Experimental study of culturing enteric neural stem cells from gut in children with Hirschsprung's disease

Objective To explore the possibility of isolating and culturing in vitro enteric neural stem cells from gut in children with Hirschsprung's disease (HD) and provide experimental rationales for treating HD with neural stem cell transplantation. Methods Human gut samples were obtained from one child aged 11 months after pull-through surgery due to HD. After enzyme digestion, single cell suspensions were isolated from postnatal human myenteric plexus. The cells were seeded into serum-free medium and generated neurosphere-like bodies in vitro. Cell proliferation was observed by the curve of optic density (OD) from CCK8 test at the timepoints of 24, 48, 72, 96, 120 and 144h and growth curve diagram plotted. And neurospheres and differentiated cells with immunofluorescent staining of nestin were identified by neuron-specific beta III tubulin (TUJ-1) and glial fibrillary acidic protein (GFAP). Results Single cells from colonic muscle formed neurospheres after culturing for 10 days and these neurospheres could be subcultured and maintained for several weeks in vitro. The OD values of CCK8 test increased progressively. Existing neurospheres, identified as nestin positive cells, could be passaged and differentiated into glial cells (GFAP positive) and neurons (TUJ-1 positive). Conclusions Enteric neural stem cells may be obtained from human postnatal gut in patients with HD. And it is a potential source of autologous neural stem cells for stem cell transplantation therapy. Key words: Hirschsprung's disease; Enteric neural stem cells; Stem cell transplantation