Differentation Of Human Umbilical Cord Mesenchymal Stem Cells Into Female Germ-like Cells

Human Umbilical Cord Mesenchymal Stem Cells (UC-MSCs) are ideal seed cells fortissue engineering because of convenient isolation, low immunogenicity, and large numbers ofcells, without controversy on social, ethical and legal aspects. Estabilishing germ celldevelopment and differentiation models in vitro by studying UC-MSCs differentiated intogerm cells has important significance to investigate the specialization and differentiation ofgerm cells and treat human infertility. Studies have proved that mesenchymal stem cells candifferentiate into germ line cells in suitable condition. The aim of the study is to explore thepotential of UC-MSCs differentiating into female germ cells in vitro.The study firstly identified the biological characteristics of UC-MSCs cultured in vitro,including the morphological characteristics, proliferation capacity, differentiation potential(fat, bone, cartilage, nerves, cardiac, and muscle cells) and characteristics of mesenchymalstem cells Furthermore, the optimal culture condition was screened and the mechanism ofPDGF promoting UC-MSCs proliferation was investigated. The optimal concentration ofPDGF was screened among different concentrations of PDGF to human UC-MSCs bycalculating cell numbers at2,4and6d respectively. Then the ninth passage of UC-MSCswere treated with PDGF in the the experimental group, with cells cultured with only fetalbovine serum (FBS) as control. The promotion of PDGF on the UC-MSCs proliferation wasstudied by counting cell numbers at2,4, and6d, and cells were cultured from the ninthpassage to the sixteenth to observe the cell viability. Next the expression of PDGF receptorwere detected by RT-PCR, and the different expression of genes related to cell proliferation,cell cycle and pluripotency were studied by immunocytochemistry and qRT-PCR. Finally, cellproliferation and were detected by BrdU staining. The rssults from the experiment show that adose-dependent function of the PDGF on the proliferation of human UC-MSCs.We sought to determine whether the critical germ cell-specific gene Figlα has impact onthe induction of oocyte-like cells in vitro. Surprisingly, some germ cell-specific genes wereup-regulated in UC-MSCs when transfcted with exogenous Figlα, and very fewer oocyte-like cells appeared, but failed to improve the induction efficiency of the follicular fluid (FF).Excitingly, we found when transfected with exogenous Figla gene, the female UC-MSCstransient transfected with a reporter plasmid which expresses GFP under the control of Figlapromoter expressed GFP. It should be emphasized that GFP was expressed only in the femaleUC-MSCs. At4d of induction with20%follicular fluid, the human UC-MSCs graduallyshaped into round from the spindle, and the size was about10μm. Oocyte-like cells wereformed at7d and reached40-50μm (female UC-MSCs) and25-40μm (male UC-MSCs).With the time extended to14d, oocyte-like cells failed to continue growing, remaining about40μm. Through QRT-PCR analysis, we found that germ cell-related genes were significantlyup-regulated compared with the control group at7d and14d. With the time extending, theexpression of related genes increased gradually (female UC-MSCs). However, in maleUC-MSCs, the expression of these genes except Stra8decreased along with the inductiontime extended to14d. Immunofluorescence analysis demonstrated that the inducedoocyte-like cells at14d expressed germ cell-specific markers OCT4, VASA, DAZL, ZP2,ZP3, and STRA8. Estrogen is key hormone in the process of follicle development andmaturation. After testing the estrogen level of induced cells, we found that the levels ofgroups in the3d,7d, and14d after induction were significantly higher than the controlgroup (0d). In addition, at3d the estrogen secretion reached the highest peak. In order toexplore the impacts of different gender of UC-MSCs on the process of germ celldifferentiation, male germ cell-specialized gene Sox9and female germ cell-specialized geneFoxl2were detected for the expression differences in the induction process. Result showedthat Sox9climbed sharply in female UC-MSCs after induced6-9days, and even more thanthe male. Until to12days its expression back to low level. However，the expression ofFOXL2has declined with induced time extend, especially the expression of Foxl2in thefemale UC-MSCs higher than male from beginning to the end.To study the potential of UC-MSCs differentiating into germ cells in vivo, wetransplanted UC-MSCs marked by BrdU into seminiferous tubule of male mice which haddamaged reproductive system and under the renal capsule of female mice. After30d oftransplantation, the evaluation of UC-MSCs differentiation in vivo was analysed by biopsyimmunofluorescence staining. The results showed the germ-like cells expressing germcell-specific markers VASA, DAZL and ZP2were also positive for BrdU.