| Background and aimsAs one of the main forms of inflammatory bowel disease(IBD),ulcerative colitis(UC)is characterized by chronic inflammation in the gut and periods of remission and intermittent relapses,which greatly reduces the quality of patients’life.To date,the main goals of available treatment are to control intestinal inflammation and to balance immune regulation.However,mucosal healing should be the therapeutic goal for IBD patients to avoid recurrence.Intestinal stem cells(ISCs)are crypt base columnar cells interspersed between Paneth cells and expressed throughout the intestine.As proliferating cells move from crypt base to the villus tip,they undergo further division and differentiation until they are committed to one lineage of the intestinal epithelium.However,it is complicated and inefficient to isolate ISCs from the intestine,which hampers its wide application in clinic.Originally isolated from bone marrow,mesenchymal stem cells(MSCs)have drawn great attention due to its multipotency and regenerative properties.Nevertheless,it is unclear how to induce BM-MSCs into intestinal stem cell-like cells in vitro and whether the induced cells could function well in vivo.Based on the published work,our study was designed to induce BM-MSCs into epithelial like stem cells under the mediation of growth factors(Activin A,FGF2)and miR-17.It is presumed that miR-17 mediated-epithelial like stem cells could be applied to treat dextran sulfate sodium(DSS)-induced experimental colitis,which would be evaluated by injecting GFP+ cells into mice through enema.Part 1 Stepwise differentiation of BM-MSCs towards intestinal stem cell-like cellsObjective:To establish a simple and direct differentiation method to induce BM-MSCs into intestinal stem cell-like cells.Methods:BM-MSCs of passage 3 were seeded in 6 well plates.After adherence,different concentrations of Activin A(0,1,5,10,20 and 100 ng/mL)were added to the medium.After 5 days,the expression of Foxa2 and Sox17 was measured by qRT-PCR and western blot.Then the optimal concentration of Activin A was used in the following experiment.For intestinal stem cell-like cells differentiation,250ng/mL FGF2 was used in the following 4 days.Lgr5 and Musashi-1,two of intestinal stem cell markers,were evaluated at the end of the protocol.Results:The expression of specific markers of endoderm—Sox17 and Foxa2,could reach highest at the treatment of 5ng/mL Activin A on day 5.Sequentially,the expression of markers for intestinal stem cells Lgr5 and Musashi-1 was induced by FGF2.However,the proportion of Lgr5-positive cells was only 30.06%.Conclusions:BM-MSCs could be induced into definitive endoderm in the treatment of Activin A,and then could be successfully transdifferentiated into intestinal stem cell-like cells by FGF2,but the efficiency was low and need to be improved.Part 2 miR-17could facilitate the differentiation of BM-MSCs towards intestinal stem cell-like cellsObjectives:To clarify the role of miR-17 in the differentiation of BM-MSCs towards intestinal stem cell-like cells as well as its mechanism.Methods:As afore mentioned,BM-MSCs could be induced into definitive endoderm by 5 ng/mL Activin A,and then cells were infected with a lentiviral system expressing miR-17 or lenti-NC according to the manufacturer’s instructions,followed by 250ng/mL FGF2 added to the medium.After 4 days of the treatment,the expression of Lgr5、Musashi-1、E2F1、WIF1and β-catenin was measured.To observe whether the intestinal stem cell-like cells could be further induced into intestinal epithelial cells,20ng/mL EGF was used in the following 16 days.The medium was changed every 3 days.Results:The proportion of Lgr5+cells could be increased to 89.14%by the overexpression of miR-17.Not only the expression of Lgr5 and Musashi-1 was elevated markedly,but the induction time could be decreased to 2 days.Exogenous miR-17 significantly knocked down E2F1 and WIF1 expression.Moreover,the intestinal stem cell-like cells could be further differentiated into epithelial absorptive cells by EGF in vitro.Conclusions:miR-17 might activate the Wnt/β-catenin signaling pathway through downregulating its direct suppressors E2F1 and WIF1,which subsequently increases the abundance of Lgr5-positive cells.Part 3 Effect of the transplantation of cultured GFP+ intestinal stem cell-like cells on the DSS-induced colitisObjective:To confirm that cultured intestinal stem cell-like cells could rescue damaged epitheliumMethods:Based on the preliminary results,3%DSS was used in the experiments.Thirty 6 week-old male C57BL/6 mice were randomly assigned into three groups.The first group was the control group,the second group was sham transplantation group,and the third group was the cell injection group.Acute colitis was induced among the second and the third groups by feeding mice with 3%DSS dissolved in drinking water for 7 days.Then the seventh and ninth day after the start of DSS administration,a total of 150 μl cell suspension containing 1×106GFP+ BM-MSCs derived-intestinal stem cell like-cells was transplanted into the third group by enema,while an equal amount of PBS was injected into colons of the second group.After the transplantation,on day 14,five mice of each group were maintained as usual before they were sacrificed and analyzed.The colon was excised for the histopathological analysis and immunofluorescene and cytokine measurements.Weight loss and bloody stool of mice were monitored daily to evaluate the severity of colitis.Then the rest of mice were sacrificed one month after the DSS administration,and colon sections were analyzed by immunofluorescene.Results:In the whole protocol,the body weights of the mice with engraftment were higher than those of sham-transplanted mice since 8d after the start of DSS administration.The mean length of colons in DSS+ISCs group was longer than that of DSS-treated mice that received PBS only.Histopathological examination of the colon sections in the distal tracts showed that ISCs treatment significantly reduced the severity of the inflamed area and the infiltration of inflammatory cells.Multiple GFP+areas concentrated in the middle and lower part of the colon,and they appeared as well-demarcated patches in the treated colons.Large cystic GFP+ structures below the surface of the treated colons were also observed.Four weeks after the DSS administration,the GFP+ cells appeared in the epithelium and differentiated into the absorptive cells expressing MUC2 and CK-18.Conclusions:The cultured intestinal stem cell-like cells readily integrated into the mouse colon,and could form self-renewing crypts.These cells are effective in ameliorating the pathological and clinical features of DSS-induced colitis.Local delivery of stem cells could be considered as a potentially safe and useful strategy for the treatment of IBD refractory to the current treatments.Conclusions1.BM-MSCs could be induced into intestinal stem cell-like cells through a sequent treatment of Activin A and FGF2.2.miR-17 could facilitate the differentiation of BM-MSCs towards intestinal stem cell-like cells through activating Wnt/β-catenin signaling pathway.3.Cultured BM-MSCs derived-intestinal stem cell-like cells could immigrate to the damaged mucosa and help rescue the damaged epithelium by differentiating into enterocyte and alleviating intestinal inflammation. |
PDF Full Text Request