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Buckley C.E.,University of Cambridge | Buckley C.E.,Summit plc. Unit 7330 | Buckley C.E.,King's College London | Marguerie A.,Summit plc. Unit 7330 | And 10 more authors.
Neuropharmacology | Year: 2010

Treatment of the autoimmune demyelinating disease multiple sclerosis (MS) requires therapies that both limit and repair damage. While several immunomodulatory treatments exist to limit damage there are currently no treatments that promote the regenerative process of remyelination. A rapid way of screening potential pro-remyelination compounds is therefore required. The use of larval zebrafish in a drug reprofiling screen allows rapid in vivo screening and has been used successfully in the past as an efficient way of identifying new indications for existing drugs. A novel screening platform for potential pro-myelination compounds was developed using zebrafish larvae. Two percent of compounds screened from reprofiling libraries altered oligodendrocyte lineage cell recruitment and/or proliferation, as measured by the numbers of dorsally migrated spinal cord olig2+ cells. Selective screening identified three compounds that altered levels of myelination, as measured by whole larvae myelin basic protein (mbp) transcript levels; the src family kinase inhibitor PP2, a biogenic amine and a thioxanthene. As well as many previously unrecognised compounds, identified compounds included those with previously known effects on myelin and/or the oligodendrocyte lineage, such as a PPAR agonist, steroid hormones and src family kinase inhibitors. As well as providing methods for further assessment of potentially beneficial compounds, this screen has highlighted 25 targets that are able to alter oligodendrocyte lineage cell recruitment or proliferation and/or mbp transcript levels in vivo and are worthy of further investigation for their potential effects on remyelination. © 2010 Elsevier Ltd.


Grant
Agency: Cordis | Branch: FP7 | Program: CP-IP | Phase: HEALTH-2007-2.1.2-3 | Award Amount: 16.09M | Year: 2008

EuroSyStem brings together elite European research teams to create a unique and world-leading programme in fundamental stem cell biology. By interconnecting complementary biological and computational expertise we will drive the generation of new knowledge on the characteristics of normal and abnormal stem cells. We will pave the way for application of systems methodology by measuring and modelling stem cell properties and behaviour. Information will be mined from studies in model organisms, but our primary focus is on the paradigmatic mammalian stem cells haematopoietic, epithelial, neural and embryonic. We will compare cellular hierarchy, signalling, epigenetics, dysregulation, and plasticity. Niche dependence, asymmetric division, transcriptional circuitry and the decision between self-renewal and commitment are linked in a cross-cutting work package. A multidisciplinary approach combines transgenesis, real time imaging, multi-parameter flow cytometry, transcriptomics, RNA interference, proteomics and single cell methodologies. SMEs will contribute to the development of enhanced resolution quantitative technologies. A platform work package will provide new computational tools and database resources, enabling implementation of novel analytical and modelling approaches. EuroSyStem will engage with and provide a focal point for the European stem cell research community. The targeted collaborations within the EuroSyStem research project will be augmented by federating European research excellence in different tissues and organisms. We will organise annual symposia, training workshops, summer schools, networking and research opportunities to promote a flourishing basic stem cell research community. This network will foster interaction and synergy, accelerating progress to a deeper and more comprehensive understanding of stem cell properties. In parallel EuroSyStem will develop WEB resources, educational and outreach materials for scientists and the lay community.


Hook L.,Stem Cell science UK Ltd | Hook L.,Plasticell | Vives J.,Stem Cell science UK Ltd | Fulton N.,Stem Cell science UK Ltd | And 14 more authors.
Neurochemistry International | Year: 2011

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells (Conti et al., 2005; Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8-9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133 +CD24 -/lo cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca 2+ imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny. © 2011 Elsevier Ltd. All rights reserved.


McLaren D.,Stem Cell science UK Ltd | McLaren D.,Biologics | Gorba T.,StemCells | Marguerie De Rotrou A.,Stem Cell science UK Ltd | And 17 more authors.
Journal of Biomolecular Screening | Year: 2013

The aim of this study was to demonstrate proof-of-concept feasibility for the use of human neural stem cells (NSCs) for high-throughput screening (HTS) applications. For this study, an adherent human induced pluripotent stem (iPS) cell-derived long-term, self-renewing, neuroepithelial-like stem (lt-NES) cell line was selected as a representative NSC. Here, we describe the automated large-scale serum-free culture ("scale-up") of human lt-NES cells on the CompacT SelecT cell culture robotic platform, followed by their subsequent automated "scale-out" into a microwell plate format. We also report a medium-throughput screen of 1000 compounds to identify modulators of neural stem cell proliferation and/or survival. The screen was performed on two independent occasions using a cell viability assay with end-point reading resulting in the identification of 24 potential hit compounds, 5 of which were found to increase the proliferation and/or survival of human lt-NES on both occasions. Follow-up studies confirmed a dose-dependent effect of one of the hit compounds, which was a Cdk-2 modulator. This approach could be further developed as part of a strategy to screen compounds to either improve the procedures for the in vitro expansion of neural stem cells or to potentially modulate endogenous neural stem cell behavior in the diseased nervous system. © 2012 Society for Laboratory Automation and Screening.


The aim of this study was to demonstrate proof-of-concept feasibility for the use of human neural stem cells (NSCs) for high-throughput screening (HTS) applications. For this study, an adherent human induced pluripotent stem (iPS) cell-derived long-term, self-renewing, neuroepithelial-like stem (lt-NES) cell line was selected as a representative NSC. Here, we describe the automated large-scale serum-free culture (scale-up) of human lt-NES cells on the CompacT SelecT cell culture robotic platform, followed by their subsequent automated scale-out into a microwell plate format. We also report a medium-throughput screen of 1000 compounds to identify modulators of neural stem cell proliferation and/or survival. The screen was performed on two independent occasions using a cell viability assay with end-point reading resulting in the identification of 24 potential hit compounds, 5 of which were found to increase the proliferation and/or survival of human lt-NES on both occasions. Follow-up studies confirmed a dose-dependent effect of one of the hit compounds, which was a Cdk-2 modulator. This approach could be further developed as part of a strategy to screen compounds to either improve the procedures for the in vitro expansion of neural stem cells or to potentially modulate endogenous neural stem cell behavior in the diseased nervous system.


PubMed | Stem Cell science UK Ltd
Type: Journal Article | Journal: Neurochemistry international | Year: 2011

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells (Conti et al., 2005; Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8-9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133(+)CD24(-/lo) cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca(2+) imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.

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