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In vitro Assessment of Developmental Toxicity and Cardiac Pharmacology using Embryonic Stem Cells

In vitro Assessment of Developmental Toxicity and Cardiac Pharmacology using Embryonic Stem Cells


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STUMMANN, Tina C., 2008. In vitro Assessment of Developmental Toxicity and Cardiac Pharmacology using Embryonic Stem Cells

@phdthesis{Stummann2008vitro-7831, title={In vitro Assessment of Developmental Toxicity and Cardiac Pharmacology using Embryonic Stem Cells}, year={2008}, author={Stummann, Tina C.}, address={Konstanz}, school={Universität Konstanz} }

<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/rdf/resource/123456789/7831"> <dcterms:rights rdf:resource="http://nbn-resolving.org/urn:nbn:de:bsz:352-20140905103416863-3868037-7"/> <dcterms:issued>2008</dcterms:issued> <dc:rights>deposit-license</dc:rights> <dc:language>eng</dc:language> <dcterms:alternative>In-vitro Erfassung von Entwicklungstoxizität und Kardiopharmakologie mit embryonalen Stammzellen</dcterms:alternative> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:37:52Z</dc:date> <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:37:52Z</dcterms:available> <dc:creator>Stummann, Tina C.</dc:creator> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/7831"/> <dcterms:abstract xml:lang="eng">The Embryonic Stem cell Test (EST) was scientifically validated in 2001 . Methylmercury (MeHg) was the most significant outlier in the study. The EST misclassification of MeHg, and the potential environmental exposure and developmental toxic hazards of heavy metals gave us the rationale to investigate if the EST could predict the embryotoxicity of two heavy metals different from MeHg, namely trivalent and hexavalent chromium. The test correctly classified trivalent chromium to be non-embryotoxic and hexavalent chromium to be embryotoxic, while the misclassification of MeHg was confirmed. We explored the potential of a mouse embryonic stem cell (mESC) neuronal differentiation assay to improve the in vitro prediction, since MeHg induces developmental abnormalities in the brain. Appearance of neuron-like cells was shown by real-time PCR experiments, demonstrating up-regulation of a number of neuronal marker genes, and immunohistochemistry, revealing the presence of cells positive for nestin, neurofilament medium polypeptide, beta;-tubulin III and Mtap2. Mtap2 mRNA expression was down-regulated in the presence of non-cytotoxic concentrations of MeHg, identifying Mtap2 mRNA expression as a sensitive toxicological endpoint for MeHg induced neuronal embryotoxicity.The mouse assay was adapted to hESCs. Occurrence of neuronal derivatives was demonstrated by real-time PCR, showing up-regulation of several neuronal marker genes, and immunohistochemistry, revealing the appearance of neurofilament medium polypeptide, Beta-tubulin III and MAP2 positive cells. The differentiating cells were exposed to MeHg in order to address whether the hESC model could detect toxicity to embryonic neuronal induction. Effects of MeHg on formation of precursors and their further maturation were addressed in two separate sub-assays. We found the precursor induction to be sensitive to MeHg in non-cytotoxic concentrations, as the expression of several neuronal mRNA markers changed. In contrast, the mRNA marker expression in matured cells was unaffected by non-cytotoxic MeHg concentrations. Overall, our results imply that NCAM1, NEUROD1 and MAP2 are promising markers for detection of MeHg toxicity to induction of neuronal precursors and that induction of neuronal precursors is more sensitive to MeHg than their differentiation into neuron-like cells. Overall, we found that both assays provide alerts for the adverse effects of MeHg on neuronal induction. Our results indicate that mouse and human neuronal precursors exhibited similar sensitivities to MeHg, but that MeHg appeared to induce toxicity to the neuronal cytoskeleton in the mouse assay, while the metal seemed to inhibit precursor differentiation in the human assay, implying the presence of interspecies differences in the toxicity mechanisms. While differentiating ESCs provide a basis for establishment of in vitro developmental toxicity tests, matured derivatives can be used in in vitro assays addressing toxicities to adults. The request for high-throughput assays to identify potential cardiac drug candidates and to give warnings of adverse cardiac effects promoted our idea to develop automated digital movie analysis of contracting cardiomyocytes derived from ESCs. The digital movie analysis software Cardio Analyser was developed for automated quantification of beating frequencies and beating areas as well as estimation of drug induced chronotropic effects by detection of the light intensity changes in the contracting cultures. The data obtained were in accordance with the literature, showing the applicability of the method. Comparing the data to equivalent results obtained by extracellular electric field potential recordings demonstrated higher sensitivity to chronotropic effects of the beta-adrenoceptor agonist isoprenaline in the movie analysis experiments. This implies that more ESCs underwent differentiation into beta-adrenoceptor responding cardiomyocytes, making this set-up better for studying beta-adrenoceptor related effects of drugs. Overall, our study indicates that the movie analysis method may have potential to be optimised for screening in early drug discovery, aiming to identify cardiac drug candidates or to give alert for adverse effects on heart functionality or embryonic heart development. In conclusion, the experimental work presented in this Ph.D. thesis focused on development of novel in vitro testing methods based on ESCs. The results indicate the applicability of ESC models for in vitro assessment of developmental toxicity and cardiac pharmacology. Hence, the results add to the scientific knowledge on the usefulness of ESCs for establishment of toxicological and pharmacological assays. The field is, however, still quite open and needs more basic research and developments.</dcterms:abstract> <dcterms:title>In vitro Assessment of Developmental Toxicity and Cardiac Pharmacology using Embryonic Stem Cells</dcterms:title> <dc:contributor>Stummann, Tina C.</dc:contributor> <dc:format>application/pdf</dc:format> </rdf:Description> </rdf:RDF>

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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