Genome-wide expression screening in the cardiac embryonic stem cell test shows additional differentiation routes that are regulated by morpholines and piperidines.

The cardiac embryonic stem cell test (ESTc) is a well-studied non-animal alternative test method based on cardiac cell differentiation inhibition as a measure for developmental toxicity of tested chemicals. In the ESTc, a heterogenic cell population is generated besides cardiomyocytes. Using the full biological domain of ESTc may improve the sensitivity of the test system, possibly broadening the range of chemicals for which developmental effects can be detected in the test. In order to improve our knowledge of the biological and chemical applicability domains of the ESTc, we applied a hypothesis-generating data-driven approach on control samples as follows. A genome-wide expression screening was performed, using Next Generation Sequencing (NGS), to map the range of developmental pathways in the ESTc and to search for a predictive embryotoxicity biomarker profile, instead of the conventional read-out of beating cardiomyocytes. The detected developmental pathways included circulatory system development, skeletal system development, heart development, muscle and organ tissue development, and nervous system and cell development. Two pesticidal chemical classes, the morpholines and piperidines, were assessed for perturbation of differentiation in the ESTc using NGS. In addition to the anticipated impact on cardiomyocyte differentiation, the other developmental pathways were also regulated, in a concentration-response fashion. Despite the structural differences between the morpholine and piperidine pairs, their gene expression effect patterns were largely comparable. In addition, some chemical-specific gene regulation was also observed, which may help with future mechanistic understanding of specific effects with individual test compounds. These similar and unique regulations of gene expression profiles by the test compounds, adds to our knowledge of the chemical applicability domain, specificity and sensitivity of the ESTc. Knowledge of both the biological and chemical applicability domain contributes to the optimal placement of ESTc in test batteries and in Integrated Approaches to Testing and Assessment (IATA).