The Effects Of FSH,hCG On Forming Follicle-like Structures And Secreting Sex Hormones Of Bone Marrow Mesenchymal Stem Cells Cultured In Hanging Drop

Objective：The aim is to explore whether the female rat bone marrow mesenchymal stemcells (FBMSCs) can differentiate into oocyte-like cells, form functional follicle-likestructures and synthesize and secret sex hormones after hanging drop culture and toobserve the effects of all-trans retinoic acid (retinoic acid, RA), human chorionicgonadotropin (hCG) and follicle-stimulating hormone (FSH) on the differentiation ofFBMSCs. This study will provide evidences for investigating the mechanisms ofFBMSC transplantation therapy for patients with premature ovarian failure orosteoporosis and lay a foundation for forming artificial ovary.Methods：FBMSCs were separated from adult female SD rat bone marrow. FBMSCs fromthe2nd passage adherent cell fraction were cultured in hanging drop for3days andthen transferred to the plate culture. Basic culture medium was DMEM with20%fetal bovine serum. The effects of RA, FSH and hCG on FBMSC differentiation andfollicle-like structure formation were observed. In order to assess whether thefunctional oocytes and follicles were derived from FBMSCs, we tested the markers ofstem cells and specific follicle cells and measured the sex hormone levels. Themorphological changes in cells were observed under an inverted microscope. Theestradiol (E2), testosterone (T), progesterone (P) and androstenedione (A)concentrations in culture medium of FBMSCs were measured by radioimmunoassay(RIA). Dierentiated cells were analyzed by immunocytochemical staining andwestern blot for the expression of SCP3, ZP3, FSHR and LHR. The expression levelsof Stra8, Oct4, C-kit, Vasa, SCP3, ZP3, GDF-9, FSHR, follistatin, P450arom, LHRand P450scc mRNA were detected by real time RT-PCR.Results:1.The second passage FBMSCs expressed CD44and CD71, but not CD34andCD45. 2. FBMSCs could proliferate, differentiate and form cellular aggregates inhanging drop culture.3. Basic culture medium was DMEM with20%fetal bovine serum. TheFBMSCs in control, RA, FSH and hCG groups could proliferate, differentiate andform cell clusters within4weeks. After4weeks, cell mass and number of cells in RAgroup decreased, but FBMSCs in FSH group, hCG group and control groupcontinually proliferated, differentiated and formed a larger cell mass and follicle-likestructures at64days. Moreover, oocyte-like cells were extruded from the follicle-likestructures.4. FBMSCs in control, RA, FSH, hCG groups expressed Stra8, Oct4, C-kit, Vasa,SCP3, ZP3, GDF-9, FSHR, follistatin, P450arom, LHR and P450scc mRNA, andshowed SCP3, FSHR, LHR and ZP3positive cells and SCP3, FSHR and ZP3protein.The results suggested that FBMSCs differentiated into oocyte-like, granulosa cell-likeand theca-like cells.5. The levels of sex hormones in the first batch culture medium were detected byRIA at0,1,3,5,7,10,14,18,21,28,37,43days after hanging drop culture.Estradiol concentrations in the4groups significantly increased at21-43days,androstenedione and testosterone increased at10-21days, and progesterone increasedat5-7days. The levels of sex hormones in the second batch culture medium weredetected at0,30,48and64days. Estradiol concentrations in the4groups increased at48-64days, and androstenedione and testosterone increased at30-64days. Nosignificant changes in progesterone were found.Conclusions：The results show that FBMSCs in hanging drop culture can forme follicle-likestructures, differentiate into oocyte-like cells, granulosa cell-like cells and thecacell-like cells, and synthesize and secrete of sex hormones.