Isolation and Enrichment of Mouse Female Germ Line Stem Cells

Objective: the existence of female germ-line stem cells (fgscs) has been the subject ofa wide range of recent studies. successful isolation and culture of fgscs could facilitatestudies on regenerative medicine and infertility treatments in the near future. our aim inthe present study was evaluation of the most commonly used techniques in enrichment offgscs and in establishment of the best procedure.materials and methods: in this experimental study, after digesting neonate ovary fromc57bl/6 mice, we performed 2 different isolation experiments: magnetic activated cellsorting (macs) and pre-plating. macs was applied using two different antibodies againstmouse vasa homolog (mvh) and stage-specific embryonic antigen-1 (ssea1) markers.after the cells were passaged and proliferated in vitro, colony-forming cells were characterizedusing reverse transcription-polymerase chain reaction (rt-pcr) (for analysisof expression of oct4, nanog, c-kit, fragilis, mvh, dazl, scp3 and zp3), alkaline phosphatase(ap) activity test and immunocytochemistry.results: data showed that colonies can be seen more frequently in pre-plating techniquethan that in macs. using the ssea1 antibody with macs, 1.98 ± 0.49% (mean ± sdv)positive cells were yield as compared to the total cells sorted. the colonies formed afterpre-plating expressed pluripotency and germ stem cell markers (oct4, nanog, c-kit, fragilis,mvh and dazl) whereas did not express zp3 and scp3 at the mrna level. immunocytochemistryin these colonies further confirmed the presence of oct4 and mvh proteins,and ap activity measured by ap-kit showed positive reaction.conclusion: we established a simple and an efficient pre-plating technique to culture and toenrich fgscs from neonatal mouse ovaries.