Formation Of Follicle Like Structure And Sex Hormone-related Gene Expression In Hanging Drop Culture Of Female Bone Marrow Mesenchymal Stem Cells

Objective:To investigate whether female rat bone marrow stem cells (FBMSCs) can form follicle like structure and produce steroidogenic cells with the capacity for sex hormone synthesis in hanging drop culture, and to provide basis for clinical application of bone marrow stem cells (BMSCs) in cell transplantation therapy for patients with premature ovarian failure.Methods:The FBMSCs were isolated from female rats and cultured in the low-glucose DMEM supplemented with 10% fetal bovine serum (FBS). The cultured cells at passage 3 were treated with all-trans retinoic acid (ATRA) to induce differentiation in hanging drops. The formation of follicle-like structure was observed and the expression of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), P450arom and P450scc mRNA in FBMSCs was examined by RT-PCR. The expression LHR was detected by immunocytochemical staining.Results:1. FBMSCs of 3rd passage appeared morphologically as a homogeneous population of spindle-shaped cells.2. FBMSCs of 3rd passage cultured in low glucose DMEM with ATRA (10-5mol/L) formed follicle-like structure. LHR protein was found in cultured FBMSCs. The nuclei of the cluster-forming cells were oval, with a blue staining. The levels of E2,testosterone (T) and progestone(P) in the media measured by radioimmunoassay (RIA) were lower and no different in 0,1,2,3,4 day after cell sphere plated 12 well plate on hanging drop culture 3 days.3. LHR and P450scc expressed in FBMSCs after isolation from female rats aged 2,4,8 and 16 weeks. The expression of FSHR and P450arom mRNA was mainly found in rats aged 16 weeks. Conclusions:FBMSCs can form follicle-like structure in hanging drop culture and express FSHR, LHR, P450arom and P450scc in vitro. In vivo female rat bone marrow stem cells can spontaneous differentiate into sex steroidogenic cells.