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Original Article |
1 Gerontology Research Center, Stem Cell Biology Unit, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland
2 Gerontology Research Center, Stem Cell Biology Unit, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland; Laboratory of Molecular Neurobiology, Medical Biochemistry and Biophysics, Retzius Laboratory, Karolinska Institutet, Stockholm, Sweden
3 Buck Institute for Aging, Novato, California
4 SuperArray Bioscience Corporation, Frederick, Maryland
5 Gerontology Research Center, Stem Cell Biology Unit, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland; Department of Neuroscience, Johns Hopkins University, Baltimore, Maryland and Carlsbad, California
* To whom correspondence should be addressed. E-mail: LuoYo{at}grc.nia.nih.gov.
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Abstract |
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We designed oligonucleotide gene specific probes to develop a focused array that can be used to discriminate between neural phenotypes, identify biomarkers, and provide an overview of the process of dopaminergic neuron and glial differentiation. We have arrayed approximately one hundred genes expressed in dopaminergic neurons, oligodendrocytes, and astrocytes, an additional two hundred known cytokines, chemokines, and their respective receptors, as well as markers for pluripotent and progenitor cells. The gene specific 60-mer 3' biased oligonucleotides for these 281 genes were arrayed in a 25 x 12 format based on function. Using human adult brain substantia nigra, human embryonic stem cells (hESCs), and the differentiated progeny of pluripotent cells, we showed that this array was capable of distinguishing dopaminergic neurons, glial cells, and pluripotent cells by their gene expression profiles in a concentration-dependent manner. Based on linear correlation coefficients of input RNA with output intensity, we identified a list of genes that can serve as reporting genes for detecting dopaminergic neurons, glial cells and contaminating ES cells and progenitors. Finally, we monitored NTera2 differentiation towards dopaminergic neurons and have shown the ability of this array to distinguish stages of differentiation, provide important clues to factors regulating differentiation, the degree of contaminating populations, and stage of cell maturity. We suggest that this focused array will serve as a useful complement to other large-scale arrays in routine assessment of cell properties prior to their therapeutic use.
Key Words. hESCs, differentiation, oligodendrocytes, astrocytes, dopamienrgic neurons
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