SCIENCE

Project funding CENTER FOR ALTERNATIVES

TO ANIMAL TESTING (CERST-NRW) of the State NRW

 

As part of CERST-NRW, two cell-based assays are being developed which aim to determine the influence of substances on the development of unborn and infants and on the central nervous system. So-called induced pluripotent stem cells (hiPSCs) are used, which are produced from human cells provided by voluntary donors. These hiPSCs are further differentiated into cardiomyocytes and brain cells in order to identify substances that interfere with the development or function of these cells. The long-term goal of the project is to contribute to a test battery that allows chemicals to be tested for their embryo and developmental neurotoxic and acute neurotoxic potential without the use of animal experiments. Here, the development and application of biostatistical evaluations significantly contribute to the integration of our projects into large international activities, such as the ToxCast and DNT-DIVER programs run by the United States Environmental Protection Agency and the US National Toxicology Program, respectively.

Funding Project of the Ministry for Culture and Science of the State of North-Rhine Westphalia, Germany

In vitro brain cells

 

This subproject of CERST-NRW deals with the formation of neural networks (NNs) from human induced neural progenitor cells (hiNPCs), which are generated from hiPSCs. The aim is to use these NNs as an alternative method for testing for acute neurotoxicity. For this purpose, we develop reproducible protocols that include both 2D and 3D steps. We characterize the resulting NNs in terms of their different cell types, their molecular makeup, and their electrical activity on microelectrode arrays (MEAs) and thus determine their applicability domain.

Funding Project of the Ministry for Culture and Science of the State of North-Rhine Westphalia, Germany

In 3D differentiated neurosphere. Green: cytoskeleton, red: neurons, blue: nuclei

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In vitro cardiomyocytes

 

The human induced pluripotent stem cell test (hiPS-test) is based on the ability of hiPSCs to differentiate into beating cardiomyocytes, thereby representing approximately the third week of pregnancy. We are developing standardized protocols to use this test in the future for the identification of embryotoxic substances. The assay currently includes the following endpoints: gene and protein expression of cardiac maturation markers, statement “does not beat / beats”, and analysis of the beating frequency of the cardiomyocytes – in each case according to substance exposure and with established positive and negative controls. In the near future, we plan to add metabolomics as another measure.

In 3D differentiated neurosphere. Green: cytoskeleton, red: neurons, blue: nuclei

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Automated image analysis and biostatistics

 

Automated image analysis is carried out in close cooperation with the bioinformatics group (Prof. Axel Mosig) based at Ruhr University Bochum. Neuronal networks are trained with fluorescence microscopic images of neurons and glial cells in order to be able to perform artificial intelligence-based automatic evaluations of the neurotoxic analyses. This enables us to expand our in-house analysis software Omnisphero (Schmuck et al., 2017). We carry out the biostatistical evaluations of such high-content imaging analysis (HCA) data sets with the help of internationally recognized biostatistical methods. CERST collaborates with the United States' National Toxicology Program at the National Institute of Environmental Health Sciences and with Brunel University, London.

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In 3D differentiated neurosphere. Green: cytoskeleton, red: neurons, blue: nuclei

Automated image analysis and biostatistics

 

Automated image analysis is carried out in close cooperation with the bioinformatics group (Prof. Axel Mosig) based at Ruhr University Bochum. Neuronal networks are trained with fluorescence microscopic images of neurons and glial cells in order to be able to perform artificial intelligence-based automatic evaluations of the neurotoxic analyses. This enables us to expand our in-house analysis software Omnisphero (Schmuck et al., 2017). We carry out the biostatistical evaluations of such high-content imaging analysis (HCA) data sets with the help of internationally recognized biostatistical methods. CERST collaborates with the United States' National Toxicology Program at the National Institute of Environmental Health Sciences and with Brunel University, London.

GRK 2578

 

Work in progress

Novel testing strategies for endocrine disruptors in the context of developmental neurotoxicity (ENDPOINTS)

 

As a member of the ENDpoiNTs H2020 research consortium, we develop innovative, human NPC-based models to identify chemicals that disrupt brain development by interfering with human endocrine processes. The ENDpoiNTs project is divided into two parts: The first deals with the development of a testing battery for chemical screening and with the acquisition of specialist knowledge about the molecular connections between the developmental neurotoxicological observations and the underlying disorders of the endocrine system. In the second part, we combine our studies with the and data of the other ENDpoiNTs partners and evaluate our results in an epidemiological context in order to guarantee the human relevance of our test methods. The test methods developed by ENDpoiNTs should ultimately be incorporated into the European and international regulatory framework.

www.endpoints.eu

Funding European Commission (Horizon2020)

hiPSC-derived dopaminergic neurons in green, nuclei in blue

Cells migrate radially out of a sphere (lower left corner)

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An in vitro testing battery for the assessment of developmental neurotoxicity

 

Developmental neurotoxicity (DNT) is of great social relevance since most of the chemicals currently in circulation have not been tested for their DNT potential. This is mainly due to the animal-based standard method, which is very resource-intensive and therefore not suitable for testing large amounts of substances. We are part of an international consortium (University of Konstanz, United States Environmental Protection Agency, European Food Safety Authority (EFSA)) that has assembled, established, and scientifically validated an test battery for predicting the influence of substances on the developing nervous system of children more quickly and cost effectively without the need for animal testing. This data forms the basis of an OECD guidance on testing for DNT, which is currently in preparation.

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A differentiated neurosphere after 5 days in culture. Red: neurons, green: oligodendrocytes, blue: nuclei

EFSA case study on flame retardants

 

Flame retardants prevent or delay the spread of fire and can therefore be found in many everyday items and furnishings. In recent decades, the substance class of polyhalogenated flame retardants – such as polychlorinated biphenyls and polybrominated diphenyl ethers – has been classified as harmful to human health and therefore banned from the market. These were subsequently replaced by less persistent, alternative flame retardants such as organophosphates, but without knowing their full toxicological profile. In this case study, we use the EFSA DNT test battery assays developed at the IUF (see “An testing battery for the assessment of developmental neurotoxicity”) to assess the toxicological potential of flame retardants for basic brain development processes. In collaboration with the Biological Medical Research Center of Heinrich Heine University Düsseldorf (Prof. Köhrer) and the core facility of the IUF (Dr. Rossi), these are also analyzed on a molecular level.

Funding European Food Safety Authority (EFSA)

Representative immunocytochemical image of differentiated hNPCs on day 5 of differentiation.
Blue: nuclei, pink: oligodendrocytes

Representative immunocytochemical image of differentiated hNPCs on day 5 of differentiation.

Blue: nuclei , pink: oligodendrocytes

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The video shows the electrical activity of NeuCyte cells on microelectrode arrays (MEAs)

Representative spike raster plot

From (screen) hit to DNT toxicant

 

In recent years, great progress has been made in the development of animal-free test methods that map certain key processes in brain development (see “An in vitro testing battery for the assessment of developmental neurotoxicity”). Based on the test methods of such an in vitro test battery, the developmental neurotoxic (DNT) hazard potential of chemicals is assessed. To improve the prediction of these alternative test methods, we are establishing an hiPSC-based “neural network formation assay” and testing the ability of pesticides to interfere with neural network formation. Together with the University of Konstanz (Prof. Marcel Leist) we are carrying out an interlaboratory comparison of the test methods of the DNT in vitro battery in order to increase the regulatory acceptance and applicability of the DNT test methods.

Funding Danish Environmental Protection Agency (DK-EPA) – Pesticide Research Program

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Respresentative confocal images of differentiated LUHMES cells on day 6 of differentiation.

Blue: nuclei, red: cytoskeleton, green: neurons.

The establishment of an AOP-based in vitro test battery for identifying compounds including Parkinsonian motor deficits

 

Parkinson’s disease (PD) is the second most common neurodegenerative disease in the elderly and is significantly more common in Egypt than in the rest of the world. Egypt is an agricultural country with a high level of occupational exposure to pesticides. Numerous epidemiological studies support the hypothesis that exposure to pesticides could increase the risk of PD. For this reason, the European Food Safety Authority (EFSA) has re-evaluated the hypothesis that pesticides could be the cause of Parkinsonian motor deficits (PMDs). To this end, adverse outcome pathways (AOPs) were generated. In this project, a human testing battery based on these AOPs is being established to determine the hazard potential of substances that might cause PMDs.

Funding Deutscher Akademischer Austauschdienst (DAAD), Bayer AG

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Development of organ-specific in vitro models under consideration of physiological and pathophysiological characteristics

 

Within the EU Horizon 2020-funded PATROLS project (Physiologically Anchored Tools for Realistic nanOmateriaL hazard aSsessment) representatives of academia, industry and governmental agencies collaborate to develop innovative laboratory- and computer-based methods for the safety assessment of nanomaterials. These developments shall contribute to minimise – and ideally eliminate – the necessity to perform animal studies for the evaluation of nanomaterials. In the context of in vitro model development, our focus is on the intestine as entry organ for nanomaterials following oral uptake.

Our aim is to mimic the physiological complexity of the organ as well as the impact of the digestive processes on the physico-chemical properties of the nanomaterials as closely as possible to the in vivo situation. Special consideration is given to the emulation of organ-specific pathological processes, e.g. as present during chronic inflammatory conditions of the intestine. The quality and transferability of the in vitro results will be analysed by targeted comparison with findings from animal studies.   

 

Funding European Commission (Horizon 2020)

Epithelial cell layer of the intestinal co-culture (Caco-2/HT29-MTX-E12) after 21 days of differentiation.

Blue: nuclei; red: tight junction-associated protein (ZO-1); green: cytoskeleton

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