Isolation,culture and primary identification of human primordial germ cells / 第三军医大学学报

ABSTRACT

Objective To explore how to isolate and culture human primordial germ cells(PGCs).Methods Human embryonic fibroblasts(HEFs) isolated from embryos at 2 to 3 months of age received a radiation of 60Co ?-ray and then served as feeder layer cells.Cells collected from trypsinized human embryonic gonadal ridges and dorsal mesenteries(5 to 9 weeks post-fertilization) were cultured on HEF feeder layers in the medium with recombinant human leukemia inhibitory factor(LIF),recombinant human basic fibroblast growth factor(bFGF) and forskolin.Histochemical and immunocytochemical assays were used to identify the stage specific embryonic antigen-1and 4(SSEA-1/4) of the cultured cells.Reverse transcription polymerase chain reaction(RT-PCR) was used to identify the expressions of specific genes,including Oct-4,Ifitm-3,stella,Mvh and DAZL.Embryonic bodies were cultured as while.Results The collected cells were growing on the feeder layer and formed a typical nestlike multicellular colonies.The cells showed diploid chromosome in karyotype analysis and expressed SSEA-1/4 and specific genes,Oct-4,Ifitm-3,stella,Mvh and DAZL,indicating that the cells were PGCs.When cultured in hanging drop,the PGCs developed into embryonic bodies,showing multipotential ability.Conclusion Human PGCs can be isolated from genital ridges and dorsal mesenteries of embryos and cultured in vitro on HEF feeder layer.The formation of the embryonic stem cell colony can be observed,and the cells cultured by this method is confirmed to be embryonic germ cells.