Isolation, Culture And Identify Of Goat Embryonic Stem Cells And Embryonic Germ Cells

Stable embryonic stem cells (ESCs) and embryonic germ cells (EGCs) lines, which may be perfect cells for making bio-reactor, have not yet been established. The aim of the present study was to isolate and culture ESCs and EGCs from goat embryos and fetuses of different development stage, and to compare some factors influencing the efficiency of establishing those stem cell lines by adopting the methods of mouse and human. The results obtained were as follows:1. A total of 45 goats, 42 Guanzhong dairy goats and 3 Boer goats, were superovulated with a result of total 341 embryos including 26 embryos of 4-cell to 16-cell, 144 morulae, 108 blastocysts and 63 hatched blastocysts. In the study of isolation of goat ESCs from different primary materials,"morulae, blastomeres of denuded morulae, balstocysts, ICM and embryonic discs", the blastomeres, ICM and embryonic discs were more suitable for the isolation of goat ESCs than the others. Due to the low attachment rate (for example, the attachment rate of blastocyst and ICM was significantly difference, 38.1%vs 76.5%, P﹤0.05), the whole embryos of morula and blastocyst were not suitable for the isolation of goat ESCs directly after both of them were seeded on feeder layers.2. The propagation characteristics of ICM were studied both in serum-containing and serum-replacement media on a feeder layer of mitomycin-inactivated mouse embryo fibroblasts (MEF). Our result indicates that the differentiation of ICM was decreased in serum-free media. The proliferation condition of ICM (total 48) were cultured by addition of different growth factors in serum-free media, and there are no statistical difference of primary colony formation among the seven groups (added to MEF conditioned medium, STO condition medium, rHF conditon medium, LIF, bFGF, both or none cytokine) on MEF cell feeder layer (p>0.05). However, addition of 10ng/mL bFGF may slightly promote the proliferation of goat ICM. These results suggest that it is suitable to culture goat ICM in serum-free media. Three methods of subculture of goat ESCs-like colonies, including traditional enzymatic digestion, mechanical cutting and combination of the both, were compared in this study. As a result, ESCs-like colonies traditionally disassociated with enzyme could be subcultured for up to 2 passages, while the mechanically disaggregated...