An Alternative Method for Long-Term Culture of Chicken Embryonic Stem Cell In Vitro

Figure 4

Characteristics of cESCs in a long culture period. This culture system can stably maintain the stem-like character of cESCs in vitro for a long term. (a) Epitope characteristic of cultured cESCs in passages 3, 5, and 20. The pluripotency surface marker SSEA-1 (green) was stained. Meanwhile, DAPI (blue) was used to stain the nucleus. (b) SSEA-1 expression was maintained at a relatively stable level. The ImageXpress Micro XLS Widefield High-Content Analysis System was used. The bars indicated the positive SSEA-1 mean cell average intensity of cultured cESCs in passages 3, 5, and 20. (c) Lin28a, Nanog, and PouV gene mRNA relative expression level of cultured cESCs in passages 3, 6, 9, and 20. Compared to the expression level of cultured cESCs in passage 3, . (d) Nanog expression characteristic of cultured cESCs in passages 3, 5, and 20. The figure indicated the merged picture of DAPI, Nanog, and EDU staining. (e) The positive proliferation rate of cultured cESCs was maintained at a relatively high level over passages 3, 5, and 20. The bars indicated the ratio of the EDU-Nanog coexpression cell number to the Nanog expression cell number calculated by ImageXpress Micro XLS Widefield High-Content Analysis System. (f) Telomerase activity. Telomerase activities of cultured cESCs in passages 0, 3, 5, and 20 were measured in cell lysate by the TRAP assay. The cells were frozen until the TRAP was performed. HCT8 cell lysate used as a positive control and noncell lysate used as a negative control. DNA ladder (N3233 New England Biolabs) was used to indicate the size of the fragment. All results are expressed as the mean ± SD of three independent experiments. Scale bar: 100 μm.

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