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Stem Cells 2003;21:575-587 www.StemCells.com
© 2003 AlphaMed Press

Designing, Testing, and Validating a Focused Stem Cell Microarray for Characterization of Neural Stem Cells and Progenitor Cells

Yongquan Luoa, Jingli Caia, Irene Ginisa, Yanyang Sunb, Siulan Leeb, Sean X. Yub, Ahmet Hokec, Mahendra Raoa

a Laboratory of Neurosciences, Gerontology Research Center, National Institute on Aging, Baltimore, Maryland, USA;
b SuperArray Bioscience Corporation, Frederick, Maryland, USA;
c Department of Neurology, Johns Hopkins University, Baltimore, Maryland, USA

Key Words. Embryonic stem cells • Neural stem cells • Focus array • Microarray • Functional genomics

Mahendra Rao, M.D., Johns Hopkins University, Baltimore, Maryland 21224, USA. Telephone: 410-558-8204; Fax: 410-558-8249; e-mail: raomah{at}grc.nia.nih.gov

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.




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