Effects of EGF and bFGF on proliferation and differentiation of human enteric neural crest progenitors in vitro

Objective To isolate and culture human enteric neural crest progenitors (hENCP) from human enteric muscular layer in vitro and observe the effects of growth factors EGF and bFGF effect on the proliferation of HENCP so as to provide experimental rationales for therapy of Hirschsprung' s disease using autonomous cells.Methods The gut muscular layer in non drug aborted fetus was stripped from the muscular layer under microscope.After digestion,single cells were seeded into serum free medium with bFGF or bFGF + EGF to observe the size and amount of neurospheres at Days 3,5,7 and observe the curve of optic density (OD) value in 1 week by methyl thiazolyl tetrazolium (MTT).After an addition of 10％ fetal bovine sera,neurospheres and differentiated cells were identified with immunofluorescent staining of p75NTR,peripherin and GFAP.Results Few cells could be separated from interstitial muscle in human embryo gut and formed neurospheres in simplified serum free medium.There were significant differences between proliferation and number of hENCP cultured with bFGF+ EGF and bFGF (P＜0.01) or EGF (P＜0.01).The staining of neurospheres and their derivatives indicated neurospheres could generate neurons,glial cells under serum induction in vitro.The percentage of differentiation into neuron in bFGF group was significantly higher than that in other groups (P＜0.01).The percentage of differentiation into glial cells in EGF group was significantly higher than that in other groups (P＜0.01).Conclusions With the capacities of self renew and proliferation,hENCP exists in human embryonic gut.After isolation and culturing in vitro,hENCP can differentiate into neurons and glial cells essential for enteric nerve system.EGF + bFGF may significantly promote the proliferation of hENCP.And EGF and bFGF may affect cell types differentiated from hENCP. Key words: Hirschsprung's disease;  Cell transplantation;  Neural crest