Human Pluripotent Stem Cells Differentiation To Female Germ Cells

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) cultured inappropriate environment have the ability to differentiation to female germ cells, which wereuse to generate the live offspring after reconstituted with female gonadal somatic cells.However, little is known about female germ cells derived from human stem cells, because ofprecious human biological materials and ethics issue.Therefore, we try to derive female germ cells from human amniotic fluid stem cells(hAFSCs) and ESCs by adding porcine follicular fluid (pFF) into culture medium, themethods and results are as follows:1. The cultivation and identification of hAFSCsRecovery and subcultured the hAFSCs lines of070607(46, XX),070609(46, XX) and091122(46XY), which were isolated from our lab. The early passage hAFSCs were detectedpluripotency genes, cell surface antigens and the expression of specific proteins by RT-PCR,flow cytometry and immunofluorescence respectively. The results showed that the cell line070607with vigorous expressed embryonic stem cell marker genes OCT4, NANOG, andCD117, and mesenchymal stem cell marker genes CD90, CD44, and CD29, but not expressedhematopoietic stem cell gene CD45; And the cell line showed ESCs specific protein OCT4,SOX2, SSEA-4, TRA-1-60, and CD117positive; meanwhile, this cell line retained themesenchymal stem cell (MSC) markers CD29(99.2%), CD44(99.8%), and CD117(99.8%),and lacked hematopoietic stem cell markers CD34(0.1%) and CD45(1%).2. Female germ cells derived from hAFSCsTo encourage differentiation into female germ cell, the cell line070607hAFSCs werecultured by two steps. The first step: the hAFSCs was induced by adding porcine follicle fluidinto germ cell medium about10~15days, and then, cell aggregates would be found, collectedthem and cultured into oocyte growth medium containing hormones and growth factors bysecond step. Following the female germ cell-like cells would be found, such as primaryfollicles, secondary follicles, cumulus oocyte complexes and oocytes.The germ cell markers: OCT4, IFITM3, DAZL, FIGLA, FSHR, CYP17, and P450were up-regulated by real-time quantitative PCR. And the differentiation triggered meiotic geneDMC1and granular cell specific gene FOXL2at later stage. After induction, the hAFSCsshowed haploid gradually by flow cytometry. At the same time, pBMP15-EGFP wastransiently transfected into hAFSCs during different time point; the result showed big roundcell expressed green fluorescent protein after the differentiation of10days. We also detectedthe induced cell expressed germ cell specific markers OCT4, NANOG, CD117, and ZP2usingimmunofluorescence, and the expression of ZP2also showed positive by western blotting.Additionally, parthenogenetic embryonic were also generated by adding porcine follicle fluid.Above all, the hAFSCs can be directional differentiation to female germ cells undersuitable culturing.3. The cultivation and identification of human embryonic stem cell and generated tofemale germ cellThe hESCs lines H1(46XY) and H9(46, XX) were cultured based on mouse embryonicfibroblast cells feeder layer or feeder-free culture system. The stem cells marker genes OCT4and NANOG, and proteins OCT4, SOX2, TRA-1-60, TRA-1-81, and SSEA-4were showedpositive respectively by RT-PCR and immunofluorescence. Embryoid bodies with moreuniform cell structure were made from hESCs under feeder-dependent. At the same time, thefemale germ cell can derived from monolayer hESCs and expressed female germ cellsmarkers: CD29, DAZL, STELLA, DMC1, BMP15, GDF9and so on, While, Embryoid bodieswere triggered to form trophoblast cells.