Yang Amy X, Mejido Josef, Luo Yongquan, Zeng Xianmin, Schwartz Catherine, Wu Tianxia, Thies R Scott, Bhattacharya Bhaskar, Han Jing, Freed Bill, Rao Mahendra, Puri Raj K
Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
Stem Cells Dev. 2005 Jun;14(3):270-84. doi: 10.1089/scd.2005.14.270.
Human embryonic stem cells (hESC) must be differentiated before clinical use. In addition, the extent of contamination of undifferentiated cells and the efficiency of differentiation must also be assessed prior to clinical application. In this manuscript, we describe the development of a focused microarray that may be used to discriminate between hESC and their differentiated progeny. This array contains 755 genes including embryonic stem cell markers as well as markers of differentiation into neural, mesodermal, and endodermal phenotypes. In addition, we have included candidate genes belonging to families of cytokines, chemokines, receptors, signaling pathways, and homeodomain proteins that are likely to be important in the process of differentiation. Testing and validation of the focused array was performed using RNA from hESC, human embryoid body (hEB) outgrowths, and a human embryonal carcinoma (hEC) cell line. We have compared gene expression with negative background, GAPDH, beta-actin positive controls, and human universal RNA (hURNA), showing that such an array can rapidly distinguish between undifferentiated and differentiated hESC-derived cell populations. We expect that the described array will be extremely useful in evaluating the extent of differentiation and the state of the hESC-derived population utilized for therapeutic purposes.
人类胚胎干细胞（hESC）在临床应用前必须进行分化。此外，在临床应用前还必须评估未分化细胞的污染程度和分化效率。在本论文中，我们描述了一种聚焦微阵列的开发，该微阵列可用于区分hESC及其分化后代。该阵列包含755个基因，包括胚胎干细胞标志物以及向神经、中胚层和内胚层表型分化的标志物。此外，我们还纳入了属于细胞因子、趋化因子、受体、信号通路和同源域蛋白家族的候选基因，这些基因在分化过程中可能很重要。使用来自hESC、人类胚状体（hEB）生长物和人类胚胎癌细胞系（hEC）的RNA对聚焦阵列进行了测试和验证。我们将基因表达与阴性背景、GAPDH、β-肌动蛋白阳性对照以及人类通用RNA（hURNA）进行了比较，结果表明这样的阵列可以快速区分未分化和分化的hESC来源的细胞群体。我们预计所描述的阵列在评估用于治疗目的的hESC来源群体的分化程度和状态方面将非常有用。
