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    "NeuroStem Chip": a novel highly specialized tool to study neural differentiationpathways in human stem cells.

    Anisimov, Sergey; Christophersen, Nicolaj; Correia, Ana S; Li, Jia-Yi; Brundin, Patrik

    Published in:BMC Genomics

    DOI:10.1186/1471-2164-8-46

    Published: 2007-01-01

    Link to publication

    Citation for published version (APA):Anisimov, S., Christophersen, N., Correia, A. S., Li, J-Y., & Brundin, P. (2007). "NeuroStem Chip": a novel highlyspecialized tool to study neural differentiation pathways in human stem cells. BMC Genomics, 8(Feb 8), 46-46.DOI: 10.1186/1471-2164-8-46

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    https://doi.org/10.1186/1471-2164-8-46http://portal.research.lu.se/portal/en/publications/neurostem-chip-a-novel-highly-specialized-tool-to-study-neural-differentiation-pathways-in-human-stem-cells(d219b1a1-bca3-4752-9669-98210d2092e9).html

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    Open AcceMethodology article"NeuroStem Chip": a novel highly specialized tool to study neural differentiation pathways in human stem cellsSergey V Anisimov*, Nicolaj S Christophersen, Ana S Correia, Jia-Yi Li and Patrik Brundin

    Address: Neuronal Survival Unit, Wallenberg Neuroscience Center, Lund University, 221 84 Lund, Sweden

    Email: Sergey V Anisimov* - [email protected]; Nicolaj S Christophersen - [email protected]; Ana S Correia - [email protected]; Jia-Yi Li - [email protected]; Patrik Brundin - [email protected]

    * Corresponding author

    AbstractBackground: Human stem cells are viewed as a possible source of neurons for a cell-basedtherapy of neurodegenerative disorders, such as Parkinson's disease. Several protocols thatgenerate different types of neurons from human stem cells (hSCs) have been developed.Nevertheless, the cellular mechanisms that underlie the development of neurons in vitro as they aresubjected to the specific differentiation protocols are often poorly understood.

    Results: We have designed a focused DNA (oligonucleotide-based) large-scale microarrayplatform (named "NeuroStem Chip") and used it to study gene expression patterns in hSCs as theydifferentiate into neurons. We have selected genes that are relevant to cells (i) being stem cells, (ii)becoming neurons, and (iii) being neurons. The NeuroStem Chip has over 1,300 pre-selected genetargets and multiple controls spotted in quadruplicates (~46,000 spots total). In this study, wepresent the NeuroStem Chip in detail and describe the special advantages it offers to the fields ofexperimental neurology and stem cell biology. To illustrate the utility of NeuroStem Chip platform,we have characterized an undifferentiated population of pluripotent human embryonic stem cells(hESCs, cell line SA02). In addition, we have performed a comparative gene expression analysis ofthose cells versus a heterogeneous population of hESC-derived cells committed towards neuronal/dopaminergic differentiation pathway by co-culturing with PA6 stromal cells for 16 days andcontaining a few tyrosine hydroxylase-positive dopaminergic neurons.

    Conclusion: We characterized the gene expression profiles of undifferentiated and dopaminergiclineage-committed hESC-derived cells using a highly focused custom microarray platform(NeuroStem Chip) that can become an important research tool in human stem cell biology. Wepropose that the areas of application for NeuroStem microarray platform could be the following:(i) characterization of the expression of established, pre-selected gene targets in hSC lines,including newly derived ones, (ii) longitudinal quality control for maintained hSC populations, (iii)following gene expression changes during differentiation under defined cell culture conditions, and(iv) confirming the success of differentiation into specific neuronal subtypes.

    Published: 8 February 2007

    BMC Genomics 2007, 8:46 doi:10.1186/1471-2164-8-46

    Received: 21 September 2006Accepted: 8 February 2007

    This article is available from: http://www.biomedcentral.com/1471-2164/8/46

    2007 Anisimov et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Page 1 of 15(page number not for citation purposes)

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  • BMC Genomics 2007, 8:46 http://www.biomedcentral.com/1471-2164/8/46

    BackgroundModern DNA microarrays permit a comprehensive analy-sis of quantitative and qualitative changes in RNA tran-script abundance, outlining the cross-sections of geneexpression and alterations of these in response to geneticor environmental stimuli. Genome-scale microarrays(cDNA- or oligonucleotide-based) are most valuablewhen screening populations of cells for the novel genesreflecting potential diagnostic and prognostic markers orfor an identification of novel therapeutic targets. On theother hand, custom microarray platforms that focus onspecific pre-selected subset of genes relevant to a particu-lar field of investigation can be less costly and more suita-ble for detection of smaller gene expression changes.

    Microarray technology has added important informationon both normal development and pathological changesin neurons. This is well illustrated by multiple studies onsubstantia nigra dopaminergic neurons, which degeneratein Parkinson's disease (PD) [1-5]. The shortcomings ofpharmacological therapies in PD have stimulated a searchfor alternative treatment strategies. In successful cases,transplants of human embryonic mesencephalicdopaminergic neurons can both restore dopaminergicneurotransmission and provide some symptomatic relief[6-8]. A wider application of neural transplantation in PDis, however, currently not feasible due to the unpredicta-ble and variable outcome, the risks of unwanted side-effects (dyskinesias) [9,10] and ethical and practical prob-lems associated with using donor cells obtained fromaborted embryos and fetuses [11,12]. Human embryonicstem cells (hESCs) are considered a promising futuresource of cells for cell replacement therapy in PD andother neurological conditions [13]. They could constitutea virtually infinite source of self-renewing cells that can bepersuaded to differentiate into specific types of neuralcells, including dopaminergic neurons [14-16]. Themolecular mechanisms that govern development of cul-tured hESCs into specific types of neural cells are not fullyunderstood. To promote our understanding of suchmechanisms, it would be valuable to have tools that read-ily and reproducibly can help to characterize the cells asthey differentiate from pluripotent stem cells into post-mitotic neurons. This important issue was addressed inearlier studies by Luo et al. and Yang et al., who designedsmall-to-moderate scale custom microarray platforms(281 and 755 gene targets, respectively) [17,18]. In addi-tion SuperArray Bioscience Corporation (Frederick, MD,USA) have manufactured a range of small-scale arrays(263 gene targets for human array; [19]). We sought tocreate an improved and updated microarray platform forhESC/neuronal differentiation-oriented gene expressionstudies. Therefore, we generated a specialized large-scaleDNA microarray platform (the "NeuroStem Chip") thathas over 1,300 pre-selected gene targets and multiple con-

    trols spotted in quadruplicates (~46,000 spots total). Herewe introduce the platform and the advantages it can offersto neuroscientists and stem cell biologists: particularly, inthe niche of gene expression-oriented characterization ofthe samples using an assay of pre-selected, already estab-lished gene targets. In the current study, we use the Neu-roStem Chip to characterize an undifferentiatedpopulation of pluripotent hESCs (cell line SA02, CellartisAB, Gteborg, Sweden) and compare the gene expressionin those cells with that of a hESC-derived cell populationrich in neurons, including tyrosine hydroxylase-positivedopaminergic neurons.

    Results and DiscussionStem cells have unique biological characteristics, but onlya limited number of genes are currently recognized asestablished stem cell markers. Examples include POUdomain, class 5, transcription factor 1 (Oct3/4), signaltransducer and activator of transcription 3 (Stat3), terato-carcinoma-derived growth factor (Tdgf1), Enk-pending(Nanog), undifferentiated embryonic cell transcriptionfactor 1 (Utf1) and DNA methyltransferase 3B (Dnmt3b)[20]. At the same time, hundreds of genes are suggested ascandidate markers for "stemness", but their coupling tothe undifferentiated stem cell state is not y

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