title: Properties of secondary and tertiary human enteric nervous system neurospheres.
abstract: 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.
authors.affiliation: Department of Paediatric Surgery, Queens's Medical Centre, Nottingham University Hospitals NHS Trust, Nottingham, UK.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Lindley
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Hawcutt
authors.forename: M Gwen
authors.initials: MG
authors.lastname: Connell
authors.forename: David H
authors.initials: DH
authors.lastname: Edgar
authors.forename: Simon E
authors.initials: SE
authors.lastname: Kenny
mesh_terms: D000818:Animals; D017615:Enteric Nervous System / Q000502:physiology*; D006627:Hirschsprung Disease / Q000628:therapy*; D006801:Humans; D051379:Mice; D033581:Stem Cell Transplantation; D013234:Stem Cells