Isolation,Establishment And Functional Study Of The Female Germline Stem Cell Line From Human Reproductive-age Women

Reproductive health is an important component of human health.Infertility,female health disorders,birth defects and other diseases are closely connected to the development of female germ cells.The discovery of human female germline stem cells(FGSCs)has opened new ways for understanding human oogenesis,delaying menopause,treating infertility,and preserving fertility.Ovarian tissue of human reproductive-age is mainly obtained by laparotomy and laparoscopic operation,the shortage of ovaries tissues available impedes their future investigations and clinical applications.Ultrasound guided transvaginal ovarian aspiration is used for collecting oocytes in assisted reproductive center and the operating process will produce follicular aspirates(FAs).In the whole world,at least 1,000,000 FAs were discarded after collecting oocytes,which resulted in a huge waste of clinical resources.Previous studies have found that preantral follicles exist in FAs,suggesting a new source of oocytes.We therefore assume that a small amount of ovarian tissues containing FGSCs can be collected into the FAs by ovarian puncture.The goal of this study was to obtain FGSCs from the abandoned FAs and to study their developmental mechanisms in vitro and in vivo.A small amount of ovarian tissues surgary removed from patients and the abandoned FAs from IVF centers were used in our study.Firstly,the proliferation activity of germ cells in ovarian tissues was identified by immunohistochemistry,and FGSCs(srFGSCs)was isolated and cultured from these ovarian tissues surgary removed from patients.Secondly,the scarce ovarian tissues were collected from FAs using a nylon cell strainer and the FGSCs were isolated from these tissues(faFGSCs).After long-term culture in vitro,the cell lines were established and their characteristics were analysized including germline characteristics were detected by immunofluorescence and RT-PCR analysis;the proliferation activity of cell lines were detected by BrdU incorporation;karyotyping and alkaline phosphatase activity,etc.Thirdly,the developmental mechanisms of faFGSCs were studied systematically in vivo and in vitro,including?)in vitro differentiation capacity under feeder-free conditions;?)communications with granulosa cells and ?)meiotic activity after cultured onto granulosa cells monolayers and retinoic acid(RA);?)follicle formation capacity of faFGSCs using intraovarian injection and xenografting.Lastly,we also developed and optimized a strategy guiding faFGSCs differentiation in vitro.We found that the germ cells with mitotic activity exist in human ovarian cortex and srFGSCs could be obtained from these tissues,meanwhile,the cell lines could be established in vitro.We also found that a small quantity of ovarian tissues were adhered on nylon mesh when FAs filtered using a cell strainer and widely exists in FAs,which part of thssues containing preantral follicles.Consistent with the results using ovarian tissue surgery removed from patients,these scarce tissues could also be used to establish the FGSCs lines.The faFGSCs have same characteristics of germline stem cells with srFGSCs,including the gene expression profile of germ cells,cell morphology and growth features.The faFGSCs cell lines also have a normal 46,XX karyotype,express weak alkaline phosphatase activity and cytokine GDNF receptor GFRA1.In addition,faFGSCs have differentiation potentials in vitro and in vivo including ?)spontaneous differentiation into oocytes after removing feeder cells;?)communicating with granulosa cells by gap junctions and paracrine factors for further differentiation;?)entering meiosis after RA induction,and ?)forming chimeric follicles with somatic cells in human ovarian cortex.Lastly,the three-step induction system could guide faFGSCs differentiated into GV oocytes in vitro.Our data revealed that a small amount of human ovarian tissues exist in FAs.The faFGSC lines could be established from these scarce tissues that similar to the tissues surgery removed from patients and could differentiate into GV oocytes in vitro.In summary,“waste” FAs can provide a new source of human FGSCs for clinical applications and basic science.The method for achieving FGSCs from abandoned FAs can not only reduce surgical risks,but also use clinical resources thoroughly.