Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro

Abstract

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1-5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

Fig. 1

The clonal morphology of bovine embryonic stem cell-like cells in different feeder layers: mouse embryonic fibroblast feeder layers (a); bovine embryonic fibroblast feeder layers (b); mixed embryonic fibroblast feeder layer at a ratio of 1:1 (mouse embryonic fibroblast feeder layers: bovine embryonic fibroblast feeder layers) (c); mixed embryonic fibroblast feeder layers at a ratio of 2:1 (mouse embryonic fibroblast feeder layers: bovine embryonic fibroblast feeder layers) (d); mixed embryonic fibroblast feeder layers at a ratio of 1:2 (mouse embryonic fibroblast feeder layers: bovine embryonic fibroblast feeder layers) (e). STO cell feeder layers (f). Cells from the fifth passage are shown

Fig. 2

Alkaline phosphatase staining of bovine embryonic stem cell-like cells: mixed embryonic fibroblast feeder layers of 1:1 (a); STO cell feeder layers (b). Both of which were positive for AP staining. Cells from the tenth passage are shown

Fig. 3

The RT-PCR analysis of bovine embryonic stem cell-like cells. OCT4, SOX2 and NANOG gene mRNA levels were observed in bovine embryonic stem cell-like cells cultured on mixed embryonic fibroblast feeder layers at 1:1 (similar results were obtained for bovine embryonic stem cell-like cells grown on STO cell feeder layers). Cells from the eighth passage are shown. M (50 bp) maker; 1 NANOG (195 bp); 2 OCT4 (106 bp); 3 SOX2 (121 bp); 4 GAPDH (128 bp); 5–8 negative control

Fig. 4

Representative immuonofluorescence images showing expression of OCT4, SSEA-1, and SSEA-4 in the bovine embryonic stem cell-like colony. Bovine embryonic stem cell-like colonies cultured on mixed embryonic fibroblast feeder layers were stained positively for OCT4 (a2) and SSEA-1 (b2); Bovine embryonic stem cell-like cells cultured on STO cell feeder layers were also stained positively for OCT4 (c2) and SSEA-4 (d2); negative controls for OCT4 (a3, c3), SSEA-4 (d3) and SSEA-1 (b3). All cells were stained with DAPI to highlight the nucleus (a1–d1), and negative controls were stained with DAPI (a4–d4). Cells from the tenth passage are shown

Fig. 5

EBs differentiated in medium without leukemia inhibitory factor and feeder layer. Bovine embryonic stem cell-like cells had differentiated spontaneously into EBs on mixed embryonic fibroblast feeder layers (a) and STO cell feeder layers (b). Cells from the eighth passage are shown

Fig. 6

Relative expression of the major pluripotency-related transcription factors in bovine embryonic stem cell-like cells cultured on murine embryonic fibroblast feeder layers. Expression of OCT4, SOX2 and NANOG were significantly lower in the fifth passage than in the first passage. Cells from the first passage to fifth passage are shown

Fig. 7

Comparison of OCT4, SOX2, and NANOG expression in bovine embryonic stem cell-like cells cultured on murine embryonic fibroblast feeder layers, STO cell feeder layers and mixed embryonic fibroblast feeder layers. OCT4, SOX2, and NANOG expression in bovine embryonic stem cell-like cells was significantly higher when cultured on STO cell feeder layers and mixed embryonic fibroblast feeder layers than on murine embryonic fibroblast feeder layers. Cells from the tenth passage are shown