Yang Hua, Qiu Ying, Zeng Xianghui, Ding Yan, Zeng Jianye, Lu Kehuan, Li Dongsheng
Reproductive Medical Center of Nanning Second People's Hospital, Third Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530031, P.R. China; State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Animal Reproduction Institute, Guangxi University, Nanning, Guangxi 530004, P.R. China.
Reproductive Medical Center of Nanning Second People's Hospital, Third Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530031, P.R. China.
Exp Ther Med. 2016 Jun;11(6):2321-2328. doi: 10.3892/etm.2016.3204. Epub 2016 Mar 30.
The aim of the present study was to investigate the effects of feeder layers composed of various ratios of mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (hFFs) on the growth of human embryonic stem cells (hESCs). In addition, the secretion levels of basic fibroblast growth factor (bFGF) by the feeder layers was detected. MEFs and hFFs were treated with mitomycin C and seeded onto gelatin-coated plates at a density of 1×10 cells/l. The hFFs and MEFs were combined and plated at the following ratios: 0:1, 1:2, 1:1, 2:1 and 1:0. The secretion of bFGF by the various hFF/MEF ratio feeder layers was detected using an enzyme-linked immunosorbent assay. Subsequently, hESCs were cultured on top of the various feeder layers. The differences in the cellular morphology of the hESCs were observed using microscopy, and the expression levels alkaline phosphatase (AKP) and octamer-binding transcription factor 4 (OCT-4) were detected using immunohistochemical analysis as indicators of differentiation status. The results showed that the hFFs secreted substantial quantities of bFGF, while no bFGF was secreted by the MEFs. The clones of hESC growing on the feeder layer containing MEF or hFF alone were flat. By contrast, hESC clones grown on a mixed feeder layer containing hFFs + MEFs at a ratio of 1:1 exhibited an accumulated growth with a clear edge, as compared with the other ratios. In addition, hESCs growing on the feeder layer were positive for the expression of AKP and OCT-4. In summary, feeder layer hFFs secreted bFGF, while MEFs did not, indicating that bFGF is not the only factor that supports the growth and differentiation of hESCs. The optimal growth of hESCs was achieved using a mixed feeder layer composed of hFFs + MEFs at a ratio of 1:1.
本研究的目的是调查由不同比例的小鼠胚胎成纤维细胞（MEFs）和人包皮成纤维细胞（hFFs）组成的饲养层对人胚胎干细胞（hESCs）生长的影响。此外，检测了饲养层碱性成纤维细胞生长因子（bFGF）的分泌水平。用丝裂霉素C处理MEFs和hFFs，并以1×10个细胞/升的密度接种到明胶包被的培养皿上。将hFFs和MEFs按以下比例混合接种：0:1、1:2、1:1、2:1和1:0。采用酶联免疫吸附测定法检测不同hFF/MEF比例饲养层中bFGF的分泌情况。随后，将hESCs培养在不同的饲养层上。用显微镜观察hESCs细胞形态的差异，并用免疫组织化学分析检测碱性磷酸酶（AKP）和八聚体结合转录因子4（OCT-4）的表达水平，作为分化状态的指标。结果显示，hFFs分泌大量bFGF，而MEFs不分泌bFGF。单独在含有MEF或hFF的饲养层上生长的hESC克隆是扁平的。相比之下，与其他比例相比，在含有1:1比例的hFFs + MEFs的混合饲养层上生长的hESC克隆呈现出边缘清晰的聚集生长。此外，在饲养层上生长的hESCs AKP和OCT-4表达呈阳性。总之，饲养层hFFs分泌bFGF，而MEFs不分泌，这表明bFGF不是支持hESCs生长和分化的唯一因素。使用由1:1比例的hFFs + MEFs组成的混合饲养层可实现hESCs的最佳生长。
