Early neurodevelopmental disturbances during sensitive periods of stem cell differentiation

Abstract

The high need for appropriate in vitro tests for regulatory and industrial purposes as well as the focused governmental and EU support leads to a constant increase in assays addressing developmental neurotoxicity (DNT). The UKN1 assay is such a test method that has been set up already 10 years ago. By using human embryonic stem cells/induced pluripotent stem cells differentiating towards neuroectodermal precursor cells (NEP), the early neurodevelopment is modeled in vitro. The NEP state corresponds to the neural plate or the early neural tube in vivo. The readout for disturbances in the differentiation is based on the measurement of gene expression of several marker genes (PAX6, OTX2, NANOG, OCT4), it has also been assessed by transcriptome analysis using oligonucleotide microarrays. The UKN1 test has served to study processes of neurodevelopment such as epigenetic mechanisms. Moreover, the test method was used to develop framework conditions for setting up DNT methods in general, for example, strategies have been developed for compound concentration range finding and for quantifying transcriptomics datasets as a DNT endpoint. Especially, for the setup of transcrip-tome conditions for DNT testing, the UKN1-based studies have pioneered the field. In this thesis project the UKN1 assay was further developed. Firstly, the effect of acutely toxic compound concentrations on the transcriptome have been investigated. The aim was to un-derstand the mechanisms of transcriptomic regulation in a developing system in unperturbed and perturbed development. Additionally, it aimed to distinguish between an acute and a de-velopmental effect. Since the test should only identify specific DNT compounds, this represents an important basis. By using genetic markers as a toxicological fingerprint for acute cytotoxi-city, rules were established to exclude compounds tested in the cytotoxic range. In the second part of the project, the NEPs were further differentiated until the cells formed neural rosettes. These represent the in vitro equivalent of the in vivo neural tube. The rosette formation was established as a new endpoint for the UKN1 assay. The new phenotypic readout provides a robust tool to anchor transcriptomics changes to an endophenotype and to investi-gate the relevance of transcriptome changes at early neurodevelopmental stages. A reliable prediction model was setup based on the acquired information. Moreover, this approach served to define a narrow time window of toxicity of a HDAC inhibitor (trichostatin A). HDAC inhibitors are used as anticonvulsants and can induce congenital malformations such as spina bifida in the developing embryo. In the third part of the project, a computational model was developed to predict the influence of different valproic acid (VPA) concentrations on the expression of single genes at every time point during differentiation. The model was based on experimental datasets over time and on data from cells exposed to various VPA concentrations. The computational model delivered Abstract 12 information (predicted) about the pathways that were activated by VPA at intermediate (not measured) time points. The VPA-induced disturbance of rosette formation (endophenotype) was successfully rescued by blocking the wnt pathway. The three parts of this work have to-gether provided a new scientific basis for the UKN1 assay and they are expected to lead to a broader application for regulatory and for industrial purposes.