关键词: 胚胎干细胞, 血清, BrdU掺入法, 免疫荧光, 人

Abstract: Objective To evaluate the biological characteristic of human embryonic stem(hES) cells in serum-containing and serum-free culture condition. Methods Human embryonic stem cell line BG02 was cultured on mitomycin C treated mouse embryonic fibroblasts(MEF) feeder layers in medium supplied with serum or knockout serum replacement for 25-30 passages to compare their morphology. The proliferation of hES cells clones was detected by BrdU incorporation assay. The double time of hES cells was evaluated by cell counting. The pluripotency markers of hES cells were examined by immunofluorescence staining. The proportions of Oct-4 and Nanog positive cells were assessed by flow cytometry. Expressions of fibroblast growth factor(FGFs) family genes were determined by RT-PCR. Results Although BG02 cells in the serum and serum-free culture medium shared similar morphology, hES clones in serum-free culture condition showed more typical morphology and more Oct-4, Nanog positive cells than that in serum-containing condition. The attachment rate of hES cells clumps cultured in serum-free condition was obviously higher than that of serum condition ( EM>P /EM><0.05). The growth of BG02 cells in serum-containing medium was more fast (EM> P/EM><0.05). The doubling time of hES cells in the serum and serum-free condition was (45.9±5.7)hours and (33.8±4.3) hours, respectively. Additionally, BG02 cells in serum-free culture condition expressed high level of FGF2,FGFR2, and FGFR4. Conclusion BG02 cells cultured in serum or serum-free culture condition represent some variations in the growth and characteristics of hES cells. These variations might be attributed to the activation of members of FGFs family in BG02 cells.

Key words: Embryonic stem cells, Serum, BrdU incorporation, Immunofluorescence, Human