The application of mouse and human embryonic stem cells with transcriptomics in alternative developmental toxicity tests A bridge from model species to man Sjors Schulpen © Copyright All rights reserved. No part of this publication may be reproduced or transmitted in any form by any means without permission of the author. ISBN: 978-94-6203-861-5 Cover and layout: Maud van Deursen, Utrecht the Netherlands Production: CPI/ Wöhrmann Print Service, Zutphen the Netherlands The application of mouse and human embryonic stem cells with transcriptomics in alternative developmental toxicity tests A bridge from model species to man De toepassing van embryonale stamcellen van muis en mens met transcriptomics in alternatieve testen voor ontwikkelingstoxiciteit Een brug van modelorganisme naar de mens (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 7 juli 2015 des middags te 2.30 uur door Sjors Hubertus Wilhelmina Schulpen geboren op 5 januari 1983 te Sittard Promotoren: Prof. dr. A.H. Piersma Prof. dr. M. van den Berg Dit proefschrift werd mogelijk gemaakt door financiële steun van: Institute for Risk Assessment Sciences van de Universiteit Utrecht, Laboratorium voor gezondheidsbeschermingsonderzoek van het Rijksinstituut voor Volksgezondheid en Mileu, Greiner Bio-One en Stichting Proefdiervrij. Contents Abbreviations 6 Chapter 1 9 Introduction Chapter 2 23 Dose response analysis of monophthalates in the murine embryonic stem cell test assessed by cardiomyocyte differentiation and gene expression Chapter 3 39 A statistical approach towards the derivation of predictive gene sets for potency ranking of chemicals in the mouse embryonic stem cell test Chapter 4 55 An abbreviated protocol for multi-lineage neural differentiation of murine embryonic stem cells and its perturbation by methylmercury Chapter 5 77 Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol Chapter 6 93 Gene expression regulation and pathway analysis after valproic acid and carbamazepine exposure in a human embryonic stem cell based neuro- developmental toxicity assay Chapter 7 109 Comparison of gene expression regulation in mouse- and human embryonic stem cell assays during neural differentiation and in response to valproic acid exposure Chapter 8 127 Summary and general discussion Nederlandse samenvatting 139 Appendices 147 References 171 Dankwoord 195 List of publications 201 Curriculum Vitae 207 Abbreviations BSA Bovine serum Albumin CBZ Carbamazepine CM Culture medium DMEM Dulbecco’s Modified Eagle’s Medium DNT Developmental neuro toxicity testing EB Embryoid bodies ECVAM European Center for the Validation of Alternative Methods ESC embryonic stem cells ES-D3 Embryonic stem cell line D3 EST Embryonic stem cell test FBS Fetal bovine serum GO Gene Ontology hESC Human embryonic stem cells hESTn Human neural embryonic stem cell test IC50 Inhibitory concentration of cell viability 50% ID50 Inhibitory concentration of differentiation 50% iPS Induced pluripotent stem cells ITS Insulin transferrin selenium KOSR Knockout serum replacement LIF Leukemia Inhibitory Factor LOOCV Leave one out cross validation MBuP monobutyl phthalate MBzP monobenzyl phthalate MEFs Mouse embryonic fibroblasts MeHg Methylmercury MEHP mono-(2-ethylhexyl) phthalate mESC Mouse embryonic stem cells mEST Mouse embryonic stem cell test mESTc Mouse cardiac embryonic stem cell test mESTn Mouse neural embryonic stem cell test MMC Mitomycin C 6 Abbreviations NEAA Non Essential Amino Acids; NOAEL No observed adverse effect level NPC Neural precursor cells OECD Organization for economic cooperation and development PBS Phosphate-buffered saline PCA Principal component analysis PDL Poly-D-Lysine RA Retinoic acid RIN RNA Integrity number RMA Robust multichip average RT-PCR Real-time polymerase chain reaction US EPA US environmental protection agency VPA Valproic Acid WEC Whole embryo culture 7 8 CHAPTER1 Introduction 9 10 Introduction Toxicology During life, organisms including man are exposed to diverse hazardous compounds. Toxico- logical research focuses on the adverse effects of chemicals on organisms. The term ‘toxicolo- gy’ is derived from the Greek word toxidos, meaning poisonous. The route of exposure toge- ther with the dosing, which depends on the amount and exposure time, are two major factors 1 contributing to the level of effect. Additional factors which are of importance are i.e. age, genetic background, gender and diet. In toxicological test protocols, like acute, sub chronic and chronic toxicity tests, multiple toxicological endpoints are studied. In these studies, a sin- gle brief exposure (i.e. 24h), repeated exposures over a longer time (weeks or months), or life time are studied, respectively. In acute toxicity testing, classically the dose that is lethal for half of the animals (LD50) is determined [1, 2]. In sub-chronic tests, the species tested are exposed for a longer period of time, in which several end points of toxicity are studied, to determine a no observed adverse effect level (NOAEL). This is the highest dose level at which no adverse effects are observed, which is used for further risk assessment calculations [3]. Sub-chronic studies address aspects such as food consumption, body weight, behavior, ECG, EEG, hema- tology, blood chemistry, urinalysis and fecal analysis. Pathologic examination is performed on organs after sacrifice. Chronic studies are performed over a significant part of the life span or the entire lifetime of a test species. In additional mutagenicity studies, performed in cultured bacteria- and mammalian cells in vitro, the potency of a compound to induce genetic damage, in terms of genetic mutations in germ- and somatic cells is examined. In carcinogenicity stu- dies, often performed if mutagenicity studies are positive, indicating that a compound might cause cancer, the tumor incidence is determined by histological examination of organs of the test species [3, 4]. For some compounds also skin sensitization and irritancy are tested. In ad- dition, ecotoxicity studies are performed to study effects of environmental exposure. Reproductive and developmental toxicology Reproductive toxicology is the science that studies the potential of a compound to affect male and female fertility. Parental endpoints include development of the female egg- and male sperm cell, fertilization, implantation, delivery of the offspring and lactation, and post- natal growth including attainment of full reproductive function [3]. Developmental toxico- logy focuses on the development of the embryo and fetus during pregnancy, including early embryogenesis, organ formation, fetal development and growth. Teratogenicity, referring to the Greek word teratos, meaning monstrosity, relates to the study of malformations, and can be considered as an aspect of developmental toxicity [4]. Back in time, teratological sciences started as a descriptive science of mystical and scientific theories which explained congenital malformations, first introduced by I.G. de Saint-Hillaire (1805–1861) [5]. Until the 1940s it was believed that all birth defects were hereditary. However, in the 1930s and 1940s it was shown by Josef Warkany that birth defects could be induced by exogenous factors [6]. First, appearance of skeletal abnormalities in the offspring of rats were linked to a deficient diet [6]. Thereafter, congenital malformations of the eyes induced in rats was demonstrated to be caused by maternal vitamin A deficiency [7]. 11 A human counterpart was reported in 1950s when aminopterin, an analog of folic acid, ex- plored to be used for abortion, caused congenital malformations [8, 9]. Subsequently, in the early 1960s there was a catastrophe with thalidomide. Thalidomide was used against nausea and vomiting in pregnant women and effective against influenza [5]. This drug was presumed safe, but although minimal toxicity occurred in adults, a high degree of embryotoxicity occur- red, causing severe congenital malformations in more than 10,000 children [10]. Maternal exposure during pregnancy caused reduction deformities of the limbs up to total absence of all limbs. Nonetheless, the thalidomide catastrophe increased the understanding of develop- mental toxicology and demonstrated the importance of hazard identification of chemicals for development. In 1959 and in a monograph of 1973 Wilson defined teratology as the science dealing with adverse effects of the environment on the developing system, including the germ cells, embryo, fetus and immature individuals [11, 12]. He proposed 6 main principles of teratology. They included that susceptibility depends on genotype, the developmental stage at the time of exposure, the compound specific mode of action or mechanism and the nature of the compound. Furthermore, the four manifestations of deviant development are death, malformation, growth retardation, and impaired function, their frequency and severity being dose dependent [11]. In the 1980ies, the organization for Economic Cooperation and Deve- lopment (OECD) established protocols worldwide, describing test guidelines for reproductive and developmental toxicity testing of chemicals. Among these tests, are the prenatal develop- mental toxicity (OECD TG414) and the two-generation reproductive