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Updated: Oct 14, 2025
Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues
Published on: June 3, 2016
Gene regulation by morpholines and piperidines in the cardiac embryonic stem cell test
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 an in vitro embryotoxicity screen which uses cardiomyocyte formation as the main differentiation route. Studies are ongoing into whether an improved specification of the biological domain can broaden the applicability of the test, e.g. to discriminate between structurally similar chemicals by measuring expression of dedicated gene transcript biomarkers. We explored this with two chemical classes: morpholines (tridemorph; fenpropimorph) and piperidines (fenpropidin; spiroxamine). These compounds cause embryotoxicity in rat such as cleft palate. This malformation can be linked to interference with retinoic acid balance, neural crest (NC) cell migration, or cholesterol biosynthesis. Also neural differentiation within the ESTc was explored in relation to these compounds. Gene transcript expression of related biomarkers were measured at low and high concentrations on differentiation day 4 (DD4) and DD10. All compounds showed stimulating effects on the cholesterol biosynthesis related marker Msmo1 after 24 h exposure and tridemorph showed inhibition of Cyp26a1 which codes for one of the enzymes that metabolises retinoic acid. A longer exposure duration enhanced expression levels for differentiation markers for cardiomyocytes (Nkx2-5; Myh6) and neural cells (Tubb3) on DD10. This readout gave additional mechanistic insight which enabled previously unavailable in vitro discrimination between the compounds, showing the practical utility of specifying the biological domain of the ESTc.
Insights
The cardiac embryonic stem cell test (ESTc) can identify chemical toxicity by measuring gene biomarkers. Specifying the biological domain allowed for in vitro discrimination between similar chemicals, improving the test's utility.
Area of Science:
- Developmental toxicology
- In vitro toxicology
- Stem cell biology
Background:
- The cardiac embryonic stem cell test (ESTc) is an in vitro screen for embryotoxicity.
- Improving ESTc applicability may involve specifying biological domains and measuring gene biomarkers.
- Structurally similar chemicals can cause developmental toxicity, such as cleft palate in rats, linked to retinoic acid, neural crest migration, or cholesterol biosynthesis.
Purpose of the Study:
- To explore specifying the biological domain of the ESTc for discriminating between morpholine and piperidine classes of chemicals.
- To investigate the effects of these compounds on cardiomyocyte and neural differentiation markers.
- To assess the utility of gene transcript biomarkers in understanding chemical-induced embryotoxicity.
Main Methods:
- Utilized the ESTc with cardiomyocyte differentiation.
- Exposed differentiating cells to morpholines (tridemorph, fenpropimorph) and piperidines (fenpropidin, spiroxamine) at varying concentrations.
- Measured gene transcript expression of biomarkers related to cholesterol biosynthesis (Msmo1), retinoic acid metabolism (Cyp26a1), cardiomyocyte differentiation (Nkx2-5, Myh6), and neural differentiation (Tubb3) at differentiation day 4 (DD4) and DD10.
Main Results:
- All tested compounds stimulated the cholesterol biosynthesis marker Msmo1 after 24 hours.
- Tridemorph inhibited Cyp26a1, an enzyme metabolizing retinoic acid.
- Longer exposure (DD10) enhanced differentiation markers for cardiomyocytes and neural cells, enabling in vitro discrimination between the chemical classes.
Conclusions:
- Specifying the biological domain of the ESTc by measuring dedicated gene transcript biomarkers enhances its applicability.
- This approach provides mechanistic insight and allows for in vitro discrimination between structurally similar, toxic chemicals.
- The study demonstrates the practical utility of refining the ESTc for targeted toxicological assessments.
