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

  • Nadine Dreser
  • Katrin Madjar
  • Anna-Katharina Holzer
  • Marion Kapitza
  • Christopher Scholz
  • Petra Kranaster
  • Simon Gutbier
  • Stefanie Klima
  • David Kolb
  • Christian Dietz
  • Timo Trefzer
  • Johannes Meisig
  • Christoph van Thriel
  • Margit Henry
  • Michael R. Berthold
  • Nils Blüthgen
  • Agapios Sachinidis
  • Jörg Rahnenführer
  • Jan G. Hengstler
  • Tanja Waldmann
  • Marcel LeistEmail author
In vitro systems

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.

Keywords

Neural rosettes Cytotoxicity Developmental toxicity Human stem cells Differentiation Neural precursor cells Phenotypic anchoring Gene expression 

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

Notes

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.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

204_2019_2612_MOESM1_ESM.pdf (1.3 mb)
Supplementary file1 (PDF 1291 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Nadine Dreser
    • 1
  • Katrin Madjar
    • 2
  • Anna-Katharina Holzer
    • 1
  • Marion Kapitza
    • 1
  • Christopher Scholz
    • 1
  • Petra Kranaster
    • 1
    • 7
  • Simon Gutbier
    • 1
    • 8
  • Stefanie Klima
    • 1
  • David Kolb
    • 3
    • 9
  • Christian Dietz
    • 3
    • 9
  • Timo Trefzer
    • 1
    • 10
  • Johannes Meisig
    • 4
  • Christoph van Thriel
    • 5
  • Margit Henry
    • 6
  • Michael R. Berthold
    • 3
  • Nils Blüthgen
    • 4
  • Agapios Sachinidis
    • 6
  • Jörg Rahnenführer
    • 2
  • Jan G. Hengstler
    • 5
  • Tanja Waldmann
    • 1
  • Marcel Leist
    • 1
    Email author
  1. 1.In Vitro Toxicology and Biomedicine, Department Inaugurated By the Doerenkamp-Zbinden Chair FoundationUniversity of KonstanzKonstanzGermany
  2. 2.Department of StatisticsTU DortmundDortmundGermany
  3. 3.Department of Computer and Information ScienceUniversity of KonstanzKonstanzGermany
  4. 4.Institute of PathologyCharité-UniversitätsmedizinBerlinGermany
  5. 5.Leibniz Research Centre for Working Environment and Human Factors (IfADo)Technical University of DortmundDortmundGermany
  6. 6.Center of Physiology and Pathophysiology, Institute of NeurophysiologyUniversity of Cologne (UKK)CologneGermany
  7. 7.Konstanz Research School Chemical Biology (KoRS-CB)University of KonstanzKonstanzGermany
  8. 8.Roche Pharma DevelopmentBaselSwitzerland
  9. 9.KNIME GmbHKonstanzGermany
  10. 10.Digital Health Center, Berlin Institute of Health (BIH)Charité-UniversitätsmedizinBerlinGermany

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