title: Enteric neurospheres are not specific to neural crest cultures: implications for neural stem cell therapies.
abstract: OBJECTIVE
  Enteric 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.
  
  
  METHODS
  Gut 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.
  
  
  RESULTS
  Cultures 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.
  
  
  CONCLUSIONS
  Spherical 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.
authors: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, London, United Kingdom.
authors: Ellen
authors: E
authors: Binder
authors: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, London, United Kingdom.
authors: Dipa
authors: D
authors: Natarajan
authors: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, London, United Kingdom.
authors: Julie
authors: J
authors: Cooper
authors: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, London, United Kingdom.
authors: Rania
authors: R
authors: Kronfli
authors: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, London, United Kingdom.
authors: Mara
authors: M
authors: Cananzi
authors: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, London, United Kingdom.
authors: Jean-Marie
authors: JM
authors: Delalande
authors: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, London, United Kingdom.
authors: Conor
authors: C
authors: McCann
authors: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, London, United Kingdom; Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands.
authors: Alan J
authors: AJ
authors: Burns
authors: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, London, United Kingdom; Department of Gastroenterology, Great Ormond Street Hospital for Children, London, United Kingdom.
authors: Nikhil
authors: N
authors: Thapar
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002454:Cell Differentiation; D064987:Cell- and Tissue-Based Therapy; D002478:Cells, Cultured; D017615:Enteric Nervous System / Q000166:cytology* / Q000378:metabolism; D005260:Female; D041981:Gastrointestinal Tract / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009432:Neural Crest / Q000166:cytology* / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology* / Q000378:metabolism; D051155:Wnt1 Protein / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D051155:Wnt1 Protein; C495246:Wnt1 protein, mouse; C054562:yellow fluorescent protein, Bacteria