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Identification of the stem cell niche is crucial for understanding the factors that regulate these cells. Rodent enteric neural crest-derived stem cells have previously been isolated by flow cytometry and culture of cell suspensions from the outer smooth muscle layers or the entire gut wall from postnatal and adult animals. Such cell suspensions contain a mixture of cell types, including smooth muscle, fibroblasts and cells associated with the vasculature and extrinsic innervation. Thus these preparations may be contaminated by stem cells associated with extrinsic sensory and autonomic nerves and by other types of stem cell that reside in the gut. Here we describe a different approach, similar to that recently used for infant human gut, to obtain enteric ganglion-derived cells, with properties of neural progenitor cells, using isolated myenteric ganglia from postnatal rat ileum. Myenteric ganglia were separated from the gut wall, dispersed and resulting cell dissociates were plated in non-adherent culture conditions with EGF and FGF-2. Under these conditions neurosphere-like bodies (NLB) developed. Cells in NLB incorporated BrdU and expressed the stem cell marker nestin but not the pan-neuronal marker PGP 9.5. Upon growth factor withdrawal some BrdU-immunopositive cells assumed the morphology of neurons and expressed PGP 9.5; others were flattened and expressed the glial cell marker GFAP. This work therefore provides evidence that neural crest-derived progenitors in the postnatal rat gut are located in the myenteric plexus, and shows that these cells can be expanded and differentiated in NLB in vitro.