Effects Of Different Feeder Layers On Bovine Embryonic Stem Cells Culture

A lot of research on bovine embryonic stem cells (bESC) have been done, but so far it has not been established a suitable environmental conditions for bESC lines. It is important to explore suitable feeder layers for bESCs so as to maintain their undifferentiated state and to ensure the pluripotency of cells. In this research, the growth of bESCs derived from ICM of bovine IVF embryos was observed under the conditions of different feeder layers. The bESCs were evaluated by a series of detections including alkaline phosphatase staining, formation of embryoid bodies and immunofluorescence staining. The expression of pluripotency-related maker genes OCT-4、SOX2and NANOG of same and different passges of bESCs which cultured on different feeder layers were tested in the method of quantitative RT-PCR(RT-qPCR), so that to find a suitable feeder layer for bESC cultue. The results could contribute to establishment of bESC lines.1, Bovine and mouse embryonic fibroblast cells were treated for2.0-3.0h with mitomycin C respectively. Mixed feeder layers were mixed according to1:0,1:1,1:2,2:1,0:1, and then cultured in the center plate coated by gelatin at2-2.5×104per well. Growth states of bESCs cultured on different feeder layers were observed and undifferentiated phenotypes were detected, including expression of alkaline phosphatase, presence of OCT-4, SOX2, NANOG and cell marker OCT-4, SSEA-1. Without feeder layer, bESC differentiation was observed in vitro. Comparison of bESC growth state on different feeder layers, the clonal morphology on the mixed feeder layer was significantly better than that on mouse or bovine embryonic fibroblast feeder layer. When mouse and bovine embryonic fibroblasts were mixed at a ratio of1:1accumulated significantly and cloning edge was clear, obvious and full. Expression of alkaline phosphatase, antigen of OCT-4and SSEA-1were strongly positive in the bESCs, specific bands of OCT-4, SOX2and NANOG mRNA appeared respectively. bESCs cultured on mixed feeder layer were able to form embryoid bodies. Comparison with conventional feeder layers prepared by mouse or bovine embryonic fibroblasts, the mixed cells feeder layer at a ratio of1:1may be more suitable for bESC culture in vitro and obtain better bESC clonal morphology.2, In this research STO cells were treated by domestic mytomycin in different concentration, and were used as feed layer cells to culture bESC. The aim of this study was to establish a suitable culture environment for bESC. STO cells was treated with domestic mytomycin (in10、15、20、30、40ug/mL) for1.5、3、4h respectively, and growth states of STO cells were observed in difference generations. bESCs cultured on the STO feeder layers were detected, including expression of alkaline phosphatase, presence of OCT-4and SSEA-4, and embryoid body formation in vitro. The results indicated that domestic mytomycin (in15μg/mL for3h) could inhibit effectively the division of STO cells, but not affect vitality of STO cells. The STO cells from five to ten generations could be used for feeder layers. BESCs cultured on the STO feeder layer cells expressed alkaline phosphatase, antigen of OCT-4and SSEA-4in positive strongly, and formed embryoid bodies. The results suggested that domestic mytomycin could inhibit effectively the division of the STO cells, and the STO cells could be used as feeder layers for culturing bESCs in vitro.3、Recent findings identifying the transcription factors involved in the regulation of pluripotency and self-renewal in ESCs may provide keys that enable the derivation of ESC in domestic species. In order to find the fundamental reasons that bESCs cultured on MEF feeder layer,can not continuous passaging growth, the bESCs cultured on MEF feeder layers growth in the first to fifth passages were detected on differential expression of the three major pluripotency-related transcription factors of OCT-4, SOX2and NANOG by RT-qPCR. The results showed that the expression levels of OCT-4、SOX2and NANOG were significantly reduced with passage, which can cause the reduction of totipotency bESCs and the promotion of relevant differentiation gene expression. Compared cultured on STO cells feeder layer and mixed feeder layer to MEF feeder layer, the bESCs in fifth passage were detected the expression of OCT-4、SOX2、NANOG by RT-qPCR. The results indicated that the expression levels of OCT-4, SOX2and NANOG of bESCs cultured with mixed feeder layer and STO cells feeder layer were improved in varying degree. The results suggested mixed feeder layer and STO cells feeder layer were better than MEF feeder layer in regulation of pluripotency self-renewal in bESC, and more suitable for culture conditions of bESC.