Characterisation of a Neural Teratogenicity Assay Based on Human ESCs Differentiation Following Exposure to Valproic Acid

Abstract

The development of in vitro testing strategies for chemical and drug screening is a priority need in order to protect human health, to increase safety, to reduce the number of animals required for conventional testing methods and finally to meet the deadlines of current legislations. The aim of this work was to design an alternative testing method based on human embryonic stem cells for the detection of prenatal neural toxicity. For this purpose we have created a model based on the generation of neural rosettes, reproducing in vitro the gastrulation events recapitulating the formation of the neural tube in vivo. To validate the model we have exposed this complex cell system to increasing concentrations of valproic acid, a known teratogenic agent, to analyse the morphological and molecular changes induced by the toxicant. Specific assays were applied to discriminate between cytotoxicity and specific neural toxicity. Transcriptomic analysis was performed with a microarray Affimetrix platform and validated by quantitative real time RT-PCR for the expression of genes involved in early neural development, neural tube formation and neural cells migration, key biological processes in which the effect of valproic acid is most relevant. The results demonstrated that neural rosette cells respond to valproic acid exposure with molecular and morphological changes similar to those observed in vivo, indicating that this method represents a promising alternative test for the detection of human prenatal neural toxicity.

Neuronal developmental gene and miRNA signatures induced by histone deacetylase inhibitors in human embryonic stem cells

“… 10 , 11 , 12 , 13 ESC-based differentiation systems toward neuronal, cardiac, hepatic and, in general, multiple lineage differentiation have been utilized to monitor the toxic nature of known developmental toxicants either on a mechanistic or functional level. 2 , 3 , 10 , 14 , 15 , 16 , 17 …”

A method for human teratogen detection by geometrically confined cell differentiation and migration

“…Human-specific drug screening platforms for teratogenicity are needed to avoid inter-species variations 2 . However, current in vitro hPSC-based models only recapitulated temporal differentiation events 4 5 6 7 8 9 , and overlooked other key processes during embryonic development, including spatial organization of the differentiation and morphogenic processes. Our μP-hPSC model is the first in vitro human developmental toxicity screening model that recapitulated both spatially controlled differentiation and collective cell migration processes during embryogenesis.…”

“…However, the need to reduce the time and cost associated with animal testing as well as circumvent high inter-species variability (~40%) in teratogenic response 3 have galvanized the development of alternative in vitro models, especially those based on human pluripotent stem cells (hPSCs). The hPSC-based testing models developed so far employed temporally-controlled differentiating stem cell cultures using either directed differentiation ( i.e., differentiation into mesoendodermal 4 , neural 5 or cardiac cells 6 ) or random differentiation in embryoid bodies 7 . Measurements of molecular biomarkers by gene expression 4 5 7 , flow cytometry 6 , or metabolite detection 8 9 were used to determine the teratogenic potential of a compound.…”

A genomics-based framework for identifying biomarkers of human neurodevelopmental toxicity

“…Human embryonic stem cells (hESCs), which can be differentiated into neurons, have been proposed as screening tools for neurodevelopmental toxicity testing [4,5]. There are wellrecognized differences in embryonic development [6], ESC properties [7] and chemicalsensitivity [8] between rodents and humans.…”