Luo Yongquan, Cai Jingli, Ginis Irene, Sun Yanyang, Lee Siulan, Yu Sean X, Hoke Ahmet, Rao Mahendra
Laboratory of Neurosciences, Gerontology Research Center, National Institute on Aging, Baltimore, Maryland, USA.
Stem Cells. 2003;21(5):575-87. doi: 10.1634/stemcells.21-5-575.
Fetal neural stem cells (NSCs) have received great attention not only for their roles in normal development but also for their potential use in the treatment of neurodegenerative disorders. To develop a robust method of assessing the state of stem cells, we have designed, tested, and validated a rodent NSC array. This array consists of 260 genes that include cell type-specific markers for embryonic stem (ES) cells and neural progenitor cells as well as growth factors, cell cycle-related genes, and extracellular matrix molecules known to regulate NSC biology. The 500-bp polymerase chain reaction products amplified and validated by using gene-specific primers were arrayed along with positive controls. Blanks were included for quality control, and some genes were arrayed in duplicate. No cross-hybridization was detected. The quality of the arrays and their sensitivity were also examined by using probes prepared by conventional reverse transcriptase or by using amplified probes prepared by linear polymerase replication (LPR). Both methods showed good reproducibility, and probes prepared by LPR labeling appeared to detect expression of a larger proportion of expressed genes. Expression detected by either method could be verified by RT-PCR with high reproducibility. Using these stem cell chips, we have profiled liver, ES, and neural cells. The cell types could be readily distinguished from each other. Nine markers specific to mouse ES cells and 17 markers found in neural cells were verified as robust markers of the stem cell state. Thus, this focused neural stem array provides a convenient and useful tool for detection and assessment of NSCs and progenitor cells and can reliably distinguish them from other cell populations.
胎儿神经干细胞（NSCs）不仅因其在正常发育中的作用，还因其在神经退行性疾病治疗中的潜在用途而备受关注。为了开发一种评估干细胞状态的可靠方法，我们设计、测试并验证了一种啮齿动物NSC阵列。该阵列由260个基因组成，包括胚胎干细胞（ES）和神经祖细胞的细胞类型特异性标志物，以及已知调节NSC生物学的生长因子、细胞周期相关基因和细胞外基质分子。使用基因特异性引物扩增并验证的500bp聚合酶链反应产物与阳性对照一起排列。设置空白对照用于质量控制，一些基因重复排列。未检测到交叉杂交。还使用常规逆转录制备的探针或线性聚合酶复制（LPR）制备的扩增探针检查了阵列的质量及其灵敏度。两种方法均显示出良好的重现性，并且LPR标记制备的探针似乎能检测到更大比例的表达基因的表达。通过两种方法检测到的表达均可通过RT-PCR进行验证，重现性高。使用这些干细胞芯片，我们对肝脏、ES和神经细胞进行了分析。这些细胞类型很容易相互区分。验证了九个小鼠ES细胞特异性标志物和17个神经细胞中发现的标志物作为干细胞状态的可靠标志物。因此，这种聚焦的神经干细胞阵列提供了一种方便且有用的工具，用于检测和评估NSCs和祖细胞，并能可靠地将它们与其他细胞群体区分开来。
