Real-time quantitative PCR analysis of undifferentiated rat embryonic stem cells and induced pluripotent stem cells

Validation study of a focused microarray for evaluation of undifferentiated rat ESCs.Using a focused microarray, undifferentiated embryonic stem cells (ESCs) can be distinguished from differentiated ESCs and other cells derived from the early embryo since they have a unique gene expression pattern associated with pluripotency and lack of markers of differentiation. To date, however, such an array has not been developed for the rat species and differences in genomes of rat and human or rat and mouse preclude the use of ESC focused human or mouse microarrays for the rat. Here, we developed a focused microarray for screening rat ESCs and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem cells (TS), rat extraembryonic endoderm cells (XEN), mouse embryonic fibroblast feeder cells (MEFs) and rat ESCs that have been differentiated in vitro. We used this tool to compare rat ESCs which have been expanded in a conventional rat ESC medium containing two inhibitors, e.g., GSK3 and MEK inhibitors, and leukemia inhibitory factor (LIF), and found expression of Cdx2, a gene associated with trophoblast determination. The rat ESC focused microarray described in this report has utility for rapid screening rat ESCs. This will enable optimization of culture conditions in the future.用于评估未分化大鼠胚胎干细胞的聚焦型基因芯片（focused microarray）验证研究。借助聚焦型基因芯片，未分化胚胎干细胞（Embryonic Stem Cells, ESCs）可与分化态胚胎干细胞及早期胚胎来源的其他细胞相区分，这是因为未分化ESCs具备与多能性相关的独特基因表达谱，且缺乏分化相关标志物。然而迄今为止，尚未针对大鼠开发此类芯片；大鼠与人类、大鼠与小鼠的基因组差异，使得无法将人类或小鼠的ESC聚焦型基因芯片应用于大鼠样本。本研究开发了一款用于筛选大鼠ESCs的聚焦型基因芯片，并提供了验证数据，证实该芯片可将未分化大鼠ESCs与大鼠滋养层干细胞（Trophoblast Stem Cells, TS）、大鼠胚外内胚层细胞（Extraembryonic Endoderm Cells, XEN）、小鼠胚胎成纤维饲养层细胞（Mouse Embryonic Fibroblast Feeder Cells, MEFs）以及体外分化的大鼠ESCs区分开来。本研究使用该工具，对比了在含两种抑制剂（即GSK3与MEK抑制剂）及白血病抑制因子（Leukemia Inhibitory Factor, LIF）的常规大鼠ESC培养基中扩增的大鼠ESCs，发现了与滋养层细胞定向分化相关的基因Cdx2的表达。本报告中描述的大鼠ESC聚焦型基因芯片可用于大鼠ESCs的快速筛选，未来将有助于培养条件的优化。