人胚胎干细胞H1培养条件的优化

Optimization of culture condition on human embryonic stem cell line H1

目的:采用不同培养基和饲养层培养人胚胎干细胞H1,建立适合H1细胞增殖的最佳条件并分析其基本生物学特性。方法:鼠源性饲养层采用ICR品系小鼠胚胎成纤维细胞(MEF),人源性饲养层采用人胚胎成纤维细胞系(HFF-1)。H1基本培养基配制分别采用传统DMEM/F12和改良培养基Knock-outTM DMEM。实验共分为MEF+DMEM/F12、MEF+K-DMEM、HFF+DMEM/F12、HFF+K-DMEM组。H1基本生物学特性检测采用免疫荧光、RT-PCR、碱性磷酸酶和核型分析。结果:MEF+DMEM/F12组中H1克隆形态规则,不发生分化,增殖速度快;而MEF+K-DMEM组细胞克隆传代后第4日发生分化;HFF+DMEM/F12组和HFF+K-DMEM组细胞传代后第3日就显示出分化趋势,克隆变扁。MEF+DMEM/F12组中H1细胞保持正常核型和基本生物学特性。结论:不同的人胚胎干细胞系最佳培养条件是不同的,建立的MEF+DMEM/F12组培养条件最适合H1细胞增殖。

Objective： To establish the optimal culture condition for H1 line （46, XY, NIH Code WA01） according to medium and feeder cells and analyze their characteristics, and to improve clone survival after frozen-thawed. Methods： H1 as cultured on HFF-1 （human fetal fibroblast, SCRC-1041, ATCC） or MEF feeders （mouse embryonic fibroblast, MEF） derived from ICR mice embryos. The basic medium was DMEM/F12 or Knock-out^TM DMEM （K-DMEM）. The experimental groups included MEF＋DMEM/F12, MEF＋ K-DMEM, HFF＋ DMEM/F12 and HFF＋ K-DMEM. Alkaline phosphatase activity, immunofluorescence, RT-PCR and Giemsa staining were used to detect the biological characteristics of HI. Results ： The morphology of H1 clones in MEF＋DMEM/F12 group was regular and remained undifferentiated state and grew significantly fast. However, differentiation occured in clones in the MEF＋K-DMEM group at day 4 after passages. The clones became flatten and differentiated in the HFF＋DMEM/F12 and HFF＋K-DMEM groups at day 3 after passages. In MEF＋ DMEM/F12 group H1 remained normal karyotype and expressed the markers for pluripotent cells, including positive expression for Oct4, SSEA-4, TRA-1-60, TRA-1-81, alkaline phosphatase activity, and negative for SSEA-1. Conclusion： The culture system differs substantially in different hES cell lines although they appear similar in the undifferentiated state. MEF＋DMEM/F12 is sufficient for self-renewal and proliferation of H1. HFF-1 does not support H1 growth.