Identification of Thalidomide-Specific Transcriptomics and Proteomics Signatures during Differentiation of Human Embryonic Stem Cells

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.

Monocrotophos in Gandaman village: India school lunch deaths and need for improved toxicity testing

“…While there is no deus ex machina to solve all these challenges for the experimental toxicologist, the advent of pluripotent stem cells (PSCs) provides significant improvements (Baquié et al 2012;Leist et al 2008), enabling the impact of potentially toxic pesticides to be studied on virtually all types of cells without recourse to immortalized cell lines. Within the last 5 years, the European ESNATS consortium has been developing pluripotent stem cell-based toxicity tests, and first results demonstrate that such test systems may indeed become powerful tools (Krug et al 2013;Bolt 2013;Vojnits et al 2012;Kern et al 2013;Meganathan et al 2012;Kuegler et al 2010;Zimmer et al 2011a, b;Balmer et al 2012). The consortium has shown that complex functional endpoints can be transferred to in vitro test systems Kadereit et al 2012;Zimmer et al 2012).…”

Mode of Action Assignment of Chemicals Using Toxicogenomics: A Case Study with Oxidative Uncouplers

“…Toxicogenomics has been selected as an experimental approach as it enables unbiased, global profiling of the molecular biological response space (Richard, 2006;Edwards and Preston, 2008), without preselection of response parameters. For single chemicals, it has been shown that exposure results in chemical-specific, gene expression profiles (Hamadeh et al, 2002;Yang et al, 2007;Kiyosawa et al, 2010;Janssens et al, 2011;Piña and Barata, 2011;Meganathan et al, 2012;Theunissen et al, 2012). The hypothesis tested in the present study is whether specific MoAs have common gene expression profiles that can be used to classify compounds and whether compounds with multiple MoAs can be classified and distinguished from those with only one MoA.…”

The use of biomarkers of toxicity for integrating in vitro hazard estimates into risk assessment for humans

“…the ideal test system will display all of the differentiated features and cellular functions found in intact organisms of various life stages, disease states, and conditions. Potential models for a cellular test system could use human embryonic stem cells (heSCs), or other types of stem/progenitor cells, in conjunction with protocols that allow these cells to differentiate along numerous organ-specific pathways (Leist et al, 2008;Kuegler et al, 2010;Wobus and löser, 2011;Zimmer et al, 2011Zimmer et al, , 2012Balmer et al, 2012;Meganathan et al, 2012).…”