冷冻胚胎经序贯共培养用于建立人胚胎干细胞系

目的:利用长期冷冻人卵裂期胚胎经序贯共培养形成的囊胚建立人类胚胎干细胞系。方法:将冷冻≥10年的卵裂期胚胎复苏后,采用单层卵丘细胞序贯共培养至囊胚期,经辅助孵出,置鼠胚胎成纤维细胞(MEF)饲养层全胚培养,原代培养6d后加入20%MEF条件培养基培养至形成原代克隆。观察人胚胎干细胞集落的生长状态并通过碱性磷酸酶染色、Oct-4基因表达、核型分析及体外分化实验等方法进行生物学鉴定。结果:9枚冷冻卵裂期胚胎经序贯共培养,有6枚发育到囊胚期,最终获得1株胚胎干细胞系。经鉴定该细胞系具有碱性磷酸酶活性、表达Oct-4胚胎干细胞特异标记、形成拟胚体、在体外形成具有自动节律性的心肌细胞、并具有46,XY核型等5种特性。结论:冷冻胚胎经序贯共培养能够改善胚胎发育潜能,有助于建立人胚胎干细胞系。

Sequential co-culture method in developing human embryonic stem cell lines derived from frozen human blastomere

Objective: To develop embryonic stem cell lines derived from blastocyst from frozen human blastomere with the sequential co-culture method. Mehtods: Thawed human blastomeres were cultured in a single layer cumulus cell sequential co-culture system. Assisted hatching was conducted when blastocysts were developed. The hatched blastocysts were then cultured with mouse embryonic fibroblast (MEF) feeder layer cell for 6 days. And then a conditional culture medium including 20% MEF was used until the primary clone was developed. The growth status of human embryonic stem cell colony differentiation and the biology identification were confirmed with alkaline phosphatase staining, detection of Oct-4 gene expression, karyotype analysis and in vitro differentiation experiments. Results: Nine frozen human blastomere cultured with a sequential co-culture method, and six of them developed to blastocysts. And ultimately, one of them developed to a embryonic stem cell line. Characteristics identified in this cell line were as follow: showing alkaline phosphatase activity, expressing Oct-4 gene which was the cell-specific marker of embryonic stem cell, forming embryoid bodies, developing to the automatic rhythm myocardial cells in vitro and with a karyotype of 46, XY. Conclusion: The sequential co-culture system can improve embryo developmental potential of thawed-frozen embryos and contribute to the establishment of human embryonic stem cell lines.