Park Se-Pill, Lee Young Jae, Lee Keum Sil, Ah Shin Hyun, Cho Hwang Yoon, Chung Kil Saeng, Kim Eun Young, Lim Jin Ho
Maria Infertility Hospital Medical Institute/Maria Biotech, Dongdaemun-gu, Seoul 130-812, Korea.
Hum Reprod. 2004 Mar;19(3):676-84. doi: 10.1093/humrep/deh102. Epub 2004 Jan 29.
Recently, human embryonic stem (hES) cells have become very important resources for basic research on cell replacement therapy and other medical applications. The purpose of this study was to test whether pluripotent hES cell lines could be successfully derived from frozen-thawed embryos that were destined to be discarded after 5 years in a routine human IVF-embryo transfer programme and whether an STO cell feeder layer can be used for the culture of hES cells.
Donated frozen embryos (blastocysts or pronuclear) were thawed, and recovered or in vitro developed blastocysts were immunosurgically treated. All inner cell masses were cultured continuously on an STO cell feeder layer and then presumed hES cell colonies were characterized.
Seven and two cell lines were established from frozen-thawed blastocysts (7/20, 35.0%) and pronuclear stage embryos (2/20, 10.0%), respectively. The doubling time of hES cells on the immortal STO cell feeder layer was approximately 36 h, similar to that of cells grown using fresh mouse embryonic fibroblast (MEF) feeder conditions. Subcultured hES cell colonies showed strong positive immunostaining for alkaline phosphatase, stage-specific embryonic antigen-4 (SSEA-4) and tumour rejection antigen 1-60 (TRA1-60) cell surface markers. Also, the hES colonies retained normal karyotypes and Oct-4 expression in prolonged subculture. When in vitro differentiation of hES cells was induced by retinoic acid, three embryonic germ layer cells were identified by RT-PCR or indirect immunocytochemistry.
This study indicates that establishment of hES cells from frozen-thawed blastocysts minimizes the ethical problem associated with the use of human embryos in research and that the STO cell feeder layer can be used for the culture of hES cells.
最近，人类胚胎干细胞（hES）已成为细胞替代疗法基础研究及其他医学应用的重要资源。本研究旨在测试能否从常规人类体外受精 - 胚胎移植计划中5年后注定要丢弃的冻融胚胎成功获得多能性hES细胞系，以及STO细胞饲养层是否可用于hES细胞培养。
解冻捐赠的冷冻胚胎（囊胚或原核胚），对复苏或体外发育的囊胚进行免疫手术处理。所有内细胞团在STO细胞饲养层上连续培养，然后对假定的hES细胞集落进行鉴定。
分别从冻融囊胚（7/20，35.0%）和原核期胚胎（2/20，10.0%）建立了7个和2个细胞系。hES细胞在永生化的STO细胞饲养层上的倍增时间约为36小时，与使用新鲜小鼠胚胎成纤维细胞（MEF）饲养条件下生长的细胞相似。传代培养的hES细胞集落对碱性磷酸酶、阶段特异性胚胎抗原 - 4（SSEA - 4）和肿瘤排斥抗原1 - 60（TRA1 - 60）细胞表面标志物呈强阳性免疫染色。此外，hES集落在长期传代培养中保持正常核型和Oct - 4表达。当用视黄酸诱导hES细胞体外分化时，通过逆转录 - 聚合酶链反应（RT - PCR）或间接免疫细胞化学鉴定出三个胚胎胚层细胞。
本研究表明，从冻融囊胚建立hES细胞可最大限度减少与在研究中使用人类胚胎相关的伦理问题，且STO细胞饲养层可用于hES细胞培养。
