无动物源性成分人胚胎干细胞培养体系与其它人胚胎干细胞培养体系的比较  

Comparison of A Novel Xeno-free Culture System with Other Different Culture Systems for HESC

作　　者：张丹[1] 麦庆云[1] 李涛[1] 徐艳文[1] 丁晨辉[1] 宏苹苹[1] 曾艳红[1] 周灿权[1] 

机构地区：[1]中山大学附属第一医院生殖中心,广东广州510080

出　　处：《中山大学学报（医学科学版）》2012年第6期828-834,847,共8页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家"863"项目(2005AA219010);国家"973"项目(2007CB948103)

摘　　要：【目的】通过比较hESC在各培养体系中未分化表面标记物及以多能性因子的表达,进一步筛选出更优化、安全有效的hESC培养体系。【方法】四种培养体系分别为:新建立的无动物源性的人包皮饲养层(XF-HFF)联合成分确定培养基(CDM)的无动物源性培养体系(XF-HFF/CDM),对照组为XF-HFF联合添加了200 mL/L人血清(HS)的培养基的培养体系(XF-HFF/HS),HS包被的培养皿联合CDM的无饲养层培养体系(HS-Matrix/CDM),以及传统的人包皮饲养层(XC-HFF)联合添加了200 mL/L血清替代物(KSR)的培养基的培养体系。通过观察克隆形态、细胞计数、免疫荧光、流式细胞术、荧光定量PCR,来比较4种hESC培养体系中的细胞增殖率、未分化表面标记物和多能性因子的表达情况,以及hESC在XF-HFF/CDM培养体系中长期培养后,对其未分化状态、多能性和染色体完整性的检测。【结果】在几种培养体系中,同一代次的hESC表达SSEA-4、TRA-1-60、Oct3/4和hTERT的情况在XF-HFF/CDM组和XC-HFF/KSR组中相似(P>0.01),且均高于其它两组培养体系(P<0.01)。hESC在XF-HFF/CDM培养体系中长期培养超过40代次,仍能表达hESC未分化表面标记物SSEA-3,SSEA-4,TRA-1-60,TRA-1-81及多能性因子hTERT、Oct3/4,在SCID小鼠体内能分化成含3个胚层的畸胎瘤,以及保持完整的二倍体核型。【结论】新构建的完全无动物源性的hESC培养体系的培养效率与传统添加KSR的培养体系相似,且优于相同CDM下的无饲养层培养体系以及相同饲养层下的含人血清的干细胞培养条件。[ Objective ] By comparing the undifferentiated and pluripotent marks of hESC in four different culture systems to evaluate and promote more optimal hESC culture environment. [ Methods ] The four culture systems consist of xeno-free human foreskin fibroblasts （XF-HFF） combined with chemically-defined medium （CDM） （XF-HFF/CDM group）, XF-HFF combined with 200 mL/L HS medium （XF-HFF/HS group）, HS-matrix coated on plates without feeder layers combined with CDM （HS-matrix/CDM group）, and xeno-contained human foreskin fibroblasts （XC-HFF） combined with 200 mL/L KSR medium （XC-HFF/KSR group）.The culture efficiencies were evaluated by morphology, cell growth curve, flow cytometry and quantitative reverse transcription-polymerase chain reaction （qRT-PCR） among these culture systems. Further more, we characterized the sternness of hESC after long-term culture in XF-HFF/CDM culture system. [Results] The rates of characteristic surface undifferentiated markers （SSEA4, Tral-60） and proliferate factors （hTERT, Oct3/4） expression of hESC cultured in XF-HFF/CDM were significantly higher than which in XF-HFF/ HS and in HS-matrix/CDM（P 〈 0.01 ）, but were comparable to which in XC-HFF/KSR. After long-term cultured in XF-HFF/CDM,hESC still expressed surface undifferentiated markers of SSEA3, SSEA4 and pluripotent markers of hTERT, Oct3/4, and maintained pluripotency and karyotype stability, over 40 passages. [ Conclusion ] The new xeno-free culture conditions demonstrated similar culture efficiency to KSR medium with HFF, and were more effective than CDM with HS-matrix, or HS medium with HFF.

关 键 词：人胚胎干细胞 无外源性成分 成分确定 比较 培养 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]