A Protocol for In Vitro High-Throughput Chemical Susceptibility Screening in Differentiating NT2 Stem Cells

Part of the Methods in Molecular Biology book series (MIMB, volume 1601)


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.

Key words

High-throughput screening Cell-based assays Cell viability Developmental toxicity and neurotoxicity DNT Chemical susceptibility Differentiation Neurotoxicity Cell fitness Drug screening Drug discovery Target validation In vitro toxicity screening CellTiter-Glo Luminescent Cell Viability Assay 



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.


  1. 1.
    May M (2000) Disturbing behavior: neurotoxic effects in children. Environ Health Perspect 108:A262–A267CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Colborn T (2004) Neurodevelopment and endocrine disruption. Environ Health Perspect 112:944–949CrossRefPubMedGoogle Scholar
  3. 3.
    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–e1859CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Herbert MR (2010) Contributions of the environment and environmentally vulnerable physiology to autism spectrum disorders. Curr Opin Neurol 23:103–110CrossRefPubMedGoogle Scholar
  5. 5.
    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):511–533CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Grandjean P, Landrigan PJ (2006) Developmental neurotoxicity of industrial chemicals. Lancet 368:2167–2178CrossRefPubMedGoogle Scholar
  7. 7.
    Grandjean P, Landrigan PJ (2014) Neurobehavioural effects of developmental toxicity. Lancet Neurol 13:330–338CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Middaugh LD, Dow-Edwards D, Li AA et al (2003) Neurobehavioral assessment: a survey of use and value in safety assessment studies. Toxicol Sci 76:250–261CrossRefPubMedGoogle Scholar
  9. 9.
    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 117:17–25CrossRefPubMedGoogle Scholar
  10. 10.
    OECD (2007) Test Guideline 426. OECD Guideline for testing of Chemicals. In: Developmental neurotoxicity study. Organisation of Economic Co-operation and development, Paris, FranceGoogle Scholar
  11. 11.
    Smirnova L, Hogberg HT, Leist M et al (2014) Developmental neurotoxicity - challenges in the 21st century and in vitro opportunities. Altex 31:129–156PubMedPubMedCentralGoogle Scholar
  12. 12.
    NRC (2007) Toxicity testing in the 21st century: a vision and a strategy, 1st edn. National Academy Press, Washington, DCGoogle Scholar
  13. 13.
    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 249:243–250CrossRefPubMedGoogle Scholar
  14. 14.
    Couillard-Despres S, Quehl E, Altendorfer K et al (2008) Human in vitro reporter model of neuronal development and early differentiation processes. BMC Neurosci 9:31CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    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 29:239–257CrossRefPubMedGoogle Scholar
  16. 16.
    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 31:679–691CrossRefPubMedGoogle Scholar
  17. 17.
    Stern M, Gierse A, Tan S et al (2014) Human Ntera2 cells as a predictive in vitro test system for developmental neurotoxicity. Arch Toxicol 88:127–136CrossRefPubMedGoogle Scholar
  18. 18.
    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 9:51CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    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 6:514–521PubMedGoogle Scholar
  20. 20.
    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 12:1802–1815PubMedGoogle Scholar
  21. 21.
    Sandhu JK, Sikorska M, Walker PR (2002) Characterization of astrocytes derived from human NTera-2/D1 embryonal carcinoma cells. J Neurosci Res 68:604–614CrossRefPubMedGoogle Scholar
  22. 22.
    Stewart R, Christie VB, Przyborski SA (2003) Manipulation of human pluripotent embryonal carcinoma stem cells and the development of neural subtypes. Stem Cells 21:248–256CrossRefPubMedGoogle Scholar
  23. 23.
    Ozdener H (2007) Inducible functional expression of Bcl-2 in human astrocytes derived from NTera-2 cells. J Neurosci Methods 159:8–18CrossRefPubMedGoogle Scholar
  24. 24.
    Coyne L, Shan M, Przyborski SA et al (2011) Neuropharmacological properties of neurons derived from human stem cells. Neurochem Int 59:404–412CrossRefPubMedGoogle Scholar
  25. 25.
    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 338:69–76CrossRefPubMedGoogle Scholar
  26. 26.
    Gilbert DF, Erdmann G, Zhang X et al (2011) A novel multiplex cell viability assay for high-throughput RNAi screening. PLoS One 6:e28338CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Gilbert DF, Boutros M (2016) A protocol for a high-throughput multiplex cell viability assay. Methods Mol Biol 1470:75–84CrossRefPubMedGoogle Scholar
  28. 28.
    Boutros M, Bras LP, Huber W (2006) Analysis of cell-based RNAi screens. Genome Biol 7:R66CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Pelz O, Gilsdorf M, Boutros M (2010) web cellHTS2: a web-application for the analysis of high-throughput screening data. BMC Bioinf 11:185CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Internal Medicine 5University Medical Center Erlangen, Friedrich-Alexander-Universität Erlangen-NürnbergErlangenGermany
  2. 2.Friedrich-Alexander University (FAU) Erlangen-NürnbergInstitute of Medical BiotechnologyErlangenGermany

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