| Endometriosis (EM) is one of the common benign gynecological diseases and its major symptoms are dysmenorrhea, infertility, dyspareunia which seriously affect women's health and quality of life. Several theories have been addressed the origin of EM, including Sampson's theory, metaplasia theory, immune theory and so on. However, the pathogenesis of EM still remains unclear. Several studies on stem cells have indicated that the stem/progenitor cells exist in endometrium, which induce the periodic regeneration of endometrium. The endometrial stem cell hypothesis assumes that the ectopic lesions may be not capable to maintain their long-time development; meanwhile the ectopic differentiation of stem cells may be a novel mechanism of the disease. So many studies supported that endometriosis is a stem cell-related disease. In previous study, we had confirmed that the male mice bone marrow-derived stem cells could engraft and differentiate into endometrial glandular epithelium both in the eutopic and ectopic endometrium, estrogen might play a key role. In present study, bone mesenchymal stem cells (BMSCs) of mice and endometrial stromal cells (ESCs) were isolated and co-cultured with exogenous factors (17β-estradiol, TGF-β,EGF, PDGF-BB).We also studied their differentiation time and the effect of estrogen. The results suggest that BMSCs have the potential to differentiate into endometrial epithelial cells in vitro. Our data not only provide evidences for the EM stem cell theory, but also seek a new guidance for the clinical intervention on EM.Objective:1,To study the differentiation of mice bone mesenchymal stem cells into endometrial epithelial cells.2,To confirm the effect of estrogen on its differentiation.Materials & Methods:1,Isolation and culture of Mice's BMSCs and ESCs, identified by flow cytometry and immunocytochemistry.2,BMSCs were treated as followings. Group 1:BMSCs cultured alone as control (DMEM/F12,2%FCS); Group 2:BMSCs cultured in the differentiation medium (DMEM/F12,2% FCS, 1×10-7mol/L 17β-estradiol, lOng/ml TGF-β,10ng/ml EGF, lOng/ml PDGF-BB); Group 3:BMSCs co-cultured with ESCs in control medium; Group 4:BMSCs co-cultured with ESCs in the differentiation medium. Realtime RT-PCR and immunofluorescence were used to test the expression of epithelial cells'marker cytokeratin-7(CK7), cytokeratin-18 (CK18), cytokeratin-19 (CK19) and epithelial membrance antigen (EMA) after 5 days.3,BMSCs were co-cultured with ESCs for one, three, five, seven days in control media. Real-time RT-PCR was used to test the expression of epithelial cells' marker CK7, CK18, CK19 and EMAin BMSCs.4,BMSCs were co-cultured with ESCs adding different concentration of 17β-estradiol (0, 1×10-9mol/L,1 X 10-8mol/L,1 X 10-7 mol/L,1 X 10"6mol/L). Realtime RT-PCR was used to test the different expression of epithelial cells' marker CK7, CK19 and stromal cell marker vimentin (Vim) and stem cell transcription factor (Oct-4) after 5 days.Results:1,Flow cytometric analysis showed the immunophenotype of BMSCs sca-15.36%, CD1172.41%, CD29 2.63% and the hematopoietic markers CD34 0.68%. ESCs were analyzed by the immunocytochemistry for the expression of stromal cell marker vimentin positive.2,Realtime RT-PCR results showed the mRNA CK7 expression levels were significantly higher in group 2,3,4 than control and upregulated in sequence from group 1 to group 4. The mRNA expression levels of CK18, CK19 were significantly upregulated in group2,3,4 compared to group 1. As to EMA, the expression was significantly higher in group 2,4 than group 1,3. In immunofluorescence analysis, the results showed that undifferentiated BMSCs in group1 and 2 were negative for cytokeratin. However, the differentiated BMSCs in group 3 and 4 were weak and strong positive for cytokeratin.3,The epithelial cells marker CK7 expression increased with co-culture time and began to increase dramatically in the first five days. CK18, CK19 and EMA expression levels were no significant time differences.4,CK7 and CK19 expression levels were highest in 1×10-8 mol/L17β-estradiol group and CK7 expression was lowest in the 1×10-6mol/L 17β-estradiol group. The expressions of VIM and Oct-4 declined in differentiated BMSCs, but had no significant differences in different concentration of 17β-estradiol group.Conclusion:1,BMSCs could be differentiated into epithelial cells in appropriate condition in vitro. Exogenous factors (17β- estradiol and growth factors) and endogenous growth factors secreted by ESCs made the best microenvironment for BMSCs differentiate into epithelial cells.2,The epithelial cells marker CK7 expression increased with co-culture time and began to increase dramatically in the first five days.3,1×10-8mol/L17β-estradiol improved the epithelial differentiation during BMSCs into epithelial cells, but 1×10-6mol/L 17β-estradiol inhibited the epithelial differentiation.
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