Development of 3D human stem cells-microglia co-cultures for neurotoxicology studies

Abstracts / Toxicology Letters 211S (2012) S43–S216

Electrophysiology is the study of the electrical properties of biological cells and tissues. It involves measurements of voltage change or electric current on a wide variety of scales from single ion channel proteins to whole organs. In recent years, the mitochondrion has been shown as the link between different signaling pathways involved in neurodegenerative diseases. The formation of the mitochondrial permeability transition pore (PTP) involves phenomena such as the dissipation of the mitochondrial electrochemical potential and the release of substances like apoptosis inducing factor (AIF) and cytochrome c from the membrane cleft. The PTP might be a very attractive target for the development of drugs preventing apoptosis. Although the small size of the organelle has prevented the use of classical electrophysiological methods, introduction of the patch-clamp technique, which allows currents to be recorded from very small cells, has enabled us to test this hypothesis. We investigated the role of dopamine-D2-receptor agonists in blocking the PTP and thus elucidating neuroprotection by these substances. Single channel currents were recorded from mitoplasts by the patch-clamp technique. Analysis of patch clamp experiments showed that the dopamine-agonists pramipexole and ropinirole block the PTP dose-dependently. The frequently described neuroprotective effect of dopamine-D2-receptor agonists like ropinirole and pramipexole in patients suffering from neurodegenerative diseases could be due to a delayed cell death by PTP mediated apoptosis. doi:10.1016/j.toxlet.2012.03.562

P23-27 Imaging and omics in developing and juvenile rats after exposure to TBTO Didima De Groot 1 , Didima De Groot 2 , Frieke Kuper 2 , Marijana Radonjic 2 , Rob Stierum 2 , Arend Heerschap 3 , Andor Veltien 3 , Rudi Dierckx 4 , Erik De Vries 4 TNO EELS, Netherlands, 2 TNO EELS, Zeist, Netherlands, 3 Radboud Univ. Med. Center Nijmegen, Netherlands, 4 University Med. Center Groningen, Netherlands

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Exposure of the brain during development may have longlasting effects on health later in life. The severity of effects depends on dose, time-point and duration of exposure. Considering proper study designs for safety evaluation studies, it is important to learn about the vulnerability of an individual during critical phases of life. To this end we compared in rats the effects of developmental (GD8PN10) versus juvenile exposure (PN22–PN70) to tributyltin oxide (TBTO; 8 mg/kg BW), known to affect both the developing nervousand immune systems. Structural (MRI) and functional ([18F]FDG microPET) brain imaging was carried out at different postnatal days (PN) (MRI: PN21, 61; PET: PN18, 21, 35, 61) and the results were compared to conventional endpoints (neuropathology: (brainweight/-size) and behaviour (functional operational battery (FOB), motor activity, startle, learning/memory tasks)). Imaging was also combined to micro array gene expression/bio-informatics analysis (PD 21, 61). Effects of TBTO appeared largest in animal exposed during development rather than adolescence (juvenile exposure). The results demonstrated that changes in MRI imaging (reduced volume of regions in the anterior part of the brain) concurred with conventional neuropathology; PET changes (disturbed [18F]FDG uptake in brain) were found in animals with impaired behaviour. Gene expression profiling confirmed that developmental exposure to TBTO affected the developing nervous system, but the immune system as well. It was concluded that innovative technologies and

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strategies may further improve safety evaluation protocols and prediction of animal data to man, to the benefit of animal wellbeing, time and costs. doi:10.1016/j.toxlet.2012.03.563

P23-28 Development of 3D human stem cells-microglia co-cultures for neurotoxicology studies Jenny Sandström von Tobel 1 , Igor Charvet 2 , Sabrina Pagano 3 , Nicolas Vuilleumier 3 , Luc Stoppini 2 , Florianne Monnet-Tschudi 1 1 3

University of Lausanne, Switzerland, 2 HEPIA Geneva, Switzerland, University Hospital of Geneva, Switzerland

Both neurodegenerative pathology and chemically induced neurotoxicity coincides with oxidative stress and inflammatory responses. These processes depend on intricate cell networks and therefore it is of great importance to study neurotoxic effects in a system comprising of all cell types of the brain. Astrocyte- and microglial activation – hallmarks of brain inflammation that depend on cell-cell signaling – have successfully been studied in an in vitro system using primary rat embryonic brain cells. Not only do these cells differentiate, but when cultured under rotation they also form three-dimensional spheres that develop into a functional tissue-like organization. This well characterized system has been proven a valuable tool in neurotoxical analysis. Our goal is to transpose this model into a human system by using human embryonic stem cells (hESC) as a base for aggregate formation. Our experimental setup generated aggregate formation from hESC, and induced neuronal- and glial lineage, as shown by immunoreactivity for tubulin ␤ III, synaptophysin and glial fibrillary acidic protein. Furthermore, nestin detection revealed the presence of undifferentiated neural stem cells. Microglia, however, originate from hematopoietic lineage. Therefore, aggregates composed of hESCs, set for neural lineage, must be supplied with microglia. Human primary monocytes–with intrinsic capacity to form microglia – were introduced to the aggregate cultures and indeed we could observe integration in the neurospheres. We are currently characterizing this new model and although preliminary, our results indicate a good possibility of establishing a powerful tool in the testing of human neurotoxicology and neuroinflammatory responses to environmental chemicals. doi:10.1016/j.toxlet.2012.03.564

P23-29 A high throughput screening system for antifungal activity and environmental toxicity Young-Keun Kwak 1 , Chang-Hwa Jeong 2 , Seong-Mi Kim 2 , Inger Kuhn 3 , Patricia Colque-Navarro 4 , Roland Möllby 4 Karolinska Institutet/PhPlate AB, Sweden, 2 JNS Tech Co., Ltd., Busan, Republic of Korea, 3 PhPlate AB, Stockholm, Sweden, 4 Karolinska Institutet, Stockholm, Sweden

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Fungicides are very important for the maintenance of healthy fish and their eggs in intensive aquaculture operations. The demand for new fungicides for use in aquaculture has been significantly increased during the last three decades. Consequently, good screen-