Plaia Todd W, Josephson Richard, Liu Ying, Zeng Xianmin, Ording Carol, Toumadje Arazdordi, Brimble Sandii N, Sherrer Eric S, Uhl Elizabeth W, Freed William J, Schulz Thomas C, Maitra Anirban, Rao Mahendra S, Auerbach Jonathan M
Stem Cell Center, American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, USA.
Stem Cells. 2006 Mar;24(3):531-46. doi: 10.1634/stemcells.2005-0315. Epub 2005 Nov 17.
Human embryonic stem cells (hESCs) offer a renewable source of a wide range of cell types for use in research and cell-based therapies. Characterizing these cells provides important information about their current state and affords relevant details for subsequent manipulations. For example, identifying genes expressed during culture, as well as their temporal expression order after passaging and conditions influencing the formation of all three germ layers may be helpful for the production of functional beta islet cells used in treating type I diabetes. Although several hESC lines have demonstrated karyotypic instability during extended time in culture, select variant lines exhibit characteristics similar to their normal parental lines. Such variant lines may be excellent tools and abundant sources of cells for pilot studies and in vitro differentiation research in which chromosome number is not a concern, similar to the role currently played by embryonal carcinoma cell lines. It is crucial that the cells be surveyed at a genetic and proteomic level during extensive propagation, expansion, and manipulation in vitro. Here we describe a comprehensive characterization of the variant hESC line BG01V, which was derived from the karyotypically normal, parental hESC line BG01. Our characterization process employs cytogenetic analysis, short tandem repeat and HLA typing, mitochondrial DNA sequencing, gene expression analysis using quantitative reverse transcription-polymerase chain reaction and microarray, assessment of telomerase activity, methylation analysis, and immunophenotyping and teratoma formation, in addition to screening for bacterial, fungal, mycoplasma, and human pathogen contamination.
人类胚胎干细胞（hESCs）为研究和基于细胞的治疗提供了多种可再生的细胞类型来源。对这些细胞进行表征可提供有关其当前状态的重要信息，并为后续操作提供相关细节。例如，鉴定培养过程中表达的基因，以及传代后它们的时间表达顺序和影响所有三个胚层形成的条件，可能有助于生产用于治疗I型糖尿病的功能性β胰岛细胞。尽管一些hESC系在长时间培养过程中表现出核型不稳定，但选定的变异系表现出与其正常亲代系相似的特征。此类变异系可能是出色的工具和丰富的细胞来源，可用于染色体数目无关紧要的初步研究和体外分化研究，类似于目前胚胎癌细胞系所起的作用。在体外广泛传代、扩增和操作过程中，对细胞进行遗传和蛋白质组水平的检测至关重要。在此，我们描述了源自核型正常的亲代hESC系BG01的变异hESC系BG01V的全面表征。我们的表征过程除了筛查细菌、真菌、支原体和人类病原体污染外，还采用了细胞遗传学分析、短串联重复序列和HLA分型、线粒体DNA测序、使用定量逆转录聚合酶链反应和微阵列的基因表达分析、端粒酶活性评估、甲基化分析以及免疫表型分析和畸胎瘤形成。
