摘要
目的:开发一种新的培养人胚胎干细胞( hESCs)的包被基质,使hESCs的培养更加简便。方法用甲醇固定的小鼠胚胎成纤维细胞( MEF)作为包被基质,人胚胎干细胞系X-01在该基质上生长,每隔5~6 d传代一次,培养10代后,对人胚胎干细胞特性进行检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达和分化能力。结果 hESCs在新的基质上生长良好,经10次传代后仍能保持典型的hESCs克隆形态。碱性磷酸酶染色阳性,免疫荧光染色 Oct4、SSEA4、Tra-1-60为阳性,体外分化可形成拟胚体。结论此种固定的基质可以大量制备,长期保存,并可以长期维持hESCs的未分化状态,为人胚胎干细胞的体外扩增探索出了一个新的途径。
Objective To reduce the animal component contamination for human embryonic stem cells ( hESCs ) and to simplify hESCs culture process , we develop a new coating substrate which can support the hESCs growth without dif -ferentiation, and is easy to store and use. Methods Mouse embryonic fibroblasts(MEF)were fixed on the surface of plate by methanol.hESCs were cultured on this new substrate and were passaged every 5 to 6 days.After 10 passages, we checked the cell morphology , alkaline phosphatase expression , embryonic specific markers and the differentiation ability in vitro.Results After 10 passages , the hESCs grew well on this new substrate and maintained the typical hESCs morpholo -gy.Alkaline phosphatase staining was positive .Immunofluorescence staining showed that the expressions of Oct 4, SSEA4, Tra-1-60 were positive .The cells formed embryoid body in vitro .Conclusions This methanol-fixed MEF substrate can support the growth of undifferentiated hESCs .The coating material can be produced in large scale and stored for a long time.It provides a new and relatively easy way to amplify hESCs .
出处
《中国实验动物学报》
CAS
CSCD
2014年第3期88-92,I0005,共6页
Acta Laboratorium Animalis Scientia Sinica
关键词
人胚胎干细胞
小鼠胚胎成纤维细胞
甲醇固定
基质
MEF蛋白质复合物
Human embryonic stem cell
Mouse embryonic fibroblast
Methanol fixation
Mouse embryonic fibro-blast
MEF-protein complex