Derivation And Identification Of Human Embryonic Germ Cells

Human embryonic germ(EG) cells from human primordial germ cells(PGCs) are pluripotent stem cells. At present, although the derivation and culture of the mouse and human embryonic stem cells have achieved great progress, there has been little report about human EG cells'development, differentiation, isolation and culture system. So, it is important to research the human EG cells derived from PGCs.1.The origin, migration and gene expression of human PGCsWe investigated histological structure, morphology and genes(OCT4 and hTERT) expression of 6～19 week human gonadal ridges by PAS staining, RT-PCR and immunohistochemistry. The result showed that human gonadal ridges had OCT4 positive cells in human embryos after 6 weeks, but the number of positive cells was small. The number of OCT4 positive cells increased in 8～12 week embryos. 13 weeks later, the positive cells decreased demonstrating the differentation of abundant PGCs. During 6～15 week, human gonadal ridges also had hTERT positive cells. Moreover the hTERT positive cells were more abroad. But it need further study.2.The effects of the condition medium and the different LIF concentration on human PGCs cultureThere were 7 condition mediums and 5 mediums with different LIF concentration used in human PGCs culture. The results showed that Knockout serum replacement medium or STO condition medium both had best culture effect in vitro. 10 ng/mL LIF had best culture effect, but there was no significant difference between the 5 ng/mL and 15 ng/mL groups.3.The identification of human EG cellsUsing MEF feeder layer and DMEM+15%KSR+10ng/mL LIF+4ng/mL bFGF + 10μm/mL Forskolin, human EG cells were passaged 5 times. Human EG cells possessed SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, OCT4, C-kit and hTERT markers by immunohistochemistry in this study. The EG cells were AKP positive. OCT4 and hTERT expression of the EG cells could be tested by RT-PCR.