Human and mouse enteric nervous system neurosphere transplants regulate the function of aganglionic embryonic distal colon.
OBJECTIVE
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.
Institute of Child Health, University of Liverpool, Royal Liverpool Children's Hospital, Alder Hey, Liverpool, United Kingdom.
Richard M
RM
Lindley
Daniel B
DB
Hawcutt
M Gwen
MG
Connell
Sarah L
SL
Almond
Sarah N
SN
Almond
Maria-Giuliana
MG
Vannucchi
Maria Simonetta
MS
Faussone-Pellegrini
David H
DH
Edgar
Simon E
SE
Kenny
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D020013:Calcium Signaling / Q000502:physiology; D002454:Cell Differentiation; D002465:Cell Movement; D003106:Colon / Q000294:innervation*; D053595:Embryonic Stem Cells / Q000637:transplantation* / Q000648:ultrastructure; D017615:Enteric Nervous System / Q000166:cytology* / Q000502:physiology; D005260:Female; D005769:Gastrointestinal Motility; D006085:Graft Survival; D006627:Hirschsprung Disease / Q000473:pathology / Q000628:therapy; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D046529:Microscopy, Electron, Transmission; D009130:Muscle, Smooth / Q000294:innervation; D009924:Organ Culture Techniques; D011247:Pregnancy; D019009:Proto-Oncogene Proteins c-kit / Q000276:immunology; D018874:Spheroids, Cellular / Q000166:cytology / Q000637:transplantation*; D033581:Stem Cell Transplantation / Q000379:methods*
D000906:Antibodies; D019009:Proto-Oncogene Proteins c-kit