A focused microarray to assess dopaminergic and glial cell differentiation from fetal tissue or embryonic stem cells.
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 100 genes expressed in dopaminergic neurons, oligodendrocytes, and astrocytes, an additional 200 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 (ESCs), 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. Using 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 ESCs and progenitors. Finally, we monitored NTera2 differentiation toward dopaminergic neurons and have shown the ability of this array to distinguish stages of differentiation and 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.
Luo
Yongquan
Y
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, USA.
Schwartz
Catherine
C
Shin
Soojung
S
Zeng
Xianmin
X
Chen
Nong
N
Wang
Yue
Y
Yu
Xiang
X
Rao
Mahendra S
MS
D001483:Base Sequence; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004298:Dopamine / Q000378:metabolism*; D005333:Fetus / Q000166:cytology / Q000378:metabolism; D006801:Humans; D009457:Neuroglia / Q000166:cytology* / Q000378:metabolism*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards; D011786:Quality Control; D039901:Totipotent Stem Cells / Q000166:cytology* / Q000378:metabolism*
D017931:DNA Primers; D004298:Dopamine