人胚胎干细胞在人源和鼠源饲养层上的生长特性比较

背景:不同来源饲养层条件下,人胚胎干细胞是否具有相同或相似的生物学特性尚不清楚,该问题的解决有利于建立标准化的饲养层体系.目的:观察比较人胚胎干细胞在人源和鼠源饲养层上的生长特性是否相同?设计、时间及地点:细胞学体外观察,于2007-09,2009-02在中国科学院昆明动物所完成.材料:清洁级孕12.5～13.5 d的ICR小鼠2只,由昆明医学院动物中心提供.永生化人成纤维细胞由美国约翰霍普金斯大学医学院建系并赠送,人胚胎干细胞株BG02由中国科学院昆明动物所提供.方法:无菌条件下取ICR胎鼠,组织块胰酶消化法分离培养小鼠胚胎成纤维细胞,永生化人成纤维细胞按常规培养,两种细胞经γ射线处理后,以2.5×10~4/cm~3接种在明胶包被的6孔板中.取人胚胎干细胞,分别接种在小鼠胚胎成纤维细胞或永生化人成纤维细胞饲养层上,加入含β-巯基乙醇的DMEM/F12培养基,使用前添加碱性成纤维细胞生长因子.主要观察指标:两种饲养层上的人胚胎干细胞的形态、特异性标记表达、Oct-4阳性率、细胞倍增时间.结果:培养在小鼠胚胎成纤维细胞和永生化人成纤维细胞饲养层上的人胚胎干细胞,其形态相似,呈圆形或椭圆形集落;均表达SSEA-3,SSEA-4,TRA-1-60,TRA-1-81和Sct-4,但不表达SSEA-1.与小鼠胚胎成纤维细胞饲养层比较,永生化人成纤维细胞饲养层上人胚胎干细胞Oct-4阳性细胞率明显升高(P<0.05),倍增时间明显延长(P<0.05).结论:人源和鼠源成纤维细胞饲养层上的人胚胎干细胞体外培养生物学特性存在明显差异.

Characteristics of human embryonic stem cells cultured on human- and mouse- origin feeder layers

BACKGROUND: Whether human embryonic stem cells (hESCs) cultured on different feeder layers can maintain identical or similar characteristics remains unclear.OBJECTIVE: To compare the characteristics of hESCs cultured on human- and mouse- origin feeder layers.DESIGN, TIME AND SETTING: The in vitro cytology observation was performed at the Kunming Institute of Zoology, Chinese Academy of Sciences between September 2007 and February 2009.MATERIALS: Two ICR pregnant mice with 12.5-13.5 embryonic days were provided by Animal Center of Kunming Medical College. Immortalized human adult fibroblast (HAFi) cell line was presented by School of Medicine, Johns Hopkins University (USA). hESCs line was provided by Kunming Institute of Zoology, Chinese Academy of Sciences.METHODS: Mouse embryonic fibroblasts (MEF) were harvested from ICR mice by trypsinization method. HAFi were conventionally cultured. After γ ray treatment, two kinds of cells were incubated on 6-well gelatin-coated plate with density of 2.5×10~4/cm~3. hESCs were cultured on HAFi or MEF feeder cells containing β-mercaptoethanol DMEM/F12 and basic fibroblast growth factor.MAIN OUTCOME MEASURES: Morphology, expressions of specific molecular markers, Oct-4 positive rate, as well as cell doubling time of hESCs cultured by two methods were compared.RESULTS: ①BG02 cells on MEF and HAFi shared the similar morphology and characteristic pluripotency markers, which expressed SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and Oct-4, but not SSEA-1. However, the proportion of positive Oct-4 cells in hESCs colonies maintained on MEF was lower than that of HAFi (P < 0.05) with shorter doubling time (P < 0.05).CONCLUSION: hESCs cultured on MEF and HAFi represent some differences in the growth and pluripotency characteristics.