Isolation, Identification And In Vitro Culture Of Mouse Primordial Germ Cells

Mammalian primordial germ cells (PGCs), considered the gamete precursors, are segregated and specified from early embryo, with pluripotency and specified migrate potential. In recent years, PGCs were used as a research model for cell fate determination, cell migration, and epigenetic cell reprogramming. But the limited cell number and difficult to purification, makes related research remains to be deepened. This study was focused on the isolation and PGCs identification of mouse PGCs cells, and its culture in vitro.In his study, mouse embryo at 10.5 dpc and 13.5 dpc were selected for PGCs sorting. Cells were dispersed from tissues by using mechanical method and enzyme digestion method, after marked with specificity antibody to SSEA-1, cells were sorted by flow cytometry. The sorted cells were identified by alkaline phosphatase (AKP) and pluripotency specific molecular markers OCT4 by immunofluorescence staining, and also the expression of PGCs specific gene were detected by RT-PCR. Results showed that 86.80% of sorted cells has positive signal after alkaline phosphatase staining, also the sorted cells were OCT4 and SSEA-1 positive. RT-PCR results showed that the sorted cell expressed PGCs specific marker genes, such as Prdml, Lhcgr and Dppa3 and Nr6al.A culture system of embryonic germ cells (EG) were adopted for PGCs culture, cells obtained by mechanical and enzyme digestion method were cultured, and different feeding layer (MEF and STO) or Basement Membrane Matrix were compared. After culture, cells from 13.5 dpc embryos did not form colony, while cells from 10.5 dpc embryos formed colony in the three culture system, which were further examined. Results showed that it is more easier to form colony by using the Matrix, the shape of colony is bigger, nest-like and have obvious boundaries surrounded. The colony were positive for alkaline phosphatase staining, and also positive for pluripotency specific molecular mark OCT4 after immunofluorescence staining. While, by using MEF and STO feeding layer, formed colony were positive for OCT4 staining, but the rate of colony formation is less than that of Matrix, colony is smaller, and the boundaries of colony is blurred. The Results suggested that cultured with Matrix helps cultured cell maintaining their pluripotency.In summary, this study obtained cells expressing PGCs specific markers by using flow cytometry sorting. By using Matrix for culture, cell colony were formed, which expressed PGCs specific markers, the above study helps for further study the characteristics and function of PGCs.