He Zhixu, Huang Shaoliang, Li Yu, Zhang Qingxue
Centre of Stem Cell, Second Hospital Affiliated to Sun Yat-sen University, Guangzhou 510120, China.
Zhonghua Yi Xue Za Zhi. 2002 Oct 10;82(19):1314-8.
To establish embryonic stem (ES) cell lines from human fertilized ovum in China.
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.
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.
Human embryonic stem cell lines were successfully established in China.
在中国建立人受精卵来源的胚胎干细胞（ES）系。
从一名志愿者获取14枚人卵母细胞，在Earle's培养基中培养并进行体外受精。受精后的4枚桑椹胚被冷冻，5枚继续发育成囊胚。去除5枚囊胚的透明带，取出其内部细胞团（ICM），接种到经丝裂霉素C灭活的小鼠胚胎成纤维细胞饲养层上。从3个存活的ICM形成的细胞克隆中进行挑选并机械分散，将细胞接种到新的饲养层上以产生更多细胞克隆。当产生多个克隆时，用dispase将其解离成较小的细胞团，再次接种到新的饲养层上进行传代培养。在细胞培养的不同阶段，进行碱性磷酸酶活性（AKP）染色、干细胞表面标志物检测和核型分析。将传代培养5个月的干细胞悬液皮下接种到重症联合免疫缺陷（SCID）小鼠的腿部，观察畸胎瘤形成情况。
9枚受精卵中，5枚发育成囊胚。5枚囊胚中的3枚的ICM存活并发育成胚胎干细胞系，分别命名为CHE1、CHE2和CHE3。这些干细胞在传代培养7个月后仍未分化。干细胞系CHE3在体外已传代40次，CHE1传代36次，CHE2传代32次。3个细胞系在第5、15和30代时AKP染色呈阳性。人干细胞表面标志物SSEA - 4、SSEA - 3、TRA - 1 - 60和GCTM - 2在CHE3的第25、30、40代时呈阳性，在CHE1和CHE2的第21、22和30代时呈阳性。但所有细胞系均不表达小鼠ES细胞标志物SSEA - 1。所有细胞系均稳定保持正常核型。常规传代5个月后，将3个细胞系的人ES细胞皮下注射到SCID小鼠体内。接种3个细胞系6周后，所有小鼠均形成畸胎瘤。组织学检查显示，它们包含来自所有三个胚胎胚层的各种组织。
在中国成功建立了人胚胎干细胞系。
