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The embryonic stem cell test (EST) is a validated method and a useful screening tool for drug discovery. EST requires microscopic observation of beating cells to be considered cardiomyocytes as an endpoint assay. However, this procedure is time-consuming and limits the throughput performance. Instead of microscopic observation, we previously established a novel assay method based on cardiac field potential as an endpoint. However, cardiac specificity of this field potential is not yet clarified, because beating cells have not been rigorously evaluated as skeletal or cardiomyocyte. Here, we investigated the relationships between field potential, beating, and cardiac troponin T (cTnT) expression, selected as a cardiomyocyte-specific marker, and evaluated suitability of the field potential as a marker for cardiomyocyte in vehicle or 5-fluorouracil treated embryo bodies. Embryoid bodies of mouse embryonic stem cells (D3) were differentiated in a chamber with multi-electrode array for 5 days, and field potential and beating were measured at the end of differentiation. In addition, these chambers were immunohistochemically stained with anti-cTnT antibody, and the correlation between field potential, beating, and cTnT expression was examined. These results indicated the area of field potential or beating mainly coincided with that of cTnT expression. 5-fluorouracil treatment decreased not only the number of field potential detecting electrodes and beating area, but also cTnT expression, and the area of these parameters was also nearly identical. These results indicate that field potential can be used as a suitable cardiac differentiation marker, and can be a promising parameter of EST.

Usefulness of field potential as a marker of embryonic stem cell-derived cardiomyocytes, and endpoint analysis of embryonic stem cell test