Definition of transcriptome-based indices for quantitative characterization of chemically disturbed stem cell development: introduction of the STOP-Toxukn and STOP-Toxukk tests

Vaibhav Shinde,Sunniva Förster,Jörg Rahnenführer,John Antonydas Gaspar,Jürgen Hescheler,Eugen Rempel,Johannes Meisig,Christoph Van Thriel,Sureshkumar Perumal Srinivasan,Nils Bluethgen,Julia Liebing,Regina Stoeber,Kesavan Meganathan,Stefan Schildknecht,Smita Jagtap,Tanja Waldmann,Patrício Godoy ,Lisa Hoelting,Jan G Hengstler,Marcel Leist,Marianna Grinberg,Agapios Sachinidis

doi:10.1007/s00204-016-1741-8

Abstract

Stem cell-based in vitro test systems can recapitulate specific phases of human development. In the UKK test system, human pluripotent stem cells (hPSCs) randomly differentiate into cells of the three germ layers and their derivatives. In the UKN1 test system, hPSCs differentiate into early neural precursor cells. During the normal differentiation period (14 days) of the UKK system, 570 genes [849 probe sets (PSs)] were regulated >fivefold; in the UKN1 system (6 days), 879 genes (1238 PSs) were regulated. We refer to these genes as ‘developmental genes’. In the present study, we used genome-wide expression data of 12 test substances in the UKK and UKN1 test systems to understand the basic principles of how chemicals interfere with the spontaneous transcriptional development in both test systems. The set of test compounds included six histone deacetylase inhibitors (HDACis), six mercury-containing compounds (‘mercurials’) and thalidomide. All compounds were tested at the maximum non-cytotoxic concentration, while valproic acid and thalidomide were additionally tested over a wide range of concentrations. In total, 242 genes (252 PSs) in the UKK test system and 793 genes (1092 PSs) in the UKN1 test system were deregulated by the 12 test compounds. We identified sets of ‘diagnostic genes’ appropriate for the identification of the influence of HDACis or mercurials. Test compounds that interfered with the expression of developmental genes usually antagonized their spontaneous development, meaning that up-regulated developmental genes were suppressed and developmental genes whose expression normally decreases were induced. The fraction of compromised developmental genes varied widely between the test compounds, and it reached up to 60 %. To quantitatively describe disturbed development on a genome-wide basis, we recommend a concept of two indices, ‘developmental potency’ (Dp) and ‘developmental index’ (Di), whereby Dp is the fraction of all developmental genes that are up- or down-regulated by a test compound, and Di is the ratio of overrepresentation of developmental genes among all genes deregulated by a test compound. The use of Di makes hazard identification more sensitive because some compounds compromise the expression of only a relatively small number of genes but have a high propensity to deregulate developmental genes specifically, resulting in a low Dp but a high Di. In conclusion, the concept based on the indices Dp and Di offers the possibility to quantitatively express the propensity of test compounds to interfere with normal development.

Highlights

Developmental toxicity testing represents a challenging field of toxicology because the currently applied animal tests, such as second-generation reproduction or developmental neurotoxicity studies, are cost as well as labour intensive and require high numbers of animals (Adler et al 2011; Forsby and Blaauboer 2007; Leist et al 2008, 2013)
We introduce two basic developmental indices to characterize the potency of developmental toxicants: (1) developmental potency (Dp), which represents the fraction of developmental genes among all genes whose expression is influenced by a test compound, and (2) developmental index (Di), which describes the degree to which developmental genes are overrepresented among all genes deregulated by a test compound
In UKN1 and UKK test systems genome-wide expression data of 12 compounds was obtained for benchmark concentrations (BMC10) resulting in viability reduced by a maximum of 10 %

Summary

Introduction

Developmental toxicity testing represents a challenging field of toxicology because the currently applied animal tests, such as second-generation reproduction or developmental neurotoxicity studies, are cost as well as labour intensive and require high numbers of animals (Adler et al 2011; Forsby and Blaauboer 2007; Leist et al 2008, 2013). Large efforts have been made to develop human stem cell-based in vitro test systems (Hengstler et al 2006; Meganathan et al 2015; Shinde et al 2015; Weng et al 2014). These in vitro systems recapitulate the critical phases of development, during which they are exposed to test compounds (Krug et al 2013). These systems have been applied in numerous studies to identify and characterize developmental toxicants (Balmer et al 2014; Meganathan et al 2012, 2015; Sisnaiske et al 2014; Zimmer et al 2014)

Methods

Results

Conclusion