Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Lara Stevanato, Caroline Hicks, John D Sinden, Lara Stevanato, Caroline Hicks, John D Sinden

Abstract

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycER(TAM)construct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3'UTRs by dual reporter plasmid transfection and dual luciferase assay.

Figures

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References

    1. Falanga V. Stem cells in tissue repair and regeneration. J Invest Dermatol. 2012;132(6):1538–1541.
    1. Pollock K, et al. A conditionally immortal clonal stem cell line from human cortical neuroepithelium for the treatment of ischemic stroke. Exp Neurol. 2006;199(1):143–155.
    1. Hodges H, et al. Making stem cell lines suitable for transplantation. Cell Transplant. 2007;16(2):101–115.
    1. Smith EJ, et al. Implantation site and lesion topology determine efficacy of a human neural stem cell line in a rat model of chronic stroke. Stem Cells. 2012;30(4):785–796.
    1. Stroemer P, et al. The neural stem cell line CTX0E03 promotes behavioral recovery and endogenous neurogenesis after experimental stroke in a dose-dependent fashion. Neurorehabil Neural Repair. 2009;23(9):895–909.
    1. Kalladka D, Muir KW. Brain repair: cell therapy in stroke. Stem Cells Cloning. 2014;7:31–44.
    1. Fire A, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391(6669):806–811.
    1. Anokye-Danso F, et al. Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell. 2011;8(4):376–388.
    1. Li X, Jin P. Roles of small regulatory RNAs in determining neuronal identity. Nat Rev Neurosci. 2010;11(5):329–338.
    1. Badylak SF. The extracellular matrix as a scaffold for tissue reconstruction. Semin Cell Dev Biol. 2002;13(5):377–383.
    1. Buxboim A, Discher DE. Stem cells feel the difference. Nat Methods. 2010;7(9):695–697.
    1. Li WJ, et al. A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells. Biomaterials. 2005;26(6):599–609.
    1. Lutolf MP, Gilbert PM, Blau HM. Designing materials to direct stem-cell fate. Nature. 2009;462(7272):433–441.
    1. Ortinau S, et al. Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells. Biomed Eng Online. 2010;9(1):70.
    1. Saha K, Pollock JF, Schaffer DV, Healy KE. Designing synthetic materials to control stem cell phenotype. Curr Opin Chem Biol. 1016;11(4):381–387.
    1. Knight E, Murray B, Carnachan R, Przyborski S. Alvetex(R): polystyrene scaffold technology for routine three dimensional cell culture. Methods Mol Biol. 2011;695:323–340.
    1. Bokhari M, Carnachan RJ, Przyborski SA, Cameron NR. Emulsion- templated porous polymers as scaffolds for three dimensional cell culture: effect of synthesis parameters on scaffold formation and homogenity. J. Mater. Chem. 2007;17:4088–4094.
    1. Bokhari M, Carnachan RJ, Cameron NR, Przyborski SA. Novel cell culture device enabling three-dimensional cell growth and improved cell function. Biochem Biophys Res Commun. 2007;354(4):1095–1100.
    1. Stevanato L, Sinden JD. The effects of microRNAs on human neural stem cell differentiation in two- and three-dimensional cultures. Stem Cell Res Ther. 2014;5(2):49.
    1. Ippolito DM, Eroglu C. Quantifying synapses: an immunocytochemistry-based assay to quantify synapse number. J Vis Exp. 2010.
    1. Chen K, Rajewsky N. Natural selection on human microRNA binding sites inferred from SNP data. Nat Genet. 2006;38(12):1452–1456.
    1. Krek A, et al. Combinatorial microRNA target predictions. Nat Genet. 2005;37(5):495–500.
    1. Maragkakis M, et al. Accurate microRNA target prediction correlates with protein repression levels. BMC Bioinformatics. 2009;10:295.
    1. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005;120(1):15–20.
    1. Aldred SF, Collins P, Trinklein N. Identifying targets of human micrornas with the LightSwitch Luciferase Assay System using 3'UTR-reporter constructs and a microRNA mimic in adherent cells. J Vis Exp. 2011.
    1. Ponnio T, Conneely OM. nor-1 regulates hippocampal axon guidance, pyramidal cell survival, and seizure susceptibility. Mol Cell Biol. 2004;24(20):9070–9078.
    1. Martinez-Morales PL, Quiroga AC, Barbas JA, Morales AV. SOX5 controls cell cycle progression in neural progenitors by interfering with the WNT-beta-catenin pathway. EMBO Rep. 2010;11(6):466–472.
    1. Kwan KY, et al. SOX5 postmitotically regulates migration, postmigratory differentiation, and projections of subplate and deep-layer neocortical neurons. Proc Natl Acad Sci U S A. 2008;105(41):16021–16026.
    1. Lai T, et al. SOX5 controls the sequential generation of distinct corticofugal neuron subtypes. Neuron. 2008;57(2):232–247.

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