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

60

Co γ-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.