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Development of a neural rosette formation assay (RoFA) to identify neurodevelopmental toxicants and to characterize their transcriptome disturbances

  • In vitro systems
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Abstract

The first in vitro tests for developmental toxicity made use of rodent cells. Newer teratology tests, e.g. developed during the ESNATS project, use human cells and measure mechanistic endpoints (such as transcriptome changes). However, the toxicological implications of mechanistic parameters are hard to judge, without functional/morphological endpoints. To address this issue, we developed a new version of the human stem cell-based test STOP-tox(UKN). For this purpose, the capacity of the cells to self-organize to neural rosettes was assessed as functional endpoint: pluripotent stem cells were allowed to differentiate into neuroepithelial cells for 6 days in the presence or absence of toxicants. Then, both transcriptome changes were measured (standard STOP-tox(UKN)) and cells were allowed to form rosettes. After optimization of staining methods, an imaging algorithm for rosette quantification was implemented and used for an automated rosette formation assay (RoFA). Neural tube toxicants (like valproic acid), which are known to disturb human development at stages when rosette-forming cells are present, were used as positive controls. Established toxicants led to distinctly different tissue organization and differentiation stages. RoFA outcome and transcript changes largely correlated concerning (1) the concentration-dependence, (2) the time dependence, and (3) the set of positive hits identified amongst 24 potential toxicants. Using such comparative data, a prediction model for the RoFA was developed. The comparative analysis was also used to identify gene dysregulations that are particularly predictive for disturbed rosette formation. This ‘RoFA predictor gene set’ may be used for a simplified and less costly setup of the STOP-tox(UKN) assay.

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Abbreviations

AOP:

Adverse outcome pathway

BMC25:

Benchmark concentration leading to 25% decrease compared to control

BPA:

Bisphenol A

BR:

Borderline range

CNS:

Central nervous system

CsA:

Cyclosporin A

DMSO:

Dimethylsulfoxide

DNT:

Developmental neurotoxicity

FGF:

Fibroblast growth factor

HDACi:

Histone deacetylase inhibitor

IFNbeta:

Interferon beta

KE:

Key events

KNDP:

Key neurodevelopmental processes

mEST:

Mouse embryonic stem cell test

NAM:

New approach methods

NCAM:

Neural cell adhesion molecule

NDD:

Neurodevelopmental distance

NEP:

Neuroepithelial precursor

OECD:

Organization for economic co-operation and development

PCMB:

P-chloromercuribenzoic acid

PMA:

Phorbol 12-myristate 13-acetate

PSC:

Pluripotent stem cells

RA:

Retinoic acid

REACH:

Registration, evaluation, authorisation and restriction of chemicals (EC 1907/2006)

RoFA:

Rosette formation assay

SD:

Standard deviation

STOP-tox(UKN) :

Stem cell-based teratogenic omics prediction-UKN toxicity assay (Shinde et al. 2016a), previously named:

UKN1:

University of Konstanz (1) assay (Krug et al. 2013b)

T:

Threshold

TSA:

Trichostatin A

U(T):

Uncertainty of threshold

VPA:

Valproic acid

Wnta:

Wnt activators

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Acknowledgements

This work was supported by the Land BW, the Doerenkamp-Zbinden foundation, the DFG (RTG1331, KoRS-CB) and the Projects from the European Union's Horizon 2020 research and innovation programme EU-ToxRisk (Grant agreement No 681002) and ENDpoiNTs (Grant agreement No 825759). We are grateful to H. Leisner and D. Fischer and the staff of the bioimaging center (BIC) for invaluable experimental support.

Author information

Author notes

  1. Simon Gutbier

    Present address: Roche Pharma Development, Grenzacherstrasse, 4070, Basel, Switzerland

  2. David Kolb & Christian Dietz

    Present address: KNIME GmbH, 78467, Konstanz, Germany

  3. Timo Trefzer

    Present address: Digital Health Center, Berlin Institute of Health (BIH), Charité-Universitätsmedizin, 10117, Berlin, Germany

  4. Tanja Waldmann and Marcel Leist shared senior authorship.

Authors and Affiliations

  1. In Vitro Toxicology and Biomedicine, Department Inaugurated By the Doerenkamp-Zbinden Chair Foundation, University of Konstanz, Box 657, 78457, Konstanz, Germany

    Nadine Dreser, Anna-Katharina Holzer, Marion Kapitza, Christopher Scholz, Petra Kranaster, Simon Gutbier, Stefanie Klima, Timo Trefzer, Tanja Waldmann & Marcel Leist

  2. Department of Statistics, TU Dortmund, 44221, Dortmund, Germany

    Katrin Madjar & Jörg Rahnenführer

  3. Department of Computer and Information Science, University of Konstanz, 78457, Konstanz, Germany

    David Kolb, Christian Dietz & Michael R. Berthold

  4. Institute of Pathology, Charité-Universitätsmedizin, 10117, Berlin, Germany

    Johannes Meisig & Nils Blüthgen

  5. Leibniz Research Centre for Working Environment and Human Factors (IfADo), Technical University of Dortmund, 44139, Dortmund, Germany

    Christoph van Thriel & Jan G. Hengstler

  6. Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of Cologne (UKK), 50931, Cologne, Germany

    Margit Henry & Agapios Sachinidis

  7. Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, 78457, Konstanz, Germany

    Petra Kranaster

Authors
  1. Nadine Dreser
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  2. Katrin Madjar
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  3. Anna-Katharina Holzer
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  4. Marion Kapitza
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  5. Christopher Scholz
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  6. Petra Kranaster
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  7. Simon Gutbier
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  8. Stefanie Klima
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  9. David Kolb
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  10. Christian Dietz
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  11. Timo Trefzer
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  12. Johannes Meisig
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  13. Christoph van Thriel
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  14. Margit Henry
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  15. Michael R. Berthold
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  16. Nils Blüthgen
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  17. Agapios Sachinidis
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  18. Jörg Rahnenführer
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  19. Jan G. Hengstler
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  20. Tanja Waldmann
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  21. Marcel Leist
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Corresponding author

Correspondence to Marcel Leist.

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Dreser, N., Madjar, K., Holzer, AK. et al. Development of a neural rosette formation assay (RoFA) to identify neurodevelopmental toxicants and to characterize their transcriptome disturbances. Arch Toxicol 94, 151–171 (2020). https://doi.org/10.1007/s00204-019-02612-5

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