Dynamic changes in energy metabolism upon embryonic stem cell differentiation support developmental toxicant identification Dorien A.M. van Dartel a, *, Sjors H. Schulpen b, c , Peter T. Theunissen b , Annelies Bunschoten a , Aldert H. Piersma b, c , Jaap Keijer a a Human and Animal Physiology, Wageningen University, P.O. Box 338, 6700 AH Wageningen, The Netherlands b Laboratory for Health Protection Research, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlands c Institute for Risk Assessment Sciences, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.163, 3508 TD Utrecht, The Netherlands A R T I C L E I N F O Article history: Received 24 June 2014 Received in revised form 24 July 2014 Accepted 24 July 2014 Available online 30 July 2014 Keywords: Energy metabolism Embryonic stem cells Embryonic stem cell test (EST) Toxicology Transcriptomics A B S T R A C T Embryonic stem cells (ESC) are widely used to study embryonic development and to identify developmental toxicants. Particularly, the embryonic stem cell test (EST) is well known as in vitro model to identify developmental toxicants. Although it is clear that energy metabolism plays a crucial role in embryonic development, the modulation of energy metabolism in in vitro models, such as the EST, is not yet described. The present study is among the first studies that analyses whole genome expression data to specifically characterize metabolic changes upon ESC early differentiation. Our transcriptomic analyses showed activation of glycolysis, truncated activation of the tricarboxylic acid (TCA) cycle, activation of lipid synthesis, as well as activation of glutaminolysis during the early phase of ESC differentiation. Taken together, this energy metabolism profile points towards energy metabolism reprogramming in the provision of metabolites for biosynthesis of cellular constituents. Next, we defined a gene set that describes this energy metabolism profile. We showed that this gene set could be successfully applied in the EST to identify developmental toxicants known to modulate cellular biosynthesis (5-fluorouracil and methoxyacetic acid), while other developmental toxicants or the negative control did not modulate the expression of this gene set. Our description of dynamic changes in energy metabolism during early ESC differentiation, as well as specific identification of developmental toxicants modulating energy metabolism, is an important step forward in the definition of the applicability domain of the EST. ã 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Embryonic stem cells (ESC) are derived from the inner cell mass of the pre-implementation embryo, and are considered the prototypical stem cells (Evans and Kaufman, 1981). These cells can be cultured for a prolonged period without losing their pluripotent characteristics, and can be induced to differentiate into all foetal and adult cell types. In vitro, it has been shown that ESC can originate a wide variety of cell types, including cardiomyocyte, neural and hepatic cells. These in vitro models can provide a powerful model system to study embryonic development on the level of embryonic cell differentiation. Experiments comparing gene expression profiles of in vivo embryonic development and in vitro ESC differentiation have shown significant overlap in the regulation of genes annotated to developmental processes (Hettne et al., 2013; Robinson et al., 2011). The good correlation between modulation of processes in vivo and in vitro explains the successful initiatives of ESC differentia- tion-based in vitro approaches for identification of developmental toxic compounds (Wobus and Loser, 2011). The best known example of these approaches is the embryonic stem cell test (EST) (Spielmann et al., 1997). Initially, identification of developmental toxicants was based on disturbance of ESC differentiation into cardiomyocytes. Later, more objective molecular endpoint meas- ures were introduced (Osman et al., 2010; Seiler et al., 2004; van Dartel et al., 2009a), which improved the efficiency, objectivity, and accuracy of the EST. Gene expression changes upon ESC differentiation generally show enrichment of three processes: cell cycling, development and metabolism (van Dartel et al., 2010b). The first two themes have often been identified to be regulated upon ESC differentiation, whereas altered energy metabolism has been less extensively described. Recently, a de novo analysis of ESC differentiation * Corresponding author. Tel.: +31 317 484136. E-mail addresses: dorien.vandartel@wur.nl, dorienvandartel@hotmail.com (D.A.M. van Dartel). http://dx.doi.org/10.1016/j.tox.2014.07.009 0300-483X/ ã 2014 Elsevier Ireland Ltd. All rights reserved. Toxicology 324 (2014) 76–87 Contents lists available at ScienceDirect Toxicology journal homepa ge: www.elsev ier.com/locate /toxicol