用低形态学评分D3胚胎建立人胚胎干细胞系

THE CULTURE OF HUMAN EMBRYONIC STEM CELL LINES FROM D3 EMBRYOS WITH LOW MORPHOLOGICAL SCORES

目的寻找人胚胎干细胞(hESC)建系材料来源。方法选用IVF低形态学评分的D3胚胎行序贯囊胚培养,用免疫外科的方法去除滋养细胞,将得到的内细胞团(ICM)接种于丝裂霉素C灭活的小鼠胚胎成纤维细胞(MEFs)上培养5-8d,每4-7d传代1次,分别取不同代的hESC进行碱性磷酸酶(AKP)染色、转录因子OCT-4、阶段特异性胚胎抗原(SSEA)SSEA-4、SSEA-1、肿瘤排斥抗原(TRA)TRA-1-60、TAR-1-81、核型及体内外分化全能性鉴定。结果130枚废弃的D3低形态学评分(评分<16)的胚胎培养出囊胚19枚,获得原代克隆5个,成功培养出两株hESC系,它们具有hESC的共同的生物学特性。结论部分低形态学评分的D3废弃胚胎可发育成囊胚。囊胚形成率与形态学评分相关,这些胚胎可作为建立hESC系的材料来源之一。

Objective To find a new source to produce hESC lines. Methods D3 embryos with low morphological scores were cultured to blastocyst stage. Trophectoderm cells were separated from the ICMs by immunosurgery and isolated ICMs were cultured for 5-8 days on mitomycin-treated mouse embryonic fibroblasts （MEFs）. Colonies derived from the ICMs were passed every 4-7 days and evaluated for cell surface markers including AKP, OCT-4, SSEA-4, SSEA-1, TRA-1-60, TRA-1-81, differentiation potentials and karyotypes. Results A total of 19 blastocysts were obtained from 130 embryos （quality score 〈 16） and blastulation rate was 14.62%. Immunosurgery resulted in the formation of 5 primary colonies, which produced 2 cell lines. Both cell lines satisfied the criteria that characterize pluripotent hESC. Conclusion A subset with poor quality D3 embryos judged on the basis of morphological assessment can form blastocysts. The blastulation rate is related to D3 scores and poor quality D3 embryos could be a new source for establishment of hES cell banks.