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Migration and differentiation of enteric neural crest cells in the embryonic gut explant cultured ex vivo

Hirschsprung's disease (HSCR) is a common congenital gut motility disorder characterized by the reduction or absence of enteric neurons in the distal region of the colon. The Dominant megacolon (Dom) mutant mouse, which carries a spontaneous mutation in Sox10, exhibits HSCR‐like phenotypes, and hence has been used as an animal model of HSCR. It has been known that in Sox10Dom/+ mouse embryos, the migration and differentiation of neural crest cells (NCCs), which are progenitors of enteric neurons and glial cells, are abnormal, leading to hypoganglionosis or aganglionosis in the hindgut. In Sox10Dom/Dom homozygous embryos, the entire gut tube is aganglionic and the embryo dies before E13.5. However, it is still unknown whether hypoganglionic and aganglionic phenotypes exhibited by Dom mice is a result of abnormalities in NCCs only (cell‐autonomous effects), alterations of gut microenvironment through which NCCs migrate (niche effects), or both. Previously, an organotypic gut explant system has already been established, which allows ex vivo co‐culture of donor NCCs of different genotypes with a recipient embryonic gut segment isolated from embryos of the same or other genotypes. In the present study, E13.5 Dom mouse hindgut explants were used as recipient gut segments for transplantation of donor NCCs. Five days after cell transplantation, the hindgut explant cultured ex vivo appeared morphologically and histologically normal with rhythmic contractions and relaxations, indicating that the development of the hindgut explant ex vivo was similar to that in vivo. To obtain donor enteric neural crest cells (ENCCs), midgut segments which were genetically labeled with GFP were dissected from E11.5 mouse embryos and cultured on a fibronectin‐coated surface. ENCCs emigrated from the midgut segments were collected and transplanted to the hindgut explant, which was then cultured ex vivo for 5 days. We found that transplanted ENCCs survived, migrated, and differentiated into TuJ1‐immunoreative enteric neurons in normoganglionic and hypoganglionic segments of the hindgut explant. When transplanted to aganglionic segments of the hindgut, ENCCs were still able to differentiate into TuJ1‐immunoreactive neurons, but they were not able to migrate as far as those transplanted cells in the normoganglionic and hypoganglionic segments, and most of them stayed near the transplantation site without forming a normal neuronal network. Our results indicated that endogenous enteric neurons were required for the normal migration and integration of transplanted ENCCs within the hindgut explant.