利用冷冻胚胎建立人类胚胎干细胞系

Derivation of human embryonic stem cell lines from frozen embryos

背景：目前人胚胎干细胞建系的技术路线主要有囊胚法、核移植法和胚胎单卵裂球法，但由于破坏胚胎，涉及诸多伦理问题。目的：探讨利用冷冻胚胎建立人类胚胎干细胞系的可行性。设计、时间及地点：细胞学体内外实验，于200501／200608在沈阳市妇婴医院生殖中心完成。材料：妊娠13．5d的ICR鼠20只用于制各小鼠胚胎成纤维细胞饲养层，10周龄SICD鼠2只用于人胚胎干细胞体内分化实验。行IVF／ICSI助孕患者自愿捐献的冷冻胚胎，由沈阳市妇婴医院生殖中心提供。方法：复苏冷冻的胚胎，采用序贯培养法进行囊胚培养，用免疫外科方法去除滋养细胞，将得到的胚胎内细胞团接种于丝裂霉素C灭活的小鼠胚胎成纤维细胞上培养并传代。主要观察指标：取不同代次的培养细胞，分别进行生长特性、碱性磷酸酶染色、转录因子OCT-4、阶段特异性胚胎抗原SSEA-4、SSEA-1、肿瘤排斥抗原TRA-1-60、TAR-1-81、核型及体内分化全能性鉴定。结果：20个解冻卵裂期胚胎，获得9个囊胚，分离完整的内细胞团共7个，从7个内细胞团培养出2个人胚胎干细咆系，其中一个细胞系连续培养76代（20个月）。所培养的细胞具有人胚胎干细胞的共同生物学特性，即细胞呈扁平或圆形，可见1—3个核仁：碱性磷酸酶以及SSEA．3，SSEA．4，TRA-1．81，TRA-1-60，OCT-4均呈阳性表达，而SSEA-1呈阴性：核型正常，为46，XX核型；将细胞注射入SCID鼠皮下形成肿瘤，病理切片显示为成熟畸胎瘤：短串联重复分型结果显示所形成的畸胎瘤与人胚胎干细胞为相同的遗传背景。结论：实施体外受精-胚胎移植的夫妇怀孕后，多余的冷冻保存胚胎所分离培养的细胞具备人胚胎干细胞的所有特性，是建立人胚胎干细胞系很好的材料来源。

BACKGROUND： Presently, the method of establishing human embryonic stem cells mainly contains blastula method, nuclear transplantation method and embryo blastomere method. Embryo destruction is involved in many ethical problems. OBJECTIVE： To explore the feasibility of establishing a new human embryonic stem cell lines using frozen cleavage human embryos. DESIGN, TIME AND SETTING： The cytological in vivo or vitro study was performed at the Reproduction Center, Shenyang Gynecology and Obstetrics Hospital from January 2005 to August 2006. MATERIALS： A total of 20 ICR mice at pregnancy of 13.5 days were used to prepare mouse embryo fibroblast feeder layer. Two SICD mice aged 10 weeks were used for human embryonic stem cell in vivo differentiation study. Frozen-thawed cleavage stage human embryos donated by couples undergoing in vitro fertilization （IVF）/intracytoplasmic sperm injection （ICSI） were supplied by Reproduction Center, Shenyang Gynecology and Obstetrics Hospital. METHODS： Frozen-thawed cleavage stage human embryos were cultured to the blastocyst stage by sequential culture method. Trophocytes were removed by immunosurgical method. Obtained inner cell mass was plated on mouse embryonic fibroblast feeder layer following mitomycin-C inactivation. MAIN OUTCOME MEASURES： Cultured cells at various passages were determined in following parameters： growth characteristics, alkaline phosphatase staining, transcription factor OCT-4, phase specific embryonal antigen SSEA-4, SSEA-1, tumor rejection antigen TRA-1-60, TRA-1-81, differentiation potentials and karyotypes. RESULTS： Nine blastocysts were obtained from 20 frozen thawed human cleavage stage embryos, and 7 inner cell masses were isolated by immunosurgery, and established two human embryonic stem cell lines. One line was cultured for 76 passages （20 months）. Cultured human embryonic stem cell colonies were compact, flat and round, with 1-3 nuclei, and positive for alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-81, TRA-1-60, OCT-4, but negative for SSEA-1. Karyotype was normal, 46, XX type. These cells were subcutaneously infused into SCID mice to form tumors. Pathological sections showed mature teratoma. Short tandem repeat typing results demonstrated that formed teratoma had similar genetic background as human embryonic stem cells. CONCLUSION： Established human embryonic stem cells from frozen-thawed blastocysts have the characteristics of human embryonic stem cells, and may be a good source of human embryonic stem cell establishment.