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Updated: Aug 27, 2025
Modified Mouse Embryonic Stem Cell based Assay for Quantifying Cardiogenic Induction Efficiency
Published on: April 22, 2011
Genome-wide expression screening in the cardiac embryonic stem cell test shows additional differentiation routes that
R H Mennen1, N Hallmark2, M Pallardy3
1Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands.
Abstract:
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).
Insights
The cardiac embryonic stem cell test (ESTc) can detect developmental toxicity by analyzing gene expression in various pathways, not just cardiomyocyte differentiation. This improves understanding of chemical impacts and test applicability.
Area of Science:
- Developmental toxicology
- Stem cell biology
- Genomics and transcriptomics
Background:
- The cardiac embryonic stem cell test (ESTc) is an established non-animal method for assessing chemical developmental toxicity.
- Current ESTc methods primarily focus on cardiomyocyte differentiation, potentially limiting sensitivity and applicability.
- Expanding the biological domain of ESTc could enhance its ability to detect a broader range of chemical effects.
Purpose of the Study:
- To explore the full biological applicability domain of the ESTc using a data-driven approach.
- To identify a predictive embryotoxicity biomarker profile beyond cardiomyocyte beating.
- To assess the impact of morpholine and piperidine pesticides on ESTc developmental pathways.
Main Methods:
- Genome-wide expression screening using Next Generation Sequencing (NGS) on ESTc control samples.
- Analysis of developmental pathways affected by chemical exposure.
- Assessment of gene expression perturbations in response to morpholine and piperidine pesticides.
Main Results:
- NGS identified multiple developmental pathways in ESTc, including circulatory, skeletal, heart, muscle, organ tissue, and nervous system development.
- Morpholine and piperidine pesticides regulated these pathways in a concentration-dependent manner.
- Gene expression patterns showed both comparable and chemical-specific regulations, offering mechanistic insights.
Conclusions:
- The ESTc's biological domain encompasses diverse developmental pathways, enhancing its sensitivity for detecting chemical toxicity.
- Gene expression profiling provides a broader read-out than cardiomyocyte differentiation alone.
- This study expands the knowledge of ESTc's chemical applicability domain, aiding its integration into testing strategies and Integrated Approaches to Testing and Assessment (IATA).
