Abstract
ObjectivesEnteric neural stem cells provide hope of curative treatment for enteric neuropathies. Current protocols for their harvesting from humans focus on the generation of ‘neurospheres’ from cultures of dissociated gut tissue. The study aims to better understand the derivation, generation and composition of enteric neurospheres.DesignGut tissue was obtained from Wnt1-Cre;Rosa26Yfp/Yfp transgenic mice (constitutively labeled neural crest cells) and paediatric patients. Gut cells were cultured either unsorted (mixed neural crest/non-neural crest), or following FACS selection into neural crest (murine-YFP+ve/human-p75+ve) or non-neural crest (YFP-ve/p75-ve) populations. Cultures and resultant neurospheres were characterized using immunolabelling in vitro and following transplantation in vivo.ResultsCultures of (i) unsorted, (ii) neural crest, and (iii) non-neural crest cell populations generated neurospheres similar in numbers, size and morphology. Unsorted neurospheres were highly heterogeneous for neural crest content. Neural crest-derived (YFP+ve/p75+ve) neurospheres contained only neural derivatives (neurons and glia) and were devoid of non-neural cells (i.e. negative for SMA, c-Kit), with the converse true for non-neural crest-derived (YFP-ve/p75-ve) ‘neurospheres’. Under differentiation conditions only YFP+ve cells gave rise to neural derivatives. Both YFP+ve and YFP-ve cells displayed proliferation and spread upon transplantation in vivo, but YFP-ve cells did not locate or integrate within the host ENS.ConclusionsSpherical accumulations of cells, so-called ‘neurospheres’ forming in cultures of dissociated gut contain variable proportions of neural crest-derived cells. If they are to be used for ENS cell replacement therapy then improved protocols for their generation, including cell selection, should be sought in order to avoid inadvertent transplantation of non-therapeutic, non-ENS cells.
Highlights
-called ‘neurospheres’ forming in cultures of dissociated gut contain variable proportions of neural crest-derived cells. If they are to be used for enteric nervous system (ENS) cell replacement therapy improved protocols for their generation, including cell selection, should be sought in order to avoid inadvertent transplantation of non-therapeutic, nonENS cells
Severe gut motility disorders result from developmental or acquired abnormalities of the enteric nervous system (ENS) and include aganglionosis in Hirschsprung disease (HSCR), dysganglionosis or neuronal dysfunction seen in chronic intestinal pseudo-obstruction (CIPO) and immune-mediated neuronal destruction in conditions such as oesophageal achalasia [1,2,3,4]
Over the last few years, several groups have been working towards developing enteric nervous system (ENS) cell replacement therapy strategies, which have been greatly aided by advances in the field of neural stem cell biology [10,11,12,13]
Summary
Severe gut motility disorders result from developmental or acquired abnormalities of the enteric nervous system (ENS) and include aganglionosis in Hirschsprung disease (HSCR), dysganglionosis or neuronal dysfunction seen in chronic intestinal pseudo-obstruction (CIPO) and immune-mediated neuronal destruction in conditions such as oesophageal achalasia [1,2,3,4]. In order to maximise the efficiency of replenishing the ENS, and given its entire derivation from neural crest, transplanting a purer population of neural crest-derived cells would seem to be most favourable [22,23] This ideal requirement appears to have been satisfied when many of the above mentioned studies demonstrated that neural crest cells, that retain characteristics of early ENS progenitors, could be isolated as single cells and cultured to form large cellular aggregates termed neurospheres or neurosphere-like bodies (NLBs). These neurospheres have been shown to form from mouse gut when cultured in enriched medium [17] and from human gut cultures [18,20,24]. They have been regarded as niches for enteric neural crest stem cells and their formation has been established as an initial method of selecting and enriching for large numbers of neural crest cells in culture, especially from human gut tissue [18,20,24,25]
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