Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers
Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers
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DOI：
10.1093/humrep/deh102
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发表时间：
2004-03-01
影响因子：
6.1
通讯作者：
Lim, JH
中科院分区：
文献类型：
--
作者：
Park, SP;Lee, YJ;Lim, JH
背景资料：近年来，人胚胎干细胞已成为细胞替代疗法基础研究和其他医学应用的重要资源。本研究的目的是测试多能hES细胞系是否可以成功地从冻融胚胎中获得，这些胚胎在常规人类IVF胚胎移植计划中5年后注定要被丢弃，以及STO细胞饲养层是否可以用于hES细胞的培养。研究方法：将捐献的冷冻胚胎（囊胚或原核）解冻，并对回收的或体外发育的囊胚进行免疫手术处理。将所有内细胞团在STO细胞饲养层上连续培养，然后表征假定的hES细胞集落。结果如下：分别从冻融囊胚（7/20，35.0%）和原核期胚胎（2/20，10.0%）中建立了7个和2个细胞系。hES细胞在永生STO细胞饲养层上的倍增时间类似于36小时，与使用新鲜小鼠胚胎成纤维细胞（MEF）饲养条件生长的细胞的倍增时间类似。传代培养的hES细胞集落显示碱性磷酸酶、阶段特异性胚胎抗原-4（SSEA-4）和肿瘤排斥抗原1-60（TRA 1 -60）细胞表面标志物的强阳性免疫染色。此外，hES集落在延长的传代培养中保留了正常的核型和Oct-4表达。用维甲酸诱导hES细胞体外分化，通过RT-PCR或间接免疫细胞化学鉴定出3个胚胎生殖层细胞。结论：这项研究表明，从冻融囊胚建立hES细胞最大限度地减少了与研究中使用人类胚胎相关的伦理问题，STO细胞饲养层可用于培养hES细胞。
Background: 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. Methods: 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. Results: 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 similar to36 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. Conclusions: 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.