Osteoblast differentiation of murine embryonic stem cells as a model to study the embryotoxic effect of compounds

The embryonic stem cell test (ESTc), in which the effect of chemical compounds on cardiomyocyte differentiation is evaluated, is one of the most studied in vitro alternatives for developmental toxicity testing. Because the assay readout is restricted to a single endpoint of differentiation, compounds that affect alternative differentiation pathways might be overlooked. It has therefore been suggested that the predictive value of the EST may be improved by including alternative differentiation endpoints. The aim of the present study was to evaluate the effect of five teratogenic compounds as well as one non-teratogenic compound on the differentiation of murine embryonic stem cells into osteoblasts (ESTo) and to compare results with those in the classical ESTc. We established an ESTo assay which proved robust, stable and reproducible. In this study, we showed that the evaluated compounds affected osteoblast differentiation both at the level of calcium concentrations in the culture as well as on multiple gene expression. Furthermore, we showed that the effect on calcium concentrations appeared to be primarily mediated by a general apoptotic effect and not by a specific effect on differentiation. The compounds tested showed little difference in their potency in the ESTo as compared to the ESTc. Before a definitive statement can be made regarding the added value of including an osteoblast differentiation endpoint into the EST, more compounds need to be evaluated. (C) 2012 Elsevier Ltd. All rights reserved.