Investigation Of Pluripotency In Mouse Bone Marrow Mesenchymal Stem Cells And The Interaction Between MSCs And Granulosa Cells

Objective:To explore the cause of multi-differentiation potential of mouse bone marrow mesenchymal stem cell(MSCs) and the effect of MSCs on mouse ovarian granulosa cells (GC) in vitro co-cultured.Methods:MSCs were isolated from the bone of the adult mouse and cultured in vitro.Total RNA were extracted from the mouse bone marrow (Day 0) and cultured mouse MSCs at 2,4,6,8 and10 days. The pluripotency marker genes Oct-4 and nanog, pluripotency related transcription factors Klf4 and c-Myc and the marker genes of three embryonal layers, such as CYP51, SM22αand nestin, which are typical genes specific for endoderm, mesoderm and ectoderm respectively, were detected by RT-PCR.MSCs and GC from mouse were cultured and purified in vitro using their adherent characteristics. A co-culture system was established by culturing MSCs in the Transwell insert and GC on the out plastic plates (6 wells). The cells were divided into 3 groups:alone cultured MSCs, alone cultured GC and MSCs co-cultured with GC. Total RNA were extracted from the cells of these 3 groups at the 4th day and the 6th day respectively. The ovarian marker genes mRNA were detected by RT-PCR. The pluripotency related genes and the three embryonal layers genes were also detected at the 4th day and the 6th days respectively. Cell proliferation of these 3 groups were detected by MTT assay after 3 days and 5 days respectively.Results:The mRNA of Oct-4, nanog, Klf4 and c-Myc were detected in mouse MSCs. Furthermore, in addition to SM22a, RT-PCR revealed the presence of mRNA for CYP51 and nestin during the primary culture of mouse MSCs. the mRNA of ZP1, ZP2 and ZP3, FSHR were detected in both alone cultured and co-cultured GC at the 4th day and the 6th day. But the mRNA of ZP3, FSHR and BMP-15 were different between co-cultured GC and alone cultured GC.The mouse MSCs promoted the proliferation of the mouse ovarian GC. No mRNA of ovarian characteristics genes were detected in alone cultured or co-cultured mouse MSCs. The mRNA of pluripotency marker genes and pluripotency related transcription factors were different between co-cultured MSCs and alone cultured MSCs, while the mRNA of genes of the three embryonal layers did not significantly change. In addition, the mouse ovarian GC promoted the proliferation of mouse MSCs.Conclusion:Mouse MSCs, which were isolated by adhesion purification, expressed the mRNA of the pluripotency marker genes and the marker genes of three embryonal layers. In vitro co-culture, the proliferation of the mouse ovarian GC and the mouse MSCs were promoted mutually and affected the quantity of mRNA for some genes.