Stem cellge arrayposter

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Stem cellge arrayposter

  1. 1. The development of focused microarray to assess dopaminergic and glial cell differentiation derived from human stem cells Catherine M. Schwartz1,5, Yongquan Luo1, Soojung Shin1, Xianmin Zeng2, Nong Chen3, YueWang3, Xiang Yu3and Mahendra S. Rao 1,4 Abstract Embryonic stem cells and their derived progeny have received a great attention for their potential use in cell replacement therapy for the treatment of neural deficit neurodegenerative disorders. We have designed, tested, and validated a dopaminergic neuron-glial focused array for the purpose of assessing the state of oligodendrocyte or dopaminergic neuron differentiation. This array consists of 281 genes that include cell type-specific markers for dopaminergic neurons, oligodendrocytes, astrocytes, embryonic stem (ES) cells, and progenitor cells as well as cytokines, chemokines, and their respective receptors in order to give an overview of potential pathways that may regulate differentiation. The gene specific 60-mer 3' biased oligonucleotides were arrayed in a 25 x 12 format based on function along with positive controls, negative controls, duplicates, and blanks. We have profiled human adult brain substantia niagra, human ES cells, human ES derived neural stem cells, and the differentiated progeny of pluripotent stem cells, and have shown these diverse populations could be readily distinguished 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 a set of reporting genes for detecting dopaminergic neurons, glial, and contaminating ES cells or progenitor cells. Furthermore, we have used this array to monitor NTera2 differentiation towards dopaminergic neurons and have shown the capacity of this array to distinguish stages of differentiation, assess cell maturity, determine the degree of contaminating populations, and provide important clues to mechanisms regulating differentiation. Thus, this focused dopaminergicglial array provides a useful tool in the routine assessment of cell properties prior to their therapeutic use. A A A B C C B D B Validation Experiments. RNA from samples known to express genes included on the array served as experimental controls to test the array. cRNAs from human substantia niagra, BG01V, and BG03 derived NSCs were hybridized to arrays and easily distinguished based on gene expression patterns (A). RT-PCR confirmed the presence of several genes detected by the array in substantia niagra (column 1), BG01V (column 2), and NSCs derived from BG03 (column 3). Tissue specific genes were identified and summarized in the above table (C). Application Experiments. cRNAs derived from the undifferentiated human embryonal carcinoma stem cell line NTera2 (NT2), NTera2 cells induced towards dopaminergic neurons by co-culture with the mouse stromal cell line PA6 for 12 days followed by flow cytometry enrichment for PSA-NCAM expressing cells (NT2-NCAM), and FAB5 positive/NCAM negative cells from human brain at embryonic 20 weeks (FA2B5+) were hybridized to the array (A-images, B-relative intensity normalized to GAPDH). NTera2 cells differentiated for 12 days on PA6 cells and immunocytochemistry co-localization staining of PSA-NCAM with tyrosine hydroxolase (TH) or Oct4 (C). Gene expression was confirmed by RT-PCR (D). A Conclusions •We designed a focused dopaminergic glial array containing a total of 281 known genes, housekeeping genes to serve as positive controls, and negative controls arranged by gene function. C D B C D •The designed focused dopaminergic glial array was able to adequately detect and distinguish dopaminergic, neural, and pluripotent stem cell populations in a concentration dependent manner. •We utilized this array to develop a list of candidate genes that could be used to distinguish various populations as well as for the purpose of testing the purity of NCAM selected cells differentiated towards dopaminergic neurons. Array format and quality control. A total of 281 genes were arrayed in 25x12 format based on function, including positive detection controls (biotinylated articficial sequence 2 complementary sequences), positive hybridization controls (housekeeping genes: GAPDH, beta-Actin, RPS27A, and B2M), negative hybridization controls (blanks, plasmid PUC1B DNA or artificial sequences not expected to be present in cDNA) (A). Two array experiments were run using cDNA generated from equal RNA sample mixtures (B). The spot intensity was measured and normalized to GAPDH. A linear plot of these two experiments (C) resulted in a correlation coefficient value of 0.9728. The average (Mean), standard deviation (SD), and coefficient of variance expressed as a percent (CV%) were calculated (D). Titration experiments. Hybridization and labeling titration experiments were performed in order to test the ability of the array to detect specific genes in a concentration dependent manner. For titration experiments, biotionylated cRNA targets were generated from human substania niagra and BG01V. The total RNA was held constant while the ratio of human substantia niagra (SN) to BG01V was altered (A). The results showed a positive linear relationship of detected hybridization signals of tissue specifiic gene to cRNA input with correlation coefficients ranging from 0.60 to 0.99. The amplification labeling system was tested by using the same ratio of RNA used for hybridization experiments and examined the intensity changes of specific genes (B-D). The correlation coefficients for the amplification experiments ranged from 0.51-1.00. Aknowledgements: This research was supported by the Intramural Research Traing Program of the NIH, National Institute on Aging (NIA). 1Gerontology Research Center; Stem Cell Biology Unit; Laboratory of Neurosciences; NIA; NIH; DHHS; Baltimore, MD 2Buck Institute for Age Research, Novato, CA. 3SuperArray Bioscience Corporation, Frederick, MD 4Department of Neuroscience, Johns Hopkins University; Baltimore, MD and CRL, 1600 Faraday Drive, Carlsbad, CA 92008 5Laboratory of Molecular Neurobiology; Medical Biochemistry and Biophysics;Karolinska Institute; Stockholm, Sweden

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