SummaryNeuron and glia generating progenitors of the mammalian enteric nervous system isolated from foetal and postnatal gut cultures

Normal organogenesis requires co-ordinate development and interaction of multiple cell types, and is seemingly governed by tissue specific factors. Lymphoid organogenesis during embryonic life is dependent on molecules the temporal expression of which is tightly regulated. During this process, haematopoietic 'inducer' cells interact with stromal 'organizer' cells, giving rise to the lymphoid organ primordia. Here we show that the haematopoietic cells in the gut exhibit a random pattern of motility before aggregation into the primordia of Peyer's patches, a major component of the gut-associated lymphoid tissue. We further show that a CD45+CD4-CD3-Il7Ralpha-c-Kit+CD11c+ haematopoietic population expressing lymphotoxin has an important role in the formation of Peyer's patches. A subset of these cells expresses the receptor tyrosine kinase RET, which is essential for mammalian enteric nervous system formation. We demonstrate that RET signalling is also crucial for Peyer's patch formation. Functional genetic analysis revealed that Gfra3-deficiency results in impairment of Peyer's patch development, suggesting that the signalling axis RET/GFRalpha3/ARTN is involved in this process. To support this hypothesis, we show that the RET ligand ARTN is a strong attractant of gut haematopoietic cells, inducing the formation of ectopic Peyer's patch-like structures. Our work strongly suggests that the RET signalling pathway, by regulating the development of both the nervous and lymphoid system in the gut, has a key role in the molecular mechanisms that orchestrate intestine organogenesis.

The transcriptional regulator SOX10 and the signalling molecule endothelin 3 have important roles in the development of the mammalian enteric nervous system (ENS). Using a clonal cell culture system, we show that SOX10 inhibits overt neuronal and glial differentiation of multilineage ENS progenitor cells (EPCs), without interfering with their neurogenic commitment. We also demonstrate that endothelin 3 inhibits reversibly the commitment and differentiation of EPCs along the neurogenic and gliogenic lineages, suggesting a role for this factor in the maintenance of multilineage ENS progenitors. Consistent with such a role, the proportion of Sox10-expressing progenitors in the total population of enteric neural crest cells is reduced in the gut of endothelin 3-deficient embryos. This reduction may be related to the requirement of endothelin signalling for the proliferation of ENS progenitors. The dependence of ENS progenitors on endothelin 3 is more pronounced at the migratory front of enteric neural crest cells, which is associated with relatively high levels of endothelin 3 mRNA. Our findings indicate that SOX10 and endothelin 3 have a crucial role in the maintenance of multilineage enteric nervous system progenitors.

ObjectivesEnteric neuropathies are severe gastrointestinal disorders with unsatisfactory outcomes. We aimed to investigate the potential of enteric neural stem cell therapy approaches for such disorders by transplanting mouse enteric neural crest cells (ENCCs) into ganglionic and aganglionic mouse gut in vivo and analysing functional integration and long-term safety.DesignNeurospheres generated from yellow fluorescent protein (YFP) expressing ENCCs selected from postnatal Wnt1-cre;R26R-YFP/YFP murine gut were transplanted into ganglionic hindgut of wild-type littermates or aganglionic hindgut of Ednrbtm1Ywa mice (lacking functional endothelin receptor type-B). Intestines were then assessed for ENCC integration and differentiation using immunohistochemistry, cell function using calcium imaging, and long-term safety using PCR to detect off-target YFP expression.ResultsYFP+ ENCCs engrafted, proliferated and differentiated into enteric neurons and glia within recipient ganglionic gut. Transplanted cells and their projections spread along the endogenous myenteric plexus to form branching networks. Electrical point stimulation of endogenous nerve fibres resulted in calcium transients (F/F0 = 1.16±0.01;43 cells, n = 6) in YFP+ transplanted ENCCs (abolished with TTX). Long-term follow-up (24 months) showed transplanted ENCCs did not give rise to tumours or spread to other organs (PCR negative in extraintestinal sites). In aganglionic gut ENCCs similarly spread and differentiated to form neuronal and glial networks with projections closely associated with endogenous neural networks of the transition zone.ConclusionsTransplanted ENCCs successfully engrafted into recipient ganglionic and aganglionic gut showing appropriate spread, localisation and, importantly, functional integration without any long-term safety issues. This study provides key support for the development and use of enteric neural stem cell therapies.

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.