Embryotoxicity assessment of developmental neurotoxicants using a neuronal endpoint in the embryonic stem cell test

Abstract

The embryonic stem cell test (EST) is a validated in vitro embryotoxicity test; however, as the inhibition of cardiac differentiation alone is used as a differentiation endpoint in the EST, it may not be a useful test to screen embryotoxic chemicals that affect the differentiation of noncardiac tissues. Previously, methylmercury (MeHg), cadmium and arsenic compounds, which are heavy metals that induce developmental neurotoxicity in vivo, were misclassified as nonembryotoxic with the EST. The aim of this study was to improve the EST to correctly screen such developmental neurotoxicants. We developed a neuronal endpoint (Tuj-1 ID₅₀) using flow cytometry analysis of Tuj-1-positive cells to screen developmental neurotoxicants (MeHg, valproic acid, sodium arsenate and sodium arsenite) correctly using an adherent monoculture differentiation method. Using Tuj-1 ID₅₀ in the EST instead of cardiac ID₅₀, all of the tested chemicals were classified as embryotoxic, while the negative controls were correctly classified as nonembryotoxic. To support the validity of Tuj-1 ID₅₀) , we compared the results from two experimenters who independently tested MeHg using our modified EST. An additional neuronal endpoint (MAP2 ID₅₀), obtained by analyzing the relative quantity of MAP2 mRNA, was used to classify the same chemicals. There were no significant differences in the three endpoint values of the two experimenters or in the classification results, except for isoniazid. In conclusion, our results indicate that Tuj-1 ID₅₀ can be used as a surrogate endpoint of the traditional EST to screen developmental neurotoxicants correctly and it can also be applied to other chemicals.

Prospects and Frontiers of Stem Cell Toxicology

“…Therefore, to address this concern, the neural EST was introduced and successfully validated with six classical neurotoxicants with diverse mechanisms of toxicities [ 16 ]. Similar refinements were obtained by other groups [ 13 , 29 , 32 ]. There were also EST-inspired tests based on the differentiation of mESCs into endothelial cells [ 33 ] and osteoblasts [ 14 , 31 ], which showed to be effective and responsive to novel embryotoxic compounds [ 33 ], strongly validating the choice of using additional differentiation protocols in the EST.…”

Hand1-Luc Embryonic Stem Cell Test (Hand1-Luc EST): A novel rapid and highly reproducible <i>in vitro</i> test for embryotoxicity by measuring cytotoxicity and differentiation toxicity using engineered mouse ES cells

“…It would be considered as a great improvement to be able to gather experiments together to evaluate embryotoxicity. Other laboratories proposed new protocols considering the neuronal (Baek et al, 2012;Hayess et al, 2013) or the bone tissue (de Jong et al, 2012;zur Nieden et al, 2010). However, although showing promise and representing real advances in the domain, those protocols still require complicated manipulations that can hardly be automated with the current available technologies.…”

Toxic effects of methylmercury, arsanilic acid and danofloxacin on the differentiation of mouse embryonic stem cells into neural cells

“…In our investigation, assessing developmental neurotoxicity of chemicals was done with whole populations of ESCs rather than pure subpopulations ( i.e. , Tuj1- or MAP2-positive cells) [2,30]. Therefore, our study provided an overview of chemical-induced neurotoxic effects observed during in vitro neural differentiation.…”