Preliminary identification of enteric nervous system stem cells in intestinal mucosa of the patients with Hirschsprung' s disease

Abstract

Objective To identify the enteric nervous system (ENS) stem cells in intestinal mucosa of the patients with Hirschsprung's disease (HD). Methods Fifteen patients were pathologically diagnosed with Hirschsprung's disease. They underwent surgery to remove the aganglionic intestines.The removed intestines were collected for this study. Of the 15 patients, 8 were infants (age≤1 year)and 7 were toddlers (age＞ 1year). Two were long-segment HD, and 13 were short-segment HD.Three pieces of tissues were collected from the stenotic segment, transitional zone and dilated segment of the removed intestines. The intestines proximate to the dilated segment were selected as controls.Immunohistochemical staining of stem cell markers including SOX10, Nestin, TuJ1 and GFAP was performed to indentify the ENS stem cells in intestinal mucosa. The TBS buffer was used as the negative control of the primary antibody. The S-P method was applied to analyze the expressions of the 4stem cell markers through the intestine. Results The expressions of SOX10, Nestin, and TUJ1 in the mucosa of the transitional zone and dilated segment were strong. And scattered SOX10, Nestin, and TUJ1 positive cells were observed in the lamina propria. But most of the SOX10, Nestin, and TUJ1 positive cells located in mucosa closed to the muscularis mucosa. The expression of GFAP in the mucosa of the transitional zone and dilated segment was negative. The expressions of SOX10, Nestin,TUJ1 and GFAP in myenteric plexus were stronger than those of submucosal plexus (SOX10 expression, infant 50% vs 31.3%, toddler 35. 7% vs 21.4%; Nestin expression, infant 43. 8% vs 37. 5%,toddler 28. 6% vs 14. 2%; TUJ1 expression, infant 37. 5% vs 18. 5%; toddler 28. 6% vs 28. 6%;GFAP expression 25.0 % vs 18. 5 % ; toddler 57. 1% vs 35. 7 % ; P ＜ 0. 05 ). The expressions of SOX10, Nestin and TUJ1 were stronger in submucosal plexus than those in mucosa (SOX10 expression, infant 31.3% vs 25. 0%, toddler 21.4% vs 7. 1% ; Nestin expression, infant 25. 0% vs 18. 8%,toddler 14. 3% vs 14. 3%; TUJ1 expression, infant 18. 5% vs 12. 5 %, toddler 28. 6% vs 7. 1%; P＜0. 05). The expressions of SOX10 and Nestin in myenteric plexus of the dilated segment in infants were stronger than thoset in toddler (SOX10, 75. 0% vs 42. 8% ; Nestin, 62. 5% vs 42. 8%, P＜0. 05). The expressions SOX10 and Nestin in mucosa of the the dilated segment in infants were stronger than those in toddler (SOX10, 37. 5% vs 14. 3%; Nestin, 25. 0% vs 14. 3%, P＜0. 05). Conclusions The ENS stem cells may exist in the mucosa of the ganglionic intestines of HD patients, whichare scattered and mostly located in the mucosa near muscularis mucosa. In HD patients, the expressions of SOX10, Nestin, TUJ1 and GFAP in myenteric plexus are higher than those in submucosal plexus. However, the expressions of SOX10, Nestin, TUJ1 and GFAP in the submucosal plexus are higher than those in the mucous layer. The expressions of SOX10 and Nestin in mucosa of the dilated segment in infants are higher than those in toddlers, and it decreases with age. These observations suggest the ENS stem cells or precursor cells in the gut mucosa may be derived from the gut neural crest cells. Key words: Hirschsprung's disease; ENS stem cells; SOX10; Nestin; TUJ1