Stem cell toxicology assays for cardiac differentiation

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Congenital cardiac defects (CCDs) are among the most common birth defects and fetal cardiac defects likely contribute to miscarriages, which terminate up to 25% of all pregnancies. Environmental factors may influence up to 80% of all CCDs, but little is known about how chemicals from the environment may affect formation of the heart. We propose to develop a cardiopoiesis (formation of the heart) assay to screen chemicals using stem cells, which recapitulate early event in heart formation, in which cardiac myocytes, vascular endothelial cells, and vascular smooth muscle cells differentiate from mesodermal multipotent cardiac precursors. The assay is performed in 384 well plates and uses murine embryonic stem cells (mESCs) bioengineered with reporter genes to report the proportion of cardiac myocytes, vascular endothelial cells, and vascular smooth that emerge from the cultures. In preliminary experiments, 550 known chemical pathway modulators were screened with the cardiopoiesis assay, and inhibitors of the Wnt pathway were found to strongly upregulate production of cardiac myocytes. This is consistent with the known influence of the Wnt pathway on cardiac development and the suspected role of dysregulated Wnt activity in producing CCDs. Phase I goals will be to further improve the assay by identifying compounds or genomic constructs that can serve as reliable positive or negative effectors in the system, to develop methods to maximize the information that can be garnered from each screening run, and to develop methods to improve the consistency and further miniaturize the assay.