Skip to main content

In Vitro Techniques for Assessing Neurotoxicity Using Human iPSC-Derived Neuronal Models

Part of the Neuromethods book series (NM,volume 145)

Abstract

The central nervous system consists of a multitude of different neurons and supporting cells that form networks for transmitting neuronal signals. Proper function of the nervous system depends critically on a wide range of highly regulated processes including intracellular calcium homeostasis, neurotransmitter release, and electrical activity. Due to the diversity of cell types and complexity of signaling processes, the (central) nervous system is very vulnerable to toxic insults.

Nowadays, a broad range of approaches and cell models is available to study neurotoxicity. In this chapter we show the applicability of human induced pluripotent stem cell (hiPSC)-derived neuronal co-cultures for in vitro neurotoxicity testing. We demonstrate that immunocytochemistry can be used to visualize networks of cultured cells and to differentiate between different cell types. Live cell imaging and electrophysiology techniques demonstrate that the neuronal networks develop spontaneous activity, including synchronized calcium oscillations that coincide with spontaneous changes in membrane potential as well as spontaneous electrical activity with defined (network) bursting. Importantly, as shown in this chapter, spontaneously active human iPSC-derived neuronal co-cultures are suitable for in vitro neurotoxicity assessment. Future application of live imaging and electrophysiological techniques on hiPSC from different donors and/or patients differentiated in different cell types holds great promise for personalized neurotoxicity assessment and safety screening.

Key words

  • In vitro neurotoxicity screening
  • Human induced pluripotent stem cell-derived neuronal models
  • Single-cell fluorescent microscopy
  • Calcium homeostasis
  • Membrane potential
  • Spontaneous neuronal activity
  • Immunocytochemistry
  • Multi-well microelectrode array

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-9228-7_2
  • Chapter length: 19 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   119.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-9228-7
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   159.99
Price excludes VAT (USA)
Hardcover Book
USD   219.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Clapham DE (2007) Calcium signaling. Cell 131:1047–1058. https://doi.org/10.1016/J.CELL.2007.11.028

    CAS  CrossRef  PubMed  Google Scholar 

  2. Westerink R (2006) Targeting exocytosis: ins and outs of the modulation of quantal dopamine release. CNS Neurol Disord Drug Targets 5:57–77. https://doi.org/10.2174/187152706784111597

    CAS  CrossRef  PubMed  Google Scholar 

  3. Barclay JW, Morgan A, Burgoyne RD (2005) Calcium-dependent regulation of exocytosis. Cell Calcium 38:343–353

    CAS  CrossRef  PubMed  Google Scholar 

  4. Südhof TC (2014) The molecular machinery of neurotransmitter release (nobel lecture). Angew Chem Int Ed 53:12696–12717. https://doi.org/10.1002/anie.201406359

    CAS  CrossRef  Google Scholar 

  5. Kuijlaars J, Oyelami T, Diels A et al (2016) Sustained synchronized neuronal network activity in a human astrocyte co-culture system. Sci Rep 6:36529. https://doi.org/10.1038/srep36529

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  6. Görtz P, Fleischer W, Rosenbaum C et al (2004) Neuronal network properties of human teratocarcinoma cell line-derived neurons. Brain Res 1018:18–25. https://doi.org/10.1016/j.brainres.2004.05.076

    CAS  CrossRef  PubMed  Google Scholar 

  7. Odawara A, Katoh H, Matsuda N, Suzuki I (2016) Physiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture. Sci Rep 6:26181. https://doi.org/10.1038/srep26181

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  8. Paavilainen T, Pelkonen A, Mäkinen ME-L et al (2018) Effect of prolonged differentiation on functional maturation of human pluripotent stem cell-derived neuronal cultures. Stem Cell Res 27:151–161. https://doi.org/10.1016/j.scr.2018.01.018

    CAS  CrossRef  PubMed  Google Scholar 

  9. Johnstone AFM, Gross GW, Weiss DG et al (2010) Microelectrode arrays: a physiologically based neurotoxicity testing platform for the 21st century. Neurotoxicology 31:331–350

    CAS  CrossRef  PubMed  Google Scholar 

  10. Robinette BL, Harrill JA, Mundy WR, Shafer TJ (2011) In vitro assessment of developmental neurotoxicity: use of microelectrode arrays to measure functional changes in neuronal network ontogeny1. Front Neuroeng 4:1. https://doi.org/10.3389/fneng.2011.00001

    CrossRef  PubMed  PubMed Central  Google Scholar 

  11. Cotterill E, Hall D, Wallace K et al (2016) Characterization of early cortical neural network development in multiwell microelectrode array plates. J Biomol Screen 21:510–519. https://doi.org/10.1177/1087057116640520

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  12. Hondebrink L, Verboven AHA, Drega WS et al (2016) Neurotoxicity screening of (illicit) drugs using novel methods for analysis of microelectrode array (MEA) recordings. Neurotoxicology 55:1–9. https://doi.org/10.1016/j.neuro.2016.04.020

    CAS  CrossRef  PubMed  Google Scholar 

  13. Novellino A, Scelfo B, Palosaari T et al (2011) Development of micro-electrode array based tests for neurotoxicity: assessment of interlaboratory reproducibility with neuroactive chemicals. Front Neuroeng 4:4. https://doi.org/10.3389/fneng.2011.00004

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  14. Vassallo A, Chiappalone M, De Camargos Lopes R et al (2017) A multi-laboratory evaluation of microelectrode array-based measurements of neural network activity for acute neurotoxicity testing. Neurotoxicology 60:280–292. https://doi.org/10.1016/j.neuro.2016.03.019

    CAS  CrossRef  PubMed  Google Scholar 

  15. McConnell ER, McClain MA, Ross J et al (2012) Evaluation of multi-well microelectrode arrays for neurotoxicity screening using a chemical training set. Neurotoxicology 33:1048–1057. https://doi.org/10.1016/j.neuro.2012.05.001

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  16. Valdivia P, Martin M, LeFew WR et al (2014) Multi-well microelectrode array recordings detect neuroactivity of ToxCast compounds. Neurotoxicology 44:204–217. https://doi.org/10.1016/j.neuro.2014.06.012

    CAS  CrossRef  PubMed  Google Scholar 

  17. Nicolas J, Hendriksen PJM, van Kleef RGDM et al (2014) Detection of marine neurotoxins in food safety testing using a multielectrode array. Mol Nutr Food Res 58:2369–2378. https://doi.org/10.1002/mnfr.201400479

    CAS  CrossRef  PubMed  Google Scholar 

  18. Hondebrink L, Kasteel EEJ, Tukker AM et al (2017) Neuropharmacological characterization of the new psychoactive substance methoxetamine. Neuropharmacology 123:1–9. https://doi.org/10.1016/j.neuropharm.2017.04.035

    CAS  CrossRef  PubMed  Google Scholar 

  19. Bradley JA, Luithardt HH, Metea MR, Strock CJ (2018) In vitro screening for seizure liability using microelectrode array technology. Toxicol Sci 163:240–253. https://doi.org/10.1093/toxsci/kfy029

    CAS  CrossRef  PubMed  Google Scholar 

  20. Dingemans MML, Schütte MG, Wiersma DMM et al (2016) Chronic 14-day exposure to insecticides or methylmercury modulates neuronal activity in primary rat cortical cultures. Neurotoxicology 57:194–202. https://doi.org/10.1016/j.neuro.2016.10.002

    CAS  CrossRef  PubMed  Google Scholar 

  21. Hogberg HT, Sobanski T, Novellino A et al (2011) Application of micro-electrode arrays (MEAs) as an emerging technology for developmental neurotoxicity: evaluation of domoic acid-induced effects in primary cultures of rat cortical neurons. Neurotoxicology 32:158–168. https://doi.org/10.1016/j.neuro.2010.10.007

    CAS  CrossRef  PubMed  Google Scholar 

  22. Alloisio S, Nobile M, Novellino A (2015) Multiparametric characterisation of neuronal network activity for in vitro agrochemical neurotoxicity assessment. Neurotoxicology 48:152–165. https://doi.org/10.1016/j.neuro.2015.03.013

    CAS  CrossRef  PubMed  Google Scholar 

  23. Zwartsen A, Hondebrink L, Westerink RH (2018) Neurotoxicity screening of new psychoactive substances (NPS): effects on neuronal activity in rat cortical cultures using microelectrode arrays (MEA). Neurotoxicology 66:87–97. https://doi.org/10.1016/j.neuro.2018.03.007

    CAS  CrossRef  PubMed  Google Scholar 

  24. Frank CL, Brown JP, Wallace K et al (2017) From the cover: developmental neurotoxicants disrupt activity in cortical networks on microelectrode arrays: results of screening 86 compounds during neural network formation. Toxicol Sci 160:121–135. https://doi.org/10.1093/toxsci/kfx169

    CAS  CrossRef  PubMed  Google Scholar 

  25. Tukker AM, Wijnolts FMJ, de Groot A, Westerink RHS (2018) Human iPSC-derived neuronal models for in vitro neurotoxicity assessment. Neurotoxicology 67:215–225. https://doi.org/10.1016/J.NEURO.2018.06.007

  26. Odawara A, Matsuda N, Ishibashi Y et al (2018) Toxicological evaluation of convulsant and anticonvulsant drugs in human induced pluripotent stem cell-derived cortical neuronal networks using an MEA system. Sci Rep 8:10416. https://doi.org/10.1038/s41598-018-28835-7

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  27. Tukker AM, De Groot MWGDM, Wijnolts FMJ et al (2016) Is the time right for in vitro neurotoxicity testing using human iPSC-derived neurons? ALTEX 33:261–271. https://doi.org/10.14573/altex.1510091

    CrossRef  PubMed  Google Scholar 

  28. Heusinkveld HJ, Thomas GO, Lamot I et al (2010) Dual actions of lindane (γ-hexachlorocyclohexane) on calcium homeostasis and exocytosis in rat PC12 cells. Toxicol Appl Pharmacol 248:12–19. https://doi.org/10.1016/j.taap.2010.06.013

    CAS  CrossRef  PubMed  Google Scholar 

  29. Legéndy CR, Salcman M (1985) Bursts and recurrences of bursts in the spike trains of spontaneously active striate cortex neurons. J Neurophysiol 53:926–939. https://doi.org/10.1152/jn.1985.53.4.926

    CrossRef  PubMed  Google Scholar 

  30. Hyysalo A, Ristola M, Mäkinen MEL et al (2017) Laminin α5 substrates promote survival, network formation and functional development of human pluripotent stem cell-derived neurons in vitro. Stem Cell Res 24:118–127. https://doi.org/10.1016/j.scr.2017.09.002

    CAS  CrossRef  PubMed  Google Scholar 

  31. Kasteel EEJ, Westerink RHS (2017) Comparison of the acute inhibitory effects of Tetrodotoxin (TTX) in rat and human neuronal networks for risk assessment purposes. Toxicol Lett 270:12–16. https://doi.org/10.1016/j.toxlet.2017.02.014

    CAS  CrossRef  PubMed  Google Scholar 

  32. Ishii MN, Yamamoto K, Shoji M et al (2017) Human induced pluripotent stem cell (hiPSC)-derived neurons respond to convulsant drugs when co-cultured with hiPSC-derived astrocytes. Toxicology 389:130–138. https://doi.org/10.1016/j.tox.2017.06.010

    CAS  CrossRef  PubMed  Google Scholar 

Download references

Acknowledgments

We gratefully acknowledge members of the Neurotoxicology Research Group for helpful discussions. This work was funded by a grant from the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs; project number 50308-372160), the Netherlands Organisation for Health Research and Development (ZonMW; InnoSysTox project number 114027001), and the Faculty of Veterinary Medicine (Utrecht University, The Netherlands).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Remco H. S. Westerink .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Tukker, A.M., Wijnolts, F.M.J., de Groot, A., Wubbolts, R.W., Westerink, R.H.S. (2019). In Vitro Techniques for Assessing Neurotoxicity Using Human iPSC-Derived Neuronal Models. In: Aschner, M., Costa, L. (eds) Cell Culture Techniques. Neuromethods, vol 145. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9228-7_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9228-7_2

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9227-0

  • Online ISBN: 978-1-4939-9228-7

  • eBook Packages: Springer Protocols