Directed Differentiation Of Human Parthenogenetic Embryonic Stem Cells Into Intestinal Epithelial Stem Cells

Background : Inflammatory bowel disease(IBD)is a group of intestinal inflammatory response diseases,often accompanied by complications,and the incidence of the disease is increasing year by year in the world,which seriously influences the human health.Its pathogenesis has not been studied clearly,and it is thought to be caused by many factors,including environment,infection,genetics,immunity and other factors.The conventional treatment of drugs can not meet human needs.At the same time,with the rise of regenerative medicine,stem cell therapy plays an increasingly important role.Human parthenogenetic embryonic stem cells(hPESCs)have similar pluripotency to embryonic stem cells in terms of differentiation ability,and it have differentiation omnipotence,but have no ethical controversy.It have great potential in the treatment of IBD.This experiment is the process of differentiation of hPESCs into intestinal epithelial stem cells(IESCs).Objective: To explore the feasibility of inducing differentiation of hPESCs into IESCs in vitro,and to provide a more practical treatment for patients with IBD.Experimental method:1)Established the hPESCs/MEF?hPESCs/hFF?hPESCs/3T3-L1 culture system in vitro.The morphological observation and alkaline phosphatase assay were used to detect whether hPESCs could grow stably and remain undifferentiated.2)Inducing factors Activin A induced differentiation of hPESCs into definitive endoderm(DE).By detecting the expression of Sox17,Gsc,and E-Cad,it was judged whether the induced cells were transformed into DE.3)Inducing factors epidermal growth factor(EGF)induced differentiation of DE into IESCs.By detecting the expression of Musashi1 and Hes1,it was judged whether the induced cells were transformed into IESCs.Experimental results:1)hPESCs grew well in hPESCs/MEF?hPESCs/hFF?hPESCs/3T3-L1 culture system.After more than 60 generations in vitro culture,the cells remained in anundifferentiated state.2)The expression of the DE markers Sox17,Gsc and E-Cad in the induction group was significantly higher than that in the control group,and the expression level reached the peak on the 4th day.3)The expression levels of IESCs markers Musashi1 and Hes1 in the induction group were significantly higher in the control group,and the expression level reached the peak on the 5th day.Conclusion:1)hPESCs/MEF?hPESCs/hFF?hPESCs/3T3-L1 culture system can effectively amplified hPESCs and maintain their undifferentiated state.2)The Activin A can successfully induce differentiation of hPESCs into DE.3)The EGF can successfully induce differentiation of DE into IESCs.4)The phase-induced induction method successfully differentiated hPESCs into IESCs.