Design of a test system with high sensitivity for developmental neurotoxicants based on differentiation of murine embryonic stem cells to neurons

Abstract

The understanding of mechanisms and the screening of a larger panel of environmentally-present compounds has been hampered by the lack of suitable and cost effective experimental systems. In vitro neural differentiation of embryonic stem cells (ESC) has been shown to approximately recapitulate in vivo differentiation. It may thus form the basis for a suitable in vitro system to assess DNT. Here, murine ESCs were differentiated to neurons under feeder-free conditions. The mature postmitotic neuronal phenotype was corroborated on day 20 by staining for general neuronal markers and several markers for neuronal subtypes or synaptic proteins. The differentiation kinetics was assessed by q-PCR analysis. DNT testing was performed by exposure to compounds during differentiation. DNT was evaluated by e.g. Western blot or q-PCR, and the acute neurotoxicity was assessed by the release of lactate dehydrogenase (LDH) or resazurin reduction. Test compounds included known neurodevelopmental toxicants [CH3HgCl (methylmercury), Pb(CH3COO)4], as well as known acute neurotoxicants (e.g. colchicine). The concentrations for the DNT assay were chosen so that they did not affect the overall cell viability (as assessed by e.g. LDH assays). This upper toxicity threshold was determined in acute neurotoxicity experiments. When ESC-derived, terminally differentiated neurons were exposed for three days to known neurotoxicants, we detected EC50 values that were similar to those found in murine primary neuronal cultures, but about one log lower than in non-neuronal cells. All DNT tests were performed with concentrations lower than those triggering acute neurotoxicity. Under these conditions, DNT compounds showed a pronounced adverse effect on differentiation. The mRNA levels of neural markers were significantly downregulated compared to control cultures not treated during differentiation. Notably, DNT, e.g. of CH3HgCl, was detected at more than 10000 times lower concentrations than the acute neurotoxicity. This reflects the in vivo situation where mercury is neurotoxic only at high concentrations while it is toxic for the developing fetal brain at very low concentrations. Significant effects of CH3HgCl were robustly and reproducibly detected at concentrations as low as 50 pM. To our knowledge this test system has an unprecedented sensitivity and will be used both for mechanistic studies on the background of DNT of known compounds and for the pre-screening of unknown compounds.