Properties of secondary and tertiary human enteric nervous system neurospheres.

UNLABELLED: Advances in enteric nervous system (ENS) stem cell biology have raised the possibility of treating Hirschsprung's disease with ENS stem/progenitor cell (ENSPC) transplantation. This study aimed to expand ENSPC numbers by the growth and redissociation of neurospheres and assess their differential potential. METHODS: Human ENS neurospheres were cultured as previously described and redissociated to generate secondary and tertiary neurospheres. Neurospheres were assessed for the presence of neuronal (PGP9.5), glial (S100), and stem cell (p75, nestin markers). The degree of immunofluorescence was quantified using the ImageJ program. Secondary/tertiary neurospheres were transplanted into mouse distal colon grown in tissue culture. RESULTS: Secondary/tertiary neurospheres could be generated with exponentially increasing numbers. Tertiary neurospheres showed a significant increase in the proportion of p75 staining but a significant decrease in the proportion of S100 staining. After transplantation, secondary/tertiary neurosphere-derived cells positive for PGP9.5 and S100 could be identified. CONCLUSIONS: It is possible to exponentially expand neurosphere and therefore ENSPC numbers by repeated dissociation and culture. There is a loss of S100-positive cells in secondary/tertiary neurospheres, but the ENSPCs remain capable of differentiating into neurons and glia when transplanted into an embryonic gut environment.

Human and mouse enteric nervous system neurosphere transplants regulate the function of aganglionic embryonic distal colon.

BACKGROUND & AIMS: Recent advances have raised the possibility of treating enteric nervous system (ENS) disorders with transplanted progenitor cells (ENSPC). Although these cells have been shown to migrate and differentiate after transplantation, no functional effects have been demonstrated. We therefore aimed to investigate whether embryonic mouse and neonatal human ENSPC can regulate the contractility of aganglionic bowel. METHODS: Embryonic mouse and neonatal human ENSPC were grown as neurospheres before transplantation into aganglionic embryonic mouse hindgut explants and culture for 8-12 days. Engraftment and neural differentiation were confirmed using immunofluorescence and transmission electron microscopy. The contraction frequency of transplanted bowel was measured and compared with that of embryonic day 11.5 embryonic ganglionic and aganglionic bowel cultured for the same period. Calcium movement was measured at spatially defined points in bowel wall smooth muscle. Neural modulation of bowel contractility was assessed using tetrodotoxin. RESULTS: Both mouse and human ENSPC migrated and differentiated after neurosphere transplantation. Transmission electron microscopy demonstrated the existence of synapses. Transplantation restored the high contraction frequency of aganglionic bowel to the lower rate of ganglionic bowel. Calcium imaging demonstrated that neurosphere transplantation coordinates intracellular free calcium levels. Both these effects were reversed by the addition of tetrodotoxin, indicating the functional effect of neurosphere-derived neurons. CONCLUSIONS: Neonatal human gut is a source of ENSPC that can be transplanted to restore the contractile properties of aganglionic bowel by a neurally mediated mechanism. This may aid development of a stem cell-based treatment for Hirschsprung's disease.

Characterisation and transplantation of enteric nervous system progenitor cells.

AIMS: Enteric nervous system (ENS) progenitor cells have been postulated to be an appropriate source of cells for the treatment of Hirschsprung's disease. In order for this to be successful, the techniques previously used for the isolation of rodent ENS progenitor cells need to be adapted for postnatal human tissue. In this paper, we describe a method suitable for the preparation of both mouse and human postnatal ENS progenitor cells and assess their transplantation potential. METHOD: Single cell suspensions were isolated from 11.5 days post-coitum embryonic mouse caecum and postnatal human myenteric plexus. These cells were cultured under non-adherent conditions to generate neurospheres which were implanted into aganglionic embryonic mouse hindgut explants. Cell proliferation, migration and differentiation were observed using immunofluorescence microscopy. RESULTS: Neurospheres generated from both mouse and human tissues contained proliferating neural crest-derived cells that could be expanded in tissue culture to generate both glial cells and neurons. When implanted into aganglionic murine gut, cells migrated from the neurospheres using pathways appropriate for cells derived from the neural crest, and differentiated to become glia and neurons expressing neuronal phenotypic markers characteristic of the ENS including nitric oxide synthase and vasoactive intestinal polypeptide. CONCLUSION: We have developed a technique for the isolation and expansion of ENS progenitor cells from human neonates. These cells have the ability to differentiate into neurons and glia when transplanted into aganglionic gut, this demonstration being a necessary first step for their autologous transplantation in the treatment of Hirschsprung's disease.

The diverse cardiac morphology seen in hearts with isomerism of the atrial appendages with reference to the disposition of the specialised conduction system.

Congenital cardiac malformations which include isomerism of the atrial appendages are amongst the most challenging of problems for diagnosis and also for medical and surgical management. The nomenclature for pathological description is controversial, but difficulties can be overcome by the use of a segmental approach. Such an approach sets out the morphology and the topology of the chambers of the heart, together with the types and modes of the atrioventricular, ventriculo-arterial, and venous connections. We have applied this method to a study of 35 hearts known to have isomerism of the atrial appendages. We have already published accounts of 27 of these cases, but these were reviewed for this study in the light of our increased awareness of the implications of isomerism, and 8 new cases were added. After examining, or re-examining, the morphology of every heart in detail, we grouped them together according to their ventricular topology and modes of atrioventricular connection. Then we studied the course of the specialised conduction system, by the use of the light microscope, first in each individual case, and then together in their groups. We conclude that the pathways for atrioventricular conduction in hearts with isomerism of the atrial appendages are conditioned both by ventricular topology, and by the atrioventricular connections. Based on our experience, we have been able to establish guidelines that direct the clinician to the likely location of the conduction tissues.

Analysis of the effects of endothelin-3 on the development of neural crest cells in the embryonic mouse gut.

BACKGROUND/PURPOSE: Mutations in the endothelin-3 (ET-3) and endothelin-B receptor (EDNR-B) genes cause terminal colonic aganglionosis in mice and are linked to Hirschsprung's disease. These experiments are designed to determine if the development of terminal enteric ganglia depends on changes in proliferation, apoptosis, or differentiation of enteric neural crest (NC) cells in response to ET-3. METHODS: Gut from embryonic lethal-spotted mice (lacking ET-3) and controls were investigated in vivo. NC-derived cells were identified immunohistochemically and their proliferation, apoptosis and differentiation monitored by bromodeoxyuridine incorporation, the terminal deoxytransferase poly dU nick end labelling (TUNEL) reaction, and appearance of neuronal nitric oxide synthase (NOS), respectively. RESULTS: No differences in apoptosis or proliferation of NC cells were apparent between lethal-spotted embryos and controls. Although no temporal differences in the differentiation of NOS neurones were evident, these cells appeared more cranially in the gut in the absence of ET-3 than in controls. CONCLUSIONS: ET-3 has no detectable influence on proliferation, apoptosis, or timing of differentiation of NC-derived cells in the gut. However, the more proximal location of differentiated neurones in the absence of ET-3 is consistent with a restricted role in migration of NC-derived cells.