of contraction initiation sites reduced drastically (p=0.0053), enabling the emergence of long-distance propagating contractions. Expression analyses showed the development of coordinated motor activity was coincident with increased expression of various genes involved in calcium and purinergic signalling pathways. Conclusions These findings provide the first direct mechanistic evidence of the temporal development of coordinated contractile activity in the human fetal intestine, highlighting the role of calcium dynamics, purinergic signalling and interstitial cells in early stages of human motility development, potentially informing an improved understanding of the pathogenesis of gut motility disorders. ### Competing Interest Statement The authors have declared no competing interest. All sequencing data have been deposited on NCBI’s SRA and at GEO and be made available upon publication * DEGs : Differentially expressed genes ENS : Enteric nervous system FDR : False discovery rate GI : Gastrointestinal GSEA : Gene set enrichment analysis ICC : Interstitial cells of Cajal KEGG : Kyoto encyclopaedia of genes and genomes PFA : Paraformaldehyde PDGFRα : Platelet-derived growth factor receptor alpha positive cells PCW : Post-conception week PCA : Principal Component Analysis RT : Room temperature SIP : S MCs, I CC, P DGFRα+ cells ST Map : Spatiotemporal maps SCFR+ : Stem cell factor receptor positive

Hirschsprung disease (HSCR) is a devastating congenital disorder characterised by absence of the enteric nervous system (ENS) in the distal gut. Cell therapy, using human pluripotent stem cell-derived ENS progenitors, offers an attractive alternative to treat this life-limiting disorder. Here we provide an in-depth characterisation of the HSCR phenotype in the B6;129- Ednrbtm1Ywa /J mouse model including molecular analyses highlighting the wide-ranging molecular effects of aganglionosis in HSCR colon. We show that transplantation of hPSC-derived ENS progenitors leads to significant increases in contractile function with formation of extensive donor-derived neuronal networks in colonic explants. Moreover, post-transplantation, we show that integration of ENS progenitors rescues many of the biological pathways impacted in the HSCR aganglionic microenvironment. These novel results provide strong evidence that human ENS progenitor transplantation can modulate critical signalling pathways affected in HSCR and exert positive impacts on contractile function, in aganglionic tissue, supporting further translational development of this regenerative medicine approach. ### Competing Interest Statement The authors have declared no competing interest.

Objective Hirschsprung disease (HSCR) is a severe congenital disorder affecting 1:5000 live births. HSCR results from the failure of enteric nervous system (ENS) progenitors to fully colonise the gastrointestinal tract during embryonic development. This leads to aganglionosis in the distal bowel, resulting in disrupted motor activity and impaired peristalsis. Currently, the only viable treatment option is surgical resection of the aganglionic bowel. However, patients frequently suffer debilitating, lifelong symptoms, with multiple surgical procedures often necessary. Hence, alternative treatment options are crucial. An attractive strategy involves the transplantation of ENS progenitors generated from human pluripotent stem cells (hPSCs). Design ENS progenitors were generated from hPSCs using an accelerated protocol and characterised, in detail, through a combination of single-cell RNA sequencing, protein expression analysis and calcium imaging. We tested ENS progenitors’ capacity to integrate and affect functional responses in HSCR colon, after ex vivo transplantation to organotypically cultured patient-derived colonic tissue, using organ bath contractility. Results We found that our protocol consistently gives rise to high yields of a cell population exhibiting transcriptional and functional hallmarks of early ENS progenitors. Following transplantation, hPSC-derived ENS progenitors integrate, migrate and form neurons/glia within explanted human HSCR colon samples. Importantly, the transplanted HSCR tissue displayed significantly increased basal contractile activity and increased responses to electrical stimulation compared with control tissue. Conclusion Our findings demonstrate, for the first time, the potential of hPSC-derived ENS progenitors to repopulate and increase functional responses in human HSCR patient colonic tissue.

Interneuronal transfer and brain spreading of pathogenic proteins are features of neurodegenerative diseases. Pathophysiological conditions and mechanisms affecting this spreading remain poorly understood. This study investigated the relationship between neuronal activity and interneuronal transfer of α-synuclein, a Parkinson-associated protein, and elucidated mechanisms underlying this relationship. In a mouse model of α-synuclein brain spreading, hyperactivity augmented and hypoactivity attenuated protein transfer. Important features of neuronal hyperactivity reported here were an exacerbation of oxidative and nitrative reactions, pronounced accumulation of nitrated α-synuclein, and increased protein aggregation. Data also pointed to mitochondria as key targets and likely sources of reactive oxygen and nitrogen species within hyperactive neurons. Rescue experiments designed to counteract the increased burden of reactive oxygen species reversed hyperactivity-induced α-synuclein nitration, aggregation, and interneuronal transfer, providing first evidence of a causal link between these pathological effects of neuronal stimulation and indicating a mechanistic role of oxidant stress in hyperactivity-induced α-synuclein spreading.

Clinical, pathological and experimental evidence accumulated over the past two decades has led to the increasing recognition that Parkinson's disease (PD) is a whole-body disorder. From the clinical standpoint, PD patients predominantly present with motor symptoms including an impaired ability to initiate movements, loss of fine motor control and resting tremor. The constellation of PD symptoms, however, also includes a variety of non-motor manifestations that affect patients during the progressive disease course but can also precede the occurrence of motor dysfunctions. Increasing evidence supports a key role for the protein alpha synuclein (α-syn) in disease pathology and pathogenesis. A variety of genetic and environmental factors have been identified that not only confer toxic properties to α-syn (possibly related to enhanced protein aggregation) but also increase its propensity for neuron-to-neuron spread. This spreading ability may explain, at least in part, the progression of α-syn pathology throughout the brain first noted by Braak and colleagues. Intriguingly, one of the earliest brain regions to display α-syn burden in the Braak staging model, namely the dorsal motor nucleus of the vagus nerve, is notable for its connections with the enteric nervous system via the vagus nerve. In this chapter, we will review and discuss evidence supporting the possibility that the α-syn pathology characteristic of PD may be initiated in the gastrointestinal tract and propagated to the brain via the vagus nerve. In addition, we will discuss potential risk factors present within the intestinal lumen, including bacteria and viruses of the gut microbiome, that could be involved in the initiation and/or modulation of α-syn pathology.

Neuronal nitric oxide synthase (nNOS) neurons play a fundamental role in inhibitory neurotransmission, within the enteric nervous system (ENS), and in the establishment of gut motility patterns. Clinically, loss or disruption of nNOS neurons has been shown in a range of enteric neuropathies. However, the effects of nNOS loss on the composition and structure of the ENS remain poorly understood. The aim of this study was to assess the structural and transcriptional consequences of loss of nNOS neurons within the murine ENS. Expression analysis demonstrated compensatory transcriptional upregulation of pan neuronal and inhibitory neuronal subtype targets within the Nos1−/− colon, compared to control C57BL/6J mice. Conventional confocal imaging; combined with novel machine learning approaches, and automated computational analysis, revealed increased interconnectivity within the Nos1−/− ENS, compared to age-matched control mice, with increases in network density, neural projections and neuronal branching. These findings provide the first direct evidence of structural and molecular remodelling of the ENS, upon loss of nNOS signalling. Further, we demonstrate the utility of machine learning approaches, and automated computational image analysis, in revealing previously undetected; yet potentially clinically relevant, changes in ENS structure which could provide improved understanding of pathological mechanisms across a host of enteric neuropathies.

Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease.Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. In vitro, primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence.Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin α2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air-liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using in vivo chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation.Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models.

Spinal cord injury ( SCI ) causes paralysis, multisystem impairment and reduced life expectancy, as yet with no cure. Stem cell therapy can potentially replace lost neurons, promote axonal regeneration and limit scar formation, but an optimal stem cell source has yet to be found. Enteric neural stem cells ( ENSC ) isolated from the enteric nervous system ( ENS ) of the gastrointestinal ( GI ) tract are an attractive source. Here, we used the chick embryo to assess the potential of ENSC to integrate within the developing spinal cord. In vitro , isolated ENSC formed extensive cell connections when co‐cultured with spinal cord ( SC )‐derived cells. Further, qRT ‐ PCR analysis revealed the presence of TuJ1 + neurons, S100 + glia and Sox10 + stem cells within ENSC neurospheres, as well as expression of key neuronal subtype genes, at levels comparable to SC tissue. Following ENSC transplantation to an ablated region of chick embryo SC , donor neurons were found up to 12 days later. These neurons formed bridging connections within the SC injury zone, aligned along the anterior/posterior axis, and were immunopositive for TuJ1. These data provide early proof of principle support for the use of ENSC s for SCI , and encourage further research into their potential for repair.

Enteric nervous system neuropathy causes a wide range of severe gut motility disorders. Cell replacement of lost neurons using enteric neural stem cells (ENSC) is a possible therapy for these life-limiting disorders. Here we show rescue of gut motility after ENSC transplantation in a mouse model of human enteric neuropathy, the neuronal nitric oxide synthase ( nNOS −/− ) deficient mouse model, which displays slow transit in the colon. We further show that transplantation of ENSC into the colon rescues impaired colonic motility with formation of extensive networks of transplanted cells, including the development of nNOS + neurons and subsequent restoration of nitrergic responses. Moreover, post-transplantation non-cell-autonomous mechanisms restore the numbers of interstitial cells of Cajal that are reduced in the nNOS −/− colon. These results provide the first direct evidence that ENSC transplantation can modulate the enteric neuromuscular syncytium to restore function, at the organ level, in a dysmotile gastrointestinal disease model.

The evolutionary origins of the hypoxia-sensitive cells that trigger amniote respiratory reflexes – carotid body glomus cells, and ‘pulmonary neuroendocrine cells’ (PNECs) - are obscure. Homology has been proposed between glomus cells, which are neural crest-derived, and the hypoxia-sensitive ‘neuroepithelial cells’ (NECs) of fish gills, whose embryonic origin is unknown. NECs have also been likened to PNECs, which differentiate in situ within lung airway epithelia. Using genetic lineage-tracing and neural crest-deficient mutants in zebrafish, and physical fate-mapping in frog and lamprey, we find that NECs are not neural crest-derived, but endoderm-derived, like PNECs, whose endodermal origin we confirm. We discover neural crest-derived catecholaminergic cells associated with zebrafish pharyngeal arch blood vessels, and propose a new model for amniote hypoxia-sensitive cell evolution: endoderm-derived NECs were retained as PNECs, while the carotid body evolved via the aggregation of neural crest-derived catecholaminergic (chromaffin) cells already associated with blood vessels in anamniote pharyngeal arches.