Effect Of HAMSC On Differentiation Of Mouse Ovarian Surface Epithelial Cells

Objective: The co-cultured model was established on mouse ovarian epithelial cells(OSEC) cultivation with human amniotic-derived mesenchymal stem cells(h AMSC) in vitro. To preliminarily explore affection of h AMSC on OSEC differentiation into Oocyte-like cells.Methods: Enzyme digestion method and differential velocity adherent were used for isolation and purification of h AMSC and mouse OSEC. Then the phenotypes of h AMSC and OSEC were identified by flow cytometry and immunocytochemical staining. The co-cultured model was established on OSEC cultivation with h AMSC using transwell plate. The change of mouse OSEC morphology was observed. The expressions of epithelial cells, pluripotent and germ cell markers were detected by immunohistochemical, image analysis and real time polymerase chain reaction(RT-PCR).Results: 1 The fourth generation hAMSC show long fusiform, eddy-like and adherent growth. h AMSC highly express CD44, CD90, CD73 and CD105, but not express CD45, CD34, CD19, CD11 b and HLA-DR. 2 Immunocytochemical staining results showed that the second generation of mouse OSEC expressed positive broad-spectrum keratin protein Pan-cytokeratin, suggesting that the mouse OSEC was derived from epithelial cells. The expression of epidermal keratin Pan-cytokeratin, germ cell specific marker Ddx4/MVH and pluripotent marker protein Oct4 of mouse OSEC in co-culture group and alone culture, were detected after one week. The average integral optical density values of Pan-cytokeratin, Ddx4/MVH and Oct4 protein expression in were increased compared with alone OSEC culture(P<0.05). 3 RT-PCR results suggested that the levels of Ddx4/MVH, Nanog and Gdf9 expression in co-culture group were high than alone culture group(P<0.05). However, the expressions of zona pellucid 1, 2 and 3 genes could not be detected in two groups in vitro.Conclusion: Mouse OSEC in vitro could express germ line stem cell specific markers such as Oct4, Naong, Ddx4/MVH and Gdf9. It suggested that OSEC might have the regenerative potential of oocyte. The study confirmed that h AMSC could further promot and support the differentiation of mouse OSEC into oocyte-like cells or oocyte-like structure formation in vitro coculture system. However, the specific mechanism remains to be studied.