Isolation And Culture Of Chicken Primordial Germ Cells

Recently, primordial germ cells(PGCs), which are the progenitors of the sperm or EGg cells, have provided an alternative source of pluripotent stem cells. The stem cells derived from PGCs are called embryonic germ cells(EG cells). To date, EG cells with proven germ-line transmission have been completely established only in the mouse with embryonic stem cells(ES cells).Gonadal primordial germ cells were got by isolating embryonic gonads and surrounding tissues from chicken embryos(5.5-6.5 days of incubation) and dissociating the tissues by standard trypsinization procedure(0.25%Trypin+0.02%EDTA). The EG cell culture media consisting of DMEM medium supplemented with FBS, chicken serum, beta-mercaptoethanol, L-glutamine. HEPES, chicken embryonic extract and cytokines etc. After 24 hours culture, the isolated PGCs were selectively attached on the gonadal stromall cells in the plates. However, at 3~4 days primary culture, almost all the PGCs were formed of 2~20 cell mass. After 5~7 days, the numbers of putative EG clonies increased distinctly. These clonies were uniformly round, multi-layered and well delineated. The cells did not pack strongly together in small clumps and it was not difficult to discern the individual component cells. This morphology was slightly different from that of mouse EG cells.At primary culture, PGCs co-cultured with their gonadal stromall cells were well grown . When subculture, we used primary chicken embryonic fibroblast (PCEF), primary mice embryonic fibroblast (PMEF) and SNL cells to make feeder cells. Forward research founded that the PCEF cells were the most suitable for the growth of putative EG cells when having various cytokines. In this study, the putative EG cell culture media was supplemented with LIF, bFGF, SCF and IGF-1. As a result, the idealdose of LIF, bFGF, SCF and IGF-1 was: 100IU/ml,10ng/ml, 10ng/ml, 10ng/ml. In addition, there were still many other factors were influencing on the culture of putative EG cells such as the density of FBS, the cell-dispersed liquid, the temperature and PH of the culture environments etc.In this study, the chicken putative EG cells continued to- be PAS positive and manifested some typical morphology. Under suspension culture without cytokines, the putative EG clonies could be induced to form simple embryoid bodies. After long-time culture, these bodies began to differentiated into various cell types. Moreover, chimeras when injecting of the cultivated cells into receptor's x stage blastdermal also showed that the cells were some pluripotent. These study may be useful to the production of transgenic chickens by genomic modification.