摘要
目的寻找可以维持人胚胎干细胞未分化生长的人源性细胞作为饲养层细胞,从而解决使用鼠源性细胞作为饲养层带来的安全问题。方法尝试以人脐带间充质干细胞作为饲养层细胞来培养人胚胎干细胞,检验其是否可以维持人胚胎干细胞的未分化生长状态。用胶原酶消化法分离人脐带间充质干细胞,光镜下观察细胞形态;流式细胞仪检测其表面标志;诱导人脐带间充质干细胞向成骨细胞和脂肪细胞进行分化。将人胚胎干细胞系H1接种于丝裂霉素C灭活后的人脐带间充质干细胞上,每隔5d进行一次传代。培养20代后,对人胚胎干细胞特性进行相关检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达、分化能力。结果从人脐带中分离出的间充质干细胞为梭形,呈平行排列生长或漩涡状生长;细胞高表达CD44、CD29、CD73、CD105、CD90、CD86、CD147、CD117,不表达CD14、CD38、CD133、CD34、CD45、HLA-DR;具有分化成脂肪细胞和成骨细胞的潜能。人胚胎干细胞在人脐带间充质干细胞饲养层上培养20代后,继续保持人胚胎干细胞的典型形态,碱性磷酸酶染色为阳性,免疫荧光染色显示OCT4、Nanog、SSEA4、TRA-1-81、TRA-1-60的表达为阳性,SSEA1表达为阴性,体外悬浮培养可以形成拟胚体。结论人脐带间充质干细胞可以作为人胚胎干细胞的饲养层细胞,支持其生长,并维持其未分化生长状态。
Objective To find a new source of human feeder cells which can support the undifferentiated growth of human embryonic stem cells.In this study,human umbilical cord mesenchymal stem cells（HUCMSCs） were tested as feeder cells for supporting the growth of hESCs to prevent the possibility of contamination from animal feeder cells.MethodsMesenchymal stem cells were isolated from human umbilical cord by collagenase IV digestion.Their surface markers were detected by flow cytometry.The cells were induced to differentiate into osteoblast and adipocytes.HESc line H1 was grown on inactivated human umbilical cord mesenchymal stem cells,and were passaged every five days.After many passages,the morphology,expression of specific markers and differentiation capacity of hESCs were examined.Results HUCMSCs were spindle-shaped adherent cells.They had strong proliferation ability,and showed strongly positive expression of CD44,CD29,CD73,CD105,CD90,CD86,CD147,CD117 and negative expression of CD14,CD38,CD133,CD34,CD45 and HLA-DR.After induction,oil red O staining was positive.After passaged on MSCs feeder cells for 20 passages,hESCs maintained their morphological characteristics.Immunofluorescence staining showed positive expression of OCT4,Nanog,SSEA4,TRA-1-60,and TRA-1-81 and negative SSEA1.Alkaline phosphatase staining showed positive results.Moreover,the hECSs cultured on HUCMSCs formed embryoid body in vitro confirmed their pluripotency.Conclusions HUCMSCs can be used as the feeder cells for hESCs culture and maintain their undifferentiated state.
出处
《中国实验动物学报》
CAS
CSCD
2011年第4期271-276,I0009,共7页
Acta Laboratorium Animalis Scientia Sinica
基金
国家自然科学基金(编号:30772376)
北医三院临床重点项目基金(编号:BYSY200903)
教育部高等学校博士学科点专项科研基金(编号:20100001120100)
关键词
人胚胎干细胞
小鼠胚胎成纤维细胞
人脐带间充质干细胞
饲养层细胞
临床应用
Human embryonic stem cells
Mouse embryonic fibroblast cells
Human umbilical cord mesenchymal stem cells
Feeder cells
Clinical application
