Primary Culture, Subculture And Biological Identification Of Human Embryonic Germ Cells

Objective: To determine whether the cells that we obtained from the in vitro culture of human embyonic genital ridges and dorsal mesenteries were the undifferentiated human embryonic germ cells (hEGs) , to find out the best kind of passaging method , and finally to identify these serial passaged cells .Methods: Immunocytochemistrical stain which detects four specific markers including SSEA-1 , SSEA-3 , Oct-4 and telomerase was applied to ascertain whether the cells we obtained by tissue culturing of embryonic genital ridges and dorsal mesenteries were hEGs. Flow cytometry was performed to evaluate the approximate proportion of hEGs in the culturing system. Different time and various methods were employed to passage the hEGs which included pick-up method and digestive method. And immunocytochemistry was also performed to identify the biological characteristics of the passaged cells.Results: The primary hEGs expressed 4 kinds of molecular markers positively. Flow cytometry showed that about 13% of all the cells in the culturing system we adopted were hEGs. The number of hEG colonies obtained through passaging by digestion with trypsin-EDTA was larger than that by the other method. During the passaging of hEGs, it was also observed that the hEGs were thriving at 8 days after plated while the hEG colonies began to differentiate or disappeared at 16 days after plated. Immunocytochemistry showed that the fifth generation of hEGs colonies still expressed the four kinds of molecular markers as chosen above.Conclusions: The best time for passaging of hEGs is 8 days after plating while the best method is digestion by trypsin-EDTA .It is also proved that the subcultured cells are still undifferentiated hEGs.