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1.
Gut ; 73(9): 1441-1453, 2024 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-38816188

RESUMEN

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.


Asunto(s)
Colon , Sistema Nervioso Entérico , Enfermedad de Hirschsprung , Enfermedad de Hirschsprung/cirugía , Enfermedad de Hirschsprung/terapia , Humanos , Células Madre Pluripotentes , Trasplante de Células Madre/métodos , Diferenciación Celular
2.
Eur Respir J ; 55(6)2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32444408

RESUMEN

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.


Asunto(s)
Colágeno , Matriz Extracelular , Laminina , Mucosa Respiratoria , Andamios del Tejido , Animales , Bronquios , Células Cultivadas , Células Epiteliales , Humanos , Conejos
3.
J Anat ; 233(5): 592-606, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30191559

RESUMEN

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 ENSCs for SCI, and encourage further research into their potential for repair.


Asunto(s)
Células-Madre Neurales/trasplante , Regeneración de la Medula Espinal/fisiología , Médula Espinal , Trasplante de Células Madre/métodos , Animales , Embrión de Pollo , Sistema Nervioso Entérico/citología
4.
Sci Adv ; 8(35): eabn0356, 2022 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-36044566

RESUMEN

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.


Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Animales , Encéfalo/metabolismo , Ratones , Neuronas/metabolismo , Oxidantes , Enfermedad de Parkinson/metabolismo
5.
Sci Rep ; 11(1): 17189, 2021 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-34433854

RESUMEN

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.


Asunto(s)
Sistema Nervioso Entérico/metabolismo , Óxido Nítrico Sintasa de Tipo I/genética , Animales , Sistema Nervioso Entérico/citología , Aprendizaje Automático , Ratones , Ratones Endogámicos C57BL , Red Nerviosa/citología , Red Nerviosa/metabolismo , Neuronas/citología , Neuronas/metabolismo , Óxido Nítrico Sintasa de Tipo I/deficiencia
6.
Stem Cell Res Ther ; 12(1): 10, 2021 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-33407795

RESUMEN

BACKGROUND: Spinal cord injury (SCI) presents a significant challenge for the field of neurotherapeutics. Stem cells have shown promise in replenishing the cells lost to the injury process, but the release of axon growth-inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs) by activated cells within the injury site hinders the integration of transplanted cells. We hypothesised that simultaneous application of enteric neural stem cells (ENSCs) isolated from the gastrointestinal tract, with a lentivirus (LV) containing the enzyme chondroitinase ABC (ChABC), would enhance the regenerative potential of ENSCs after transplantation into the injured spinal cord. METHODS: ENSCs were harvested from the GI tract of p7 rats, expanded in vitro and characterised. Adult rats bearing a contusion injury were randomly assigned to one of four groups: no treatment, LV-ChABC injection only, ENSC transplantation only or ENSC transplantation+LV-ChABC injection. After 16 weeks, rats were sacrificed and the harvested spinal cords examined for evidence of repair. RESULTS: ENSC cultures contained a variety of neuronal subtypes suitable for replenishing cells lost through SCI. Following injury, transplanted ENSC-derived cells survived and ChABC successfully degraded CSPGs. We observed significant reductions in the injured tissue and cavity area, with the greatest improvements seen in the combined treatment group. ENSC-derived cells extended projections across the injury site into both the rostral and caudal host spinal cord, and ENSC transplantation significantly increased the number of cells extending axons across the injury site. Furthermore, the combined treatment resulted in a modest, but significant functional improvement by week 16, and we found no evidence of the spread of transplanted cells to ectopic locations or formation of tumours. CONCLUSIONS: Regenerative effects of a combined treatment with ENSCs and ChABC surpassed either treatment alone, highlighting the importance of further research into combinatorial therapies for SCI. Our work provides evidence that stem cells taken from the adult gastrointestinal tract, an easily accessible source for autologous transplantation, could be strongly considered for the repair of central nervous system disorders.


Asunto(s)
Células-Madre Neurales , Traumatismos de la Médula Espinal , Animales , Axones , Condroitina ABC Liasa/farmacología , Proteoglicanos Tipo Condroitín Sulfato , Regeneración Nerviosa , Células-Madre Neurales/trasplante , Ratas , Médula Espinal , Traumatismos de la Médula Espinal/terapia
7.
Nat Commun ; 8: 15937, 2017 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-28671186

RESUMEN

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.


Asunto(s)
Colon/enzimología , Sistema Nervioso Entérico/citología , Seudoobstrucción Intestinal/cirugía , Células-Madre Neurales/trasplante , Óxido Nítrico Sintasa/deficiencia , Animales , Colon/fisiopatología , Sistema Nervioso Entérico/enzimología , Femenino , Motilidad Gastrointestinal , Humanos , Seudoobstrucción Intestinal/enzimología , Seudoobstrucción Intestinal/genética , Seudoobstrucción Intestinal/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/trasplante , Óxido Nítrico Sintasa/genética
8.
Elife ; 62017 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-28387645

RESUMEN

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.


Asunto(s)
Hipoxia de la Célula , Linaje de la Célula , Células Neuroendocrinas , Células Neuroepiteliales , Animales , Anuros , Evolución Biológica , Lampreas , Pez Cebra
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