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Arch Toxicol. 2020 Jan;94(1):151-171. doi: 10.1007/s00204-019-02612-5. Epub 2019 Nov 11.

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

Author information

1
In Vitro Toxicology and Biomedicine, Department Inaugurated By the Doerenkamp-Zbinden Chair Foundation, University of Konstanz, Box 657, 78457, Konstanz, Germany.
2
Department of Statistics, TU Dortmund, 44221, Dortmund, Germany.
3
Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, 78457, Konstanz, Germany.
4
Roche Pharma Development, Grenzacherstrasse, 4070, Basel, Switzerland.
5
Department of Computer and Information Science, University of Konstanz, 78457, Konstanz, Germany.
6
KNIME GmbH, 78467, Konstanz, Germany.
7
Digital Health Center, Berlin Institute of Health (BIH), Charité-Universitätsmedizin, 10117, Berlin, Germany.
8
Institute of Pathology, Charité-Universitätsmedizin, 10117, Berlin, Germany.
9
Leibniz Research Centre for Working Environment and Human Factors (IfADo), Technical University of Dortmund, 44139, Dortmund, Germany.
10
Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of Cologne (UKK), 50931, Cologne, Germany.
11
In Vitro Toxicology and Biomedicine, Department Inaugurated By the Doerenkamp-Zbinden Chair Foundation, University of Konstanz, Box 657, 78457, Konstanz, Germany. Marcel.leist@uni-konstanz.de.

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:

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

PMID:
31712839
DOI:
10.1007/s00204-019-02612-5

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