Human Pluripotent Stem Cellsdifferentiation To Female Germ Cells

Generation and maturation of human female germ cells in vitro will enable studies of female germ cell development and oogenesis.Human pluripotent stem cells(hPSCs)cultured in appropriate conditions can be differentiated into specialized cell-types and organoids in vitro,however the generation of human ovarian follicle and oocyte remains a challenge.Here,we established a three-step protocol to differentiate primordial germ cell-like cells(PGCLCs)from embryonic stem cells(ESCs)and induced pluripotent stem cells(iPSCs)to the stage of follicle-like structures(FLs)and finally obtained human oocyte-like cells(OLCs)by adding cytokines(hLIF,hSCF,EGF,and BMP4)and bovine follicular fluid(bFF)into the culture medium.Methods and results are as follows:1.hPSCs culture and hiPSCs differentiatiate into primordial germ cell-like cellsBoth iPSC-TAC153(46,XX)and hESC line H9(46,XX)were cultured based on feeder-free culture system.Quality control results showed that these lines have all characteristic of pluripotency.During the first step,hiPSCs were cultured in primordial germ cell medium(PGC-m)for 10 days to produce the induced primordial germ cells(iPGCs)in vitro.At this stage,iPGCs gradually acquired the morphology of primordial germ cells,expressing characteristic germ cell markers.The transcriptome sequencing showed that numerous genes involved in ovarian development were upregulated in iPGCs.2.Follicle-like structures derived from induced primordial germ cellsDuring the second step,iPGCs were cultured in the follicle-like cell medium(FLC-m)for an additional 5 days,where the induced follicle-like structures(iFLs)were obtained from iPGCs aggregates,with characteristic primordial oocytes-like and granulosa-like cells.And meiotic gene SCP3 and granular cell specific gene FOXL2 showed positive at this stage.3.Follicle-like structure-derived oocyte-like cells and the three-step protocol`s validationDuring the third step,the detached iFLs were harvested and cultured in the oocyte-like cell medium(OLC-m)for an additional 10-15 days.The cumulus-oocyte-complex(COC)structures and OLCs were observed in different sizes(50-150 ?m diameter)and with zona pellucida.The differentiation triggered zona pellucida markers ZP2 and ZP3.At later induction stage,hPSC-derived OLCs presented with the first polar body and arrested at MII stage after in vitro maturation.In addition,this study used the hESC line H9 to validate the three-step induction protocol.Interestingly,some OLCs were self-activated and spontaneously developed into multiple-cell structures similar to preimplantation embryos positive for DDX4 and c-KIT.This study shows that OLCs can be derived from hPSCs by a three-step protocol,which provides a platform to study human female germ cells formation and oogenesis.