Abstract
The incidence of neurological diseases including learning and developmental disorders has increased in recent years. Concurrently, the number and volume of worldwide registered and traded chemicals have also increased. There is a broad consensus that the developing brain is particularly sensitive to damage by chemicals and that evaluation of chemicals for developmental toxicity or neurotoxicity is critical to human health. Human pluripotent embryonal carcinoma (NTERA-2 or NT2) cells are increasingly considered as a suitable model for in vitro developmental toxicity and neurotoxicity (DT/DNT) studies as they undergo neuronal differentiation upon stimulation with retinoic acid (RA) and allow toxicity assessment at different stages of maturation. Here we describe a protocol for cell fitness screening in differentiating NT2 cells based on the analysis of intracellular ATP levels allowing for the identification of chemicals which are potentially harmful to the developing brain. The described method is suitable to be adapted to low-, medium-, and high-throughput screening and allows multiplexing with other cell fitness indicators. While the presented protocol focuses on cell fitness screening in human pluripotent stem cells it may also be applied to other in vitro models.
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References
May M (2000) Disturbing behavior: neurotoxic effects in children. Environ Health Perspect 108:A262–A267
Colborn T (2004) Neurodevelopment and endocrine disruption. Environ Health Perspect 112:944–949
Rauh VA, Garfinkel R, Perera FP et al (2006) Impact of prenatal chlorpyrifos exposure on neurodevelopment in the first 3 years of life among inner-city children. Pediatrics 118:e1845–e1859
Herbert MR (2010) Contributions of the environment and environmentally vulnerable physiology to autism spectrum disorders. Curr Opin Neurol 23:103–110
Rice D, Barone S Jr (2000) Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ Health Perspect 108(Suppl 3):511–533
Grandjean P, Landrigan PJ (2006) Developmental neurotoxicity of industrial chemicals. Lancet 368:2167–2178
Grandjean P, Landrigan PJ (2014) Neurobehavioural effects of developmental toxicity. Lancet Neurol 13:330–338
Middaugh LD, Dow-Edwards D, Li AA et al (2003) Neurobehavioral assessment: a survey of use and value in safety assessment studies. Toxicol Sci 76:250–261
Makris SL, Raffaele K, Allen S et al (2009) A retrospective performance assessment of the developmental neurotoxicity study in support of OECD test guideline 426. Environ Health Perspect 117:17–25
OECD (2007) Test Guideline 426. OECD Guideline for testing of Chemicals. In: Developmental neurotoxicity study. Organisation of Economic Co-operation and development, Paris, France
Smirnova L, Hogberg HT, Leist M et al (2014) Developmental neurotoxicity - challenges in the 21st century and in vitro opportunities. Altex 31:129–156
NRC (2007) Toxicity testing in the 21st century: a vision and a strategy, 1st edn. National Academy Press, Washington, DC
Hill EJ, Woehrling EK, Prince M et al (2008) Differentiating human NT2/D1 neurospheres as a versatile in vitro 3D model system for developmental neurotoxicity testing. Toxicology 249:243–250
Couillard-Despres S, Quehl E, Altendorfer K et al (2008) Human in vitro reporter model of neuronal development and early differentiation processes. BMC Neurosci 9:31
Pallocca G, Fabbri M, Sacco MG et al (2013) miRNA expression profiling in a human stem cell-based model as a tool for developmental neurotoxicity testing. Cell Biol Toxicol 29:239–257
Laurenza I, Pallocca G, Mennecozzi M et al (2013) A human pluripotent carcinoma stem cell-based model for in vitro developmental neurotoxicity testing: effects of methylmercury, lead and aluminum evaluated by gene expression studies. Int J Dev Neurosci 31:679–691
Stern M, Gierse A, Tan S et al (2014) Human Ntera2 cells as a predictive in vitro test system for developmental neurotoxicity. Arch Toxicol 88:127–136
Kuenzel K, Friedrich O, Gilbert DF (2016) A recombinant human pluripotent stem cell line stably expressing halide-sensitive YFP-I152L for GABAAR and GlyR-targeted high-throughput drug screening and toxicity testing. Front Mol Neurosci 9:51
Lee VM, Andrews PW (1986) Differentiation of NTERA-2 clonal human embryonal carcinoma cells into neurons involves the induction of all three neurofilament proteins. J Neurosci 6:514–521
Pleasure SJ, Page C, Lee VM (1992) Pure, postmitotic, polarized human neurons derived from NTera 2 cells provide a system for expressing exogenous proteins in terminally differentiated neurons. J Neurosci 12:1802–1815
Sandhu JK, Sikorska M, Walker PR (2002) Characterization of astrocytes derived from human NTera-2/D1 embryonal carcinoma cells. J Neurosci Res 68:604–614
Stewart R, Christie VB, Przyborski SA (2003) Manipulation of human pluripotent embryonal carcinoma stem cells and the development of neural subtypes. Stem Cells 21:248–256
Ozdener H (2007) Inducible functional expression of Bcl-2 in human astrocytes derived from NTera-2 cells. J Neurosci Methods 159:8–18
Coyne L, Shan M, Przyborski SA et al (2011) Neuropharmacological properties of neurons derived from human stem cells. Neurochem Int 59:404–412
Menzner AK, Abolpour Mofrad S, Friedrich O et al (2015) Towards in vitro DT/DNT testing: assaying chemical susceptibility in early differentiating NT2 cells. Toxicology 338:69–76
Gilbert DF, Erdmann G, Zhang X et al (2011) A novel multiplex cell viability assay for high-throughput RNAi screening. PLoS One 6:e28338
Gilbert DF, Boutros M (2016) A protocol for a high-throughput multiplex cell viability assay. Methods Mol Biol 1470:75–84
Boutros M, Bras LP, Huber W (2006) Analysis of cell-based RNAi screens. Genome Biol 7:R66
Pelz O, Gilsdorf M, Boutros M (2010) web cellHTS2: a web-application for the analysis of high-throughput screening data. BMC Bioinf 11:185
Acknowledgements
The authors gratefully acknowledge funding of the Staedtler Stiftung. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Menzner, AK., Gilbert, D.F. (2017). A Protocol for In Vitro High-Throughput Chemical Susceptibility Screening in Differentiating NT2 Stem Cells. In: Gilbert, D., Friedrich, O. (eds) Cell Viability Assays. Methods in Molecular Biology, vol 1601. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6960-9_5
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DOI: https://doi.org/10.1007/978-1-4939-6960-9_5
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