Sidhu Kuldip S, Ryan John P, Tuch Bernie E
Stem Cell Laboratory, Faculty of Medicine, University of New South Wales, Randwick, Australia 2052.
Stem Cells Dev. 2008 Feb;17(1):41-51. doi: 10.1089/scd.2007.0055.
Here we describe the derivation of a novel human embryonic stem (hES) cell line, Endeavour-1 (E1), its four new clonal lines (E1C1, E1C2, E1C3, E1C4), and their characterization. E1 and its clonal lines are propagated on human fetal fibroblasts (HFFs) derived and grown in a largely serum-free medium. Seven inner cell masses were isolated from 34 donated human embryos (27 survived), and one new hES cell line was obtained. E1 has been in culture for over 1 year and possesses all the typical features of stem cells, i.e., expression of stem cell surface markers (stage-specific embryonic antigens SSEA-3 and SSEA-4, and tumor recognition antigens TRA-1-60 and TRA-1-81), staining for alkaline phosphatase, and the presence of the pluripotent gene marker (nanog). This line shows pluripotency both under in vitro and in vivo conditions. E1 has a normal karyotype (46XX). Using our optimized procedure for cloning, four new clonal lines were derived from E1: E1C1, E1C2, E1C3, and E1C4. These clonal lines show normal characteristics: karyotype of that of the parent line (46XX) except for E1C3, which showed reciprocal translocation involving chromosomes 15 and 17; stem cell surface markers SSEA-4, TRA-1-60, and TRA-1-81; and gene expression for pluripotency (Nanog). All of these clonal lines formed embryoid bodies (EBs) in suspension cultures. After seeding, the EBs differentiated, forming cell lineages derived from all three germ layers as indicated by immunolocalization for the ectodermal marker beta-III tubulin, the mesodermal marker CD34, and the endodermal marker alpha-fetoprotein (AFP). There were subtle differences in the expression of these markers between clones. These clonal lines showed pluripotency in vivo. E1 and its clonal lines can differentiate to definitive endoderm after treatment with activin A, and, as indicated by expression of SOX17, FOXa2, and GATA-4 by RT-PCR, there are some subtle differences between these clonal lines. This may help in selecting clonal lines for specific lineage specification and for developing future cell therapy for various diseases.
在此，我们描述了一种新型人类胚胎干细胞（hES）系Endeavour-1（E1）及其四个新的克隆系（E1C1、E1C2、E1C3、E1C4）的衍生过程及其特性。E1及其克隆系在源自人胎儿成纤维细胞（HFFs）并在基本无血清培养基中生长的细胞上进行传代培养。从34个捐赠的人类胚胎（27个存活）中分离出7个内细胞团，获得了一个新的hES细胞系。E1已在培养中超过1年，具备干细胞的所有典型特征，即干细胞表面标志物（阶段特异性胚胎抗原SSEA-3和SSEA-4，以及肿瘤识别抗原TRA-1-60和TRA-1-81）的表达、碱性磷酸酶染色以及多能基因标志物（nanog）的存在。该细胞系在体外和体内条件下均显示出多能性。E1具有正常的核型（46XX）。使用我们优化的克隆程序，从E1衍生出四个新的克隆系：E1C1、E1C2、E1C3和E1C4。这些克隆系显示出正常特征：除E1C3显示涉及15号和17号染色体的相互易位外，其核型与亲代细胞系相同（46XX）；具有干细胞表面标志物SSEA-4、TRA-1-60和TRA-1-81；以及多能性的基因表达（Nanog）。所有这些克隆系在悬浮培养中形成胚状体（EBs）。接种后，EBs分化，形成源自所有三个胚层的细胞谱系，这通过对外胚层标志物β-III微管蛋白、中胚层标志物CD34和内胚层标志物甲胎蛋白（AFP）的免疫定位得以表明。这些标志物在克隆之间的表达存在细微差异。这些克隆系在体内显示出多能性。用激活素A处理后，E1及其克隆系可分化为确定的内胚层，并且如通过RT-PCR检测SOX17、FOXa2和GATA-4的表达所示，这些克隆系之间存在一些细微差异。这可能有助于选择用于特定谱系定向以及开发未来针对各种疾病的细胞疗法的克隆系。
