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.
- BrdU, bromodeoxyuridine
- CGRP, calcitonin gene-related peptide
- ChAT, choline acetyltransferase
- dpc, days post-coitum
- DMEM, Dulbecco’s modified Eagle medium
- ENS, enteric nervous system
- ENSPC, enteric nervous system progenitor cell
- FITC, fluorescein isothiocyanate
- GDNF, glial cell line-derived neurotrophic factor
- HRNP, human ribonucleoprotein
- NOS, nitric oxide synthase
- PBS, phosphate-buffered saline
- PGP9.5, protein gene product 9.5
- SP, substance P
- TH, tyrosine hydroxylase
- VIP, vasoactive intestinal polypeptide
- enteric nervous system
- Hirschsprung’s disease
- neural crest
- stem cell
- stem cell transplantation
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