A total of 219 embryonic-germ-cell-like (EG-like) clumps were derived from 15 selected goat fetuses. Isolation of primordialgerm cells (PGCs) based on co-culture with primary goat embryonic fibroblast showed no difference from traditional feederlayer-based culture method used in mouse and human. The putative primary EG colonies were multilayer clumps ofcompact cells with unclear cell-cell boundaries. Three subculture methods of goat EG-like colony, traditional enzymaticdigestion, mechanical cutting and combination of the both, were compared in this study. As a result, EG-like coloniestraditionally disassociated with collagenase Ⅳ could be subcultured for up to 4 passages. And the mechanicallydisaggregated EG-like colonies were successfully maintained 9-12 passages with or without enzymatic treatment. Thepluripotency of the EG-like colonies was identified by their specific marker staining, spontaneous differentiation andembryoid bodies (EBs) formation in vitro. Most goat EG-like colonies (> 80%) were AKP positive and immunocytochemicallycharacterized with positive SSEA-1, Oct-4 and c-kit staining but SSEA-4. Under the condition of delaying passage, goatEG-like cells could differentiate into fibroblast-like, epithelium-like, and neuron-like cells. In addition, EBs could beobtained successfully in routine hanging drop culture. The serum free culture system (feeder layer-based) used in thisstudy was suitable for keeping PGCs and EG-like cells in their undifferentiated condition, but failed to converse them toimmortal cells. These results indicated that mechanical cutting is an effective method for passaging goat EG cell colonies.However, the microenvironment of conversing EG cells to immortal cells is still unclear.
YANG Wei-feng GE Xiu-guo HUA Jin-lian SHEN Wen-zheng DOU Zhong-ying
