Lithium influences differentiation and tissue-specific gene expression of mouse embryonic stem (ES) cells in vitro

The effects of lithium chloride (LiCl) on differentiation of mouse embryoni c stem (ES) cells were investigated in order to evaluate the ES cell test ( EST) used in a European Union validation study for screening of embryotoxic agents in vitro, We show that LiCl inhibited concentration-dependently the differentiation of ES cells into cardiac and myogenic cells. Whereas the i nhibition of cardiac differentiation by high concentrations of LiCl was obv ious at day 5 + 5, decreased skeletal muscle cell differentiation was obser ved only at day 5 + 8. Semi-quantitative RT-PCR analyses revealed significa ntly lower levels of mRNA encoding cardiac-specific alpha -myosin heavy cha in and skeletal muscle-specific myoD. By morphological investigation, an in fluence of lithium on neuronal differentiation was not evident. However, mR NA levels of genes encoding synaptophysin and the 160 kDa neurofilament pro tein were increased by high LiCl concentrations, whereas mRNA levels of mas h-1 and Engrailed-1 were decreased, suggesting a specific influence of lith ium on neuronal differentiation, Furthermore, LiCl treatment resulted in a slight, but non-significant increase of beta -catenin levels in ES cell-der ived embryoid bodies. Our results demonstrate that the ES cell test, EST ma y be suitable to detect inhibitory effects of test compounds especially on cardiac differentiation, whereas effects on neuronal cells would not be det ected. Therefore, we propose that morphological analyses of cardiac differe ntiation alone are insufficient to detect embryotoxic effects. The assay of other cell lineages at different developmental stages, and expression anal yses of tissue-specific genes should also be employed.