Abstract：To find a suitable feeder layer for successful culture conditions of bovine embryonic stem cells is important. SIM mouse(Mus nusculus) embryo-derived thioguanine and ouabain resistant(STO) cells were treated by domestic mytomycin in different concentrations, which were used as feed layer cells to culture bovine embryonic stem cell. STO cells were treated with domestic mytomycin under 10, 15, 20, 30 and 40 μg/mL for 1.5, 3.0 and 4.0 h, respectively, and growth states of STO cells were observed in difference generations. Bovine embryonic stem cell cultured in the STO feeder layers were detected, including expression of alkaline phosphatase(AKP), octamer-binding transcription factor 4(OCT-4) and stage specific embryonic antigen 1 (SSEA-1), and formation of embryoid bodies in vitro. The results indicated that domestic mytomycin (under 15 μg/mL for 3.0 h) could inhibit effectively the division of STO cells, but not affect vitality of STO cells. Compared with conducts of Sigma, there were have the same effects, and there we not only could content the demand of preparation of feeder layers, but also could reduse the experiment costs. At the same time this research found that STO cells could be passaged proliferation in vitro long-term. However, STO cells affect the morphological structure which passed on long-term, eventually leading to the decline in the quality of feeder layers. The morphological changes of STO cells after continuous passaging indicated that of the STO cells was the better growth states than that of others generations before five generations, specially the fifth generation. Spreaded to the tenth generations, the cells occured degenerative changes and STO cells of dead rate obvious reaching to fourteenth generations. From the first to the tenth generations could been best used for feeder layers. Representative immuonofluorescence staining in the eighth passage of bovine embryonic stem cells were used. Results showed bovine embryonic stem cells cultured on STO feeder layer cells from the fifth generation to the tenth generation were in good morphology, and expressed AKP, antigen of OCT-4 and SSEA-1 in positive strongly fromed embryoid bodies. All results suggest that domestic mytomycin could inhibit effectively the division of the STO cells, and the STO cells can be used as conventional feeder layer for bovine embryonic stem cells culture.
