Abstract
Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2), that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide.
Highlights
Traditional approaches to toxicological testing typically involves exposure of chemicals to large numbers of animals during the crucial period of organ development and further investigations of foetuses for visceral and skeletal developments, these approaches are expensive and time consuming [1,2,3,4]
A highly investigated in vivo developmental toxicant, at different concentrations with a combination of sensitive transcriptomics and proteomics approaches
The present time kinetic experiment demonstrates that the optimum differentiation time point, which is relevant for developmental toxicity approaches, was reached after day 12
Summary
Traditional approaches to toxicological testing typically involves exposure of chemicals to large numbers of animals during the crucial period of organ development and further investigations of foetuses for visceral and skeletal developments, these approaches are expensive and time consuming [1,2,3,4]. In order to provide costefficient and high throughput methods, a multitude of in vitro test systems have been proposed to assess the developmental toxicity of candidate drugs and environmental toxicants in the past 20 years These platforms include primary in vitro cell cultures and ex vivo models using embryo cultures [5] Embryonic stem cells (ESCs) have the unique potential to differentiate into all somatic cell types. In this context, a mouse ESC test originally covering the three end points for predicting teratogenicity (ESC cytotoxicity, fibroblasts cytotoxicity, and the inhibition of ESC differentiation into cardiomyocytes) has been initiated [6,7].
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