人胚胎干细胞系的初步建立

目的：探讨人受精卵建立人胚胎干细胞系。方法：将体外受精获得的人受精卵发育至胚胎泡期,用机械法去除胚泡透明带后,取出内细胞团,分散后接种在小鼠胚胎成纤维细胞饲养层上,进行传代培养。通过碱性磷酸酶染色、胚胎干细胞特异性表面标志物检测、染色体核型分析和严重联合免疫缺陷（SCD）小鼠体内畸胎瘤形成实验,对连续传代的细胞进行鉴定。结果：9枚受精卵在体外培养5-7d后,5个存活并发育为胚泡,取出的内细胞团经接种培养,3个内细胞团存活,呈克隆性生长,发育为胚胎干细胞,并连续体外传代7个月保持未分化状态,建系成功。3个细胞系分别命名为CHE1、CHE3和CHE3。取3个细胞系第5、15和30代的细胞进行碱性磷酸酶染色,均为阳性。对CHE3第25、30、40代、CHE1和CHE2第21、22、30代的细胞检测人胚胎干细胞表面标志SSEA-4、SSEA-3、TRA-1-60和GCTM-2,结果均为阳性;检测小鼠胚胎干细胞表面标志SSEA-1,结果为阴性。染色体核型分析为二倍体核型。将连续传代5个月后（30代左右）的3个细胞系接种到SCID小鼠体内,6周后均形成畸胎瘤,组织病理学检查显示含有3个胚层的组织结构。结论：使用5个人受精卵来源的胚泡,成功地在体外建立了3个胚胎干细胞系,长期传代后仍保持胚胎干性和多向分化的能力。

Human embryonic stem cell lines preliminarily established in China

OBJECTIVE: To establish embryonic stem (ES) cell lines from human fertilized ovum in China. METHODS: Fourteen human oocytes obtained from a volunteer were cultured in Earlg's medium and fertilized in vitro. Four of the morulae formed from the fertilized ovi were frozen and five continued to develop into blastocysts. The pellucid zones of the 5 blastocysts were removed and their inner cell masses (ICM) were taken out and inoculated onto the feeder layer of mouse embryonic fibroblasts inactivated with mitomycin C. The cell clones formed from the three surviving ICM were selected and dispersed mechanically and the cells were inoculated onto new feeder layers to produce more cell clones. When multiple clones were produced, they were dissociated with dispase into smaller cell masses which were inoculated onto new feeder layer again for subculturing. During different periods of cell culture, alkaline phosphatase activity (AKP) staining, stem cell surface marker test, and karyotyping were conducted. The suspension of stem cells subcultured for 5 months were inoculated to the legs of severe combined immunodeficiency (SCID) mice subcutaneously to observe the teratoma formation. RESULTS: Out of the 9 fertilized oocytes, 5 developed into blastocysts. The ICM of three from the five blastocysts survived and developed into embronic stem cell lines, named CHE1, CHE2, and CHE3 respectively. These stem cells remained undifferentiated after subculturing for 7 months. The stem cell line CHE3 had been grown for 40 passages in vitro, CHE1 for 36 passages, and CHE2 for 32 passages. AKP staining was positive at the passages 5, 15, and 30 for the 3 cell lines. The surface markers of human stem cell, SSEA-4, SSEA-3, TRA-1-60, and GCTM-2 were positive at passage 25, 30, 40 for CHE3, and at the passages 21, 22 and 30 for CHE1 and CHE2. But all cell lines did not express SSEA-1, a marker for mouse ES cells. All cell lines stably retained a normal karyotype. After routine passage by 5 months, human ES cells from 3 cell lines were injected subcutaneously into SCID mice. Six weeks after inoculation of the 3 cell lines teratomas were formed in all mice. Histological examination revealed that they contained various tissues derived from all three embryonic germ layers. CONCLUSION: Human embryonic stem cell lines were successfully established in China.