In vitro Assessment of Developmental Toxicity and Cardiac Pharmacology using Embryonic Stem Cells

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In vitro Assessment of Developmental Toxicity and Cardiac Pharmacology using Embryonic Stem Cells

Dissertation

zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften

Eingereicht am Fachbereich Biologie der Universität Konstanz

vorgelegt von

Tina Charlotte Stummann

Tag der mündlichen Prüfung: 23.06.2008 Vorsitzender Prof. Dr. Dietrich

1. Referent Prof. Dr. Wendel 2. Referent Prof. Dr. Dr. T. Hartung

Konstanzer Online-Publikations-System (KOPS)

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Preface

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Preface

This Ph.D. thesis is submitted for evaluation at the Department of Biology at the University of Konstanz, Germany. The work was carried out between January 2005 and April 2008 at European Centre for Validation of Alterative Methods (ECVAM), Institute for Health and Consumer protection (IHCP) at the European Commission's Joint Research Centre (JRC) in Ispra, Italy.

My university supervisor for this thesis was Prof. Dr. Albrecht Wendel (Department of Biology, University of Konstanz, Germany), and my supervisors at ECVAM were Prof. Dr. Thomas Hartung, Dr. Susanne Bremer and Dr. Lars Hareng.

The Ph.D. thesis focuses on development and evaluation of the potential of novel mouse and human embryonic stem cell models for in vitro developmental toxicity testing and cardiac pharmacology assessments. The thesis consists of a general introduction to the fields of embryonic stem cells, development toxicity testing and cardiac pharmacology assessments, focusing on evaluations of pharmaceuticals and industrial chemicals. Moreover, the thesis discusses past and on-going research projects in applying embryonic stem cell models for in vitro assessment of developmental toxicity and cardiac pharmacology, including key-results from my own experimental work represented by the following manuscripts:

• Embryotoxicity Hazard Assessment of Methylmercury and Chromium using Embryonic Stem Cells.

Stummann, T.C. et al. Toxicology 2007; 242: 130-134.

• Hazard Assessment of Methylmercury Toxicity to Neuronal Induction in Embryogenesis using Human Embryonic Stem Cells. Stummann, T.C. et al. Toxicology 2009; 257: 117-126

• Digital movie analysis for quantification of beating frequencies, chronotropic effects and beating areas in cardiomyocyte cultures. Stummann, T.C. et al. ASSAY and Drug Development Technologies 2008; (6)3: 375-385.

Additionally, the manuscripts of a review article and a book chapter are submitted with the thesis as annexes, discussing the current state of the art in using embryonic stem cells for in vitro assessment of toxicology and safety pharmacology:

• The Possible Impact of Human Embryonic Stem Cells on Safety Pharmacological and Toxicological Assessments in Drug Discovery and Drug Development. Stummann, T.C. and Bremer, S. Current Stem Cell Research & Therapy 2008; 3(2): 117-130.

• Embryonic Stem Cells in Toxicology and Safety Pharmacology. Stummann, T.C. and Bremer, S.

(2008). Book chapter in “New Technologies for Toxicity Testing” edited by Michael Balls, Robert Combes and Nirmala Bhogal, 2008. Landes Bioscience Publishing (Georgetown, TX, USA). In press.

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Acknowledgements

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Acknowledgements

I would like to like to express my gratitude to Prof. Dr. Thomas Hartung (ECVAM) and Prof.

Dr. Albrecht Wendel (Department of Biology, University of Konstanz, Germany) for giving me the opportunity to make this thesis and for supervision of my work.

I want to address special thanks to my daily supervisor Dr. Susanne Bremer (ECVAM) for her enthusiasm, valuable scientific input, good advice and strong thrust and support in my work. I am most grateful to Dr. Lars Hareng (ECVAM) for his supervision of my activities, optimism and constructive criticism. Many thanks to all the present and former members of the Reproductive Toxicity Key-area group at ECVAM for creating an excellent working spirit and a great teamwork. I especially want to thank Dr. Cristian Pellizzer for his ironic attitude to life, which always makes me laugh, Dr. Marina Hasiwa for great lab-company and for her support in submitting this thesis, Dr. Patricia Pazos for her always brilliant mood and Benjamin Kumpfmueller for being an extremely enthusiastic and positive master student.

I am very thankful to Dr. Maurice P. Whelan, Dr Tomasz Sobanski and Mateusz Wronski (Nanotechnology and Molecular Imaging Unit, IHCP, JRC) as well as Prof. Dr. Michael Schwarz and Annette Mühleisen (University of Tübingen, Germany) for our scientific collaborations. They all contributed with positive attitudes and fruitful inputs.

I would like to thank all my present and former colleagues at ECVAM for many great laughs and discussions. Especially, I would like to thank Dr. Enrico Sabbioni, Dr. Jessica Ponti and

Massimo Farina for helpful discussions on metals and Gerard Bowe, Juan Casado Poblador and Jan de Lange for excellent technical support.

Dr. Luc Stoppini (BioCell Interface SA, Switzerland) is thanked for technical support.

Finally, I would like to thank my parents for always believing in me and supporting my studies and Frans M. Christensen for bringing me through difficult times and always being with me.

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List of Publications

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List of Publications

Manuscripts which are part of this thesis:

Hazard Assessment of Methylmercury Toxicity to Neuronal Induction in Embryogenesis using Human Embryonic Stem Cells. Stummann, T.C., Hareng, L., Bremer, S. Toxicology 2009; 257:

117-126.

Digital movie analysis for quantification of beating frequencies, chronotropic effects and beating areas in cardiomyocyte cultures. Stummann T.C., Wronski, M., Sobanski, T., Kumpfmueller, B., Hareng, L., Bremer, S. and Whelan, M.P. ASSAY and Drug Development Technologies 2008, 6(3), 375-385.

Hazard Assessment of Methylmercury Toxicity to Neuronal Induction in Embryogenesis using Human Embryonic Stem Cells. Stummann, T.C., Hareng, L., Bremer, S. Toxicology 2007; 242:

130-134.

The possible impact of human embryonic stem cells on safety pharmacological and toxicological assessments in drug discovery and drug development. Stummann, T.C. and Bremer, S. Review.

Current Stem Cell Research & Therapy, 2008, 3(2): 117-130. (Annex 1)

Embryonic Stem Cells in Toxicology and Safety Pharmacology. Stummann, T.C. and Bremer, S. Book chapter in “New Technologies for Toxicity Testing” edited by Michael Balls, Robert Combes and Nirmala Bhogal, 2008. Landes Bioscience Publishing (Georgetown, TX, USA). In press. (Annex 2)

Manuscripts relevant for this thesis:

Protein biomarkers for in vitro testing of embryotoxicity. Groebe, K., Hayess, K., Klemm, M., Steemans, M., Schwall, G., Wozny, W., Sastri, C., Jaeckel, P., Stegmann, W., Zengerling, H., Schöpf, R., Poznanovic, S., Stummann, T.C., Bremer, S., Seiler, A., Hartung, T., Spielmann, H., Schrattenholz, A. In preparation.

Report and Recommendations of the Workshop of the European Centre for the Validation of Alternative Methods for Drug-induced Cardiotoxicity. Stummann, T.C., Mandenius, C-F., Zünkler, J., Meyer, T., Duker, G., Dumotier, B., Minotti, G., Kang, Y.J., Jones, R.L., Beilmann, M. Fredriksson, M. Valentin, J-P., Hasiwa, M., Bremer, S. Submitted.

hESC Technology for Toxicology and Drug Development: Current Status and Recommendations for Best Practice and Standardization. Hasiwa, M., Adler, S., Bouvier d’Yvoir, M., Bremer, S., Buszanska, L., Hartung, T., Hescheler, J., Healy, L., Price, A., Reubinoff, B., Stojanov, T., Strehl, R., Stummann, T.C., Trosko, J., Stacey, G. In preparation.

Embryotoxicity Hazard Assessment of Cadmium and Arsenic compounds using Embryonic Stem Cells. Stummann, T.C., Hareng, L., Bremer, S. Toxicology 2008; 252: 118-122.

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Oral presentations relevant for this thesis:

Developmental toxicity testing using hESCs. Stummann, T.C., Hareng, L., Bremer, S. 1^st Status Symposium of Embryonic Stem Cell Research and Launch of the European Human Embryonic Stem Cell Registry, 18-19/01/2008, Berlin, Germany.

Use of Signalling Pathways as Toxicological Endpoints for Developmental Toxicity Testing.

Stummann, T.C., Hareng, L., Bremer, S., Herbst, F., Schwarz, M. JRC Exploratory Research Symposium 2006, 13/12/2006, Ispra, Italy.

Poster presentations relevant for this thesis:

Development of an in vitro-Assay for the Assessment of Teratogenic Effects. A. Mühleisen, Stummann, T., Herbst, F., Köhle, C., Bremer, S., Schwarz, M.. Abstract. Annual meeting of German Society of Pharmacology and Toxicology (DGPT), 11-13/03/2008, Mainz, Germany.

Neuronal Embryotoxicity Hazard Assessment of Methylmercury using Human Embryonic Stem Cells. Stummann, T.C., Hareng, L., Bremer, S. Abstract. First International Symposium on Human Embryonic Stem Cell Research, 31/01-02/02/2008, Evry (Paris area), France.

ECVAM KEY AREA Reproductive Toxicity. Bremer, S., Hasiwa, M., Jacobs, M., Pellizzer, C., Seidle, T., Stummann, T., Hartung, T. Abstract. 6th World Congress on Alternatives & Animal Use in the Life Sciences, 21-25/02/2007, Tokyo, Japan

In vitro embryotoxicity hazard assessment of heavy metals. Stummann, T.C., Bremer, S., Sabbioni, E., Ponti, J. and Hareng, L. Abstract. The 14^th International Workshop on In Vitro Toxicology (INVITOX), 02-05/10/2006, Ostend, Belgium.

Integration of Embryonic Stem Cell based Tests into a Testing Strategy for Developmental Toxicity Testing. Hareng, L., Stummann, T., Pellizzer, C., Hartung, T., Bremer,S.. Abstract.

Final Meeting of the Stem Cell Priority Program 1109 of the German Research Foundation (DFG), 24-27/09/2006, Dresden, Germany

Workshop participation relevant for this thesis:

ECVAM workshop on “In vitro tests for drug induced cardiotoxicity”. Stummann, T.C.

(moderator and organisator), Mandenius, C-F. (moderator), Zünkler, J., Meyer, T., Duker, G., Dumotier, B., Minotti, G., Kang, Y.J., Jones, R.L., Beilmann, M. Fredriksson, M. Valentin, J-P., Hasiwa, M., Bremer, S. 04-05/03/2008, Ispra, Italy.

ECVAM workshop on “Standardization of cell culture procedures for growth and differentiation of human embryonic stem cell lines for toxicity testing". Stacey, G. (moderator), Hasiwa, M.

(moderator and organisator), Adler, S., Bouvier d’Yvoir, M., Bremer, S., Buszanska, L., Hartung, T., Hescheler, J., Healy, L., Price, A., Reubinoff, B., Stojanov, T., Strehl, R.,

Stummann, T.C., Trosko, J., Allsopp, T., Pera, M., Drake, R., Mandenius, C-F., Frandsen, U., Harkness, L., Marina Hasiwa, Price, A. 25-26/02/2007, Ispra, Italy.

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Other publications:

The anti-nociceptive agent ralfinamide inhibits tetrodotoxin-resistant and tetrodotoxin-sensitive Na+ currents in dorsal root ganglion neurons. Stummann, T.C., Salvati, P., Fariello R.G., Faravelli, L. Eur J Pharmacol. 2005 Mar 14;510(3):197-208.

Pharmacological investigation of the role of ion channels in salivary secretion. Stummann, T.C., Poulsen, J.H., Hay-Schmidt, A., Grunnet, M., Klærke, D., Rasmussen, H.B., Olesen, S.P., Jørgensen, N.K. Pflugers Arch. 2003 Apr;446(1):78-87.

Other presentations:

Functional fluorescent cell based assays for screening ion channel targets. Restivo A.,

Francisconi S., Stummann, T., Faravelli, L., Caccia, C., Sabido, David C. Abstract, 4th Forum of European Neuroscience, July 2004, Lisbon, Portugal.

NW-1029, an α-aminoamide derivative with antihyperalgesic and antiallodynic properties, inhibits Na⁺ currents in rat sensory neurons. Stummann, T.C., Salvati, P., Fariello R.G., Faravelli, L. Abstract, 4^th congress of the European federation of IASP Chapters (EFIC), Sep.

2003, Prague, Czech Republic.

The Role of Ion Channels in Salivary Secretion. Stummann, T.C., Poulsen, J.H., Hay-Schmidt, A., Grunnet, M., Klærke, D., Rasmussen, H.B., Jensen, B.S., Jørgensen, T., Olesen, S.P.,

Jørgensen, N.K. Abstract, Novo Nordisk and Novozymes Scholarships mini-symposium 2002, Novo Nordisk, Denmark

The Role of Ion Channels in Salivary Secretion. Stummann, T.C., Poulsen, J.H., Hay-Schmidt, A., Grunnet, M., Klærke, D., Rasmussen, H.B., Jørgensen, T., Olesen, S.P., Jørgensen, N.K.

Abstract, Meeting of the Scandinavian Physiological Society 2001, University of Aarhus, Denmark.

The Role of K⁺ Channels in Salivary Secretion. Stummann, T.C., Poulsen, J.H., Hay-Schmidt, A., Grunnet, M., Klærke, D., Rasmussen, H.B., Jensen, B.S., Jørgensen, T., Olesen, S.P., Jørgensen, N.K. Abstract, Channelopathies 2001, University of Sheffield, Sheffield, England.

Basolateral localization of KCNQ1 potassium channels in MDCK cells and native epithelia.

Rasmussen, H., Jespersen, T., Stummann, T.C., Hay-Schmidt, A., Vogel, L., Grunnet, M., Jørgensen, N.K., Olesen, S.P., Klærke, D. Abstract, Conferences Laudat, INSERM, Aix-les- Bains, France.

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Abbreviations

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Abbreviations

18S rRNA 18S ribosomal RNA ACHE acetylcholinesterase ANOVA analysis of variance

BDNF human recombinant brain derived neurotrophic factor

bmp bitmap

CHMP (EMEAs) Committee for Medical Products for Human Use

CrIII trivalent chromium

CrVI hexavalent chromium

ECVAM European Centre for the Validation of Alternative Methods

EMEA European Medicines Agency

ES embryonic stem

ESC embryonic stem cell

EST Embryonic Stem cell Test

EU European Union

FDA U.S. Food and Drug Administration

FETAX Frog Embryo Teratogenesis Assay - Xenopus GAPDH glyceraldehyde-3-phosphate dehydrogenase GCCP Good Cell Culture Practice

GDNF human recombinant glial-derived neurotrophic factor

GLP Good Laboratory Practice

hERG human ether-a-go-go related gene

hESC human embryonic stem cell

ICa,L L-type voltage gated Ca²⁺ current

I_Ca/Na non-selective cation current

ICCVAM U.S. Interagency Coordinating Committee on the Validation of Alternative Methods

ICH International Conference on Harmonisation IHCP Institute for Health and Consumer protection IK1 inward rectifying K⁺ currents.

I_Kp persistant delayed outwardly rectifying K⁺ currents IKr rapid delayed outwardly rectifying K⁺ currents I_Ks slow delayed outwardly rectifying K⁺ currents

INa Na⁺ current

ISSCR International Society for Stem Cell Research Ito transient outward K⁺ currents

JRC Joint Jesearch Centre

LIF leukemia inhibitory factor

LQTS long QT syndrome

MAP2, Mtap2 microtubule-associated protein 2

MeHg methylmercury

mESC mouse embryonic stem cell

MM test Micromass test

NCAM1, Ncam1 neural cell adhesion molecule 1

NEFL neurofilament light polypeptide 68kDa NEFM, Nefm neurofilament medium polypeptide 150kDa

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Abbreviations

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NEUROD1 neurogenic differentiation 1 transcription factors NSF-1 neural survival factor-1

OECD Organisation for Economic Co-operation and Development PAX6, Pax6 paired box 6

PGD preimplantation genetic diagnosis

PMDA Japanese Pharmaceuticals and Medical Devices Agency POU5F1, Pou5f1 POU class 5 homeobox 1 transcription factor 1

PS-NCAM polysialylated neural cell adhesion molecule

REACH Registration, Evaluation, Authorisation and Restriction of Chemicals

S.D. standard derivation

S.E.M. standard error of mean

SSEA-1 stage-specific embryonic antigen-1 SSEA-3 stage-specific embryonic antigen-3 SSEA-4 stage-specific embryonic antigen-4

TG test guideline

TH tyrosine hydroxylase

TPH1 tryptophan hydroxylase 1

TPH2 tryptophan hydroxylase 2 TRA-1-60 Tumor rejection antigen-1-60 TRA-1-81 Tumor rejection antigen-1-81 WEC test Whole Embryo Culture test

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Table of Contents

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3A.4. Results ...49

3A.4.1. EST classification of MeHg, CrVI and CrIII ... 49

3A.4.2. Neuronal marker mRNA and protein expression profiles during ES cell neuronal differentiation ... 52

3A.4.3. Contribution of cytotoxicity to the effects of MeHg on neuronal differentiation .... 54

3A.4.4. Effects of MeHg on neuronal marker mRNA kinetics ... 55

3A.5. Discussion ...58

3A.6. Conclusions ...62

3A.7. Acknowledgements ...62

3B. Hazard Assessment of Methylmercury Toxicity to Neuronal Induction in Embryogenesis using Human Embryonic Stem Cells ...63

3B.1. Abstract ...63

3B.2. Introduction ...64

3B.3. Materials and Methods ...65

3B.3.1. Cell line and maintenance culture conditions ... 65

3B.3.2. Differentiation of hESCs into neuronal precursors ... 65

3B.3.3. Maturation of hESC derived neuronal precursors ... 66

3B.3.4. Cell sampling and MeHg exposure ... 66

3B.3.5. Cytotoxicity assays ... 66

3B.3.6. RNA purification, reverse transcription and real-time PCR analysis of relative gene expression ... 67

3B.3.7. Immunohistochemistry ... 67

3B.3.8. Data processing and statistical analyses ... 68

3B.4. Results ...68

3B.4.1. Neuronal marker mRNA expression profiles during neuronal precursor differentiation ... 68

3B.4.2. Establishment of a protocol for maturation of hESC derived neuronal precursors . 71 3B.4.3. Neuronal marker mRNA and protein expression profiles during maturation of hESC derived neuronal precursors ... 73

3B.4.4. Effects of MeHg on neuronal precursor differentiation ... 75

3B.4.5. Effects of MeHg on maturation of hESC derived neuronal precursors ... 77

3B.5. Discussion ...78

3B.7. Acknowledgements ...82

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Table of Contents

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3C. Digital Movie Analysis for Quantification of Beating Frequencies, Chronotropic Effects

and Beating Areas in Cardiomyocyte Cultures...83

3C.1. Abstract ...83

3C.2. Introduction ...83

3C.3. Materials and Methods ...85

3C.3.1. Cell culture conditions ... 85

3C.3.2. Differentiation of ES cells into contracting cardiomyocytes ... 86

3C.3.3. Digital movie recording system ... 86

3C.3.4. BioCell field potential recording system ... 87

3C.3.5. Isoprenaline ... 87

3C.3.6. Graphic illustrations and statistical analyses of data ... 87

3C.4. Results ...87

3C.4.1. Development of Cardio Analyser software for extraction of beating frequencies ... 87

3C.4.2. Characterisation of the beating frequency of ES cell derived cardiomyocytes using movie analysis ... 89

3C.4.3. Optimization of the BioCell MEA system for recordings with ES cell derived cardiomyocytes ... 90

3C.4.4. Characterisation of field potentials from ES cell derived cardiomyocytes ... 91

3C.4.5. Detection of chronotropic effects of isoprenaline ... 92

3C.4.6. Development of Cardio Analyser software for quantification of the beating areas . 93 3C.5. Discussion ...95

3C.6. Acknowledgement ...98

4. Summarising Discussion ...99

4.1. Developmental Toxicity ...99

4.2. Cardiac Pharmacology ...107

4.3. Pros and cons of in vitro testing using embryonic stem cells ...110

5. Summary ...112

6. Zusammenfassung...114

7. Aufstellung der eigenen und fremden anteile an der promotion ...117

8. References ...119

Annex I. The Possible Impact of Human Embryonic Stem Cells on Safety Pharmacological and Toxicological Assessments in Drug Discovery and Drug Development. ...128

AI.1. Abstract ...128

AI.2. Introduction ...128

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Table of Contents

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AI.3. Regulatory requirements for non-clinical safety assessment of conventional

pharmaceuticals ...131

AI.4. The possible impact of human embryonic stem cells on non-clinical safety assessment131 AI.4.1. Adverse cardiac effects ... 132

AI.4.2. Hepatotoxicity... 135

AI.4.3. Developmental toxicity ... 137

AI.4.4. How hESC based assays may restructure the product pipeline ... 139

AI.5. How can hESC assays be made practically applicable for use in regulatory safety assessments? ...141

AI.5.1. Standardisation and up-scaling of hESC maintenance cultures ... 141

AI.5.2. Safety assays based on hESCs and hESC derivatives ... 142

AI.5.3. Regulatory guidelines of relevance for in vitro safety assays based on hESCs ... 144

AI.5.4. Validation of in vitro tests based on hESCs ... 145

AI.6. Ethics ...147

AI.7. Conclusions ...150

AI.8. References ...150

Annex II. Embryonic Stem Cells in Toxicology and Safety Pharmacology ...155

AII.1. Abstract ...155

AII.2. Introduction ...155

AII.3. Major progress in the use of embryonic stem cells in safety pharmacology and toxicology ...156

AII.3.1. Screening for cardiotoxicity ... 157

AII.3.2. Screening for hepatotoxicity ... 159

AII.4. The use of embryonic stem cells in embryotoxicity tests ...159

AII.4.1. The embryonic stem cell test ... 161

AII.4.2 Ongoing research activities in improvement of embryotoxicity tests based on embryonic stem cells... 162

AII.4.3. A battery approach must be used for embryotoxicity testing in vitro ... 163

AII.5. Further considerations ...163

AII.5.1. Quality criteria for embryonic stem cells used in toxicology and safety pharmacology ... 163

AII.5.2. Ethical issues ... 164

AII.6. Conclusions ...164

AII.7. References ...165

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List of Figures

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List of Figures

Fig. 1.1 The heart and its electrophysiological activities ………..…17

Fig. 1.2 Time of susceptibility to developmental harm from teratogens ………22

Fig. 1.3 Frequency of standardised toxicological endpoints in developmental testing ………..22

Fig. 1.4 Test animals needed for different toxicological endpoint studies in REACH ………..26

Fig. 1.5 Adverse effects of cardiotoxicants ………...34

Fig. 1.6 The electrocardiogram and the underlying ventricular currents ………....35

Fig. 3A.1 EST dose-response curves for CrIII, CrVI and MeHg ……….51

Fig. 3A.2 Gene expression kinetics during neuronal precursor differentiation of mESCs ………..53

Fig. 3A.3 Immunohistochemical characterization of neuron-like cells differentiated from ESCs …….54

Fig. 3A.4 Cytotoxic effects of MeHg exposure during mESC neuronal precursor differentiation …….55

Fig. 3A.5 Effects of 10^-7M MeHg on mRNA expression in mESC neuronal precursor differentiation...57

Fig. 3A.6 Effects of non-cytotoxic concentrations of MeHg on Mtap2 mRNA expression ………..58

Fig. 3B.1 Gene expression kinetics during neuronal precursor differentiation of hESCs ………..70

Fig. 3B.2 Establishment of a protocol for maturation of human neuronal precursors ………..72

Fig. 3B.3 Gene expression kinetics of precursors maturating into human neuron-like cells ………...…...74

Fig. 3B.4 Immunohistochemical characterisation of neuron-like cells derived from hESCs …….75

Fig. 3B.5 Effects of MeHg on neuronal precursor differentiation from hESCs ………...76

Fig. 3B.6 Effects of MeHg on maturation of hESC derived neuronal precursors ………...78

Fig. 3C.1 Determination of beating frequencies from pixel light intensity changes ………...89

Fig. 3C.2 Noise filtering of beating frequencies determined from pixel light intensity changes …….89

Fig. 3C.3 Characteristics of the contracting activity in the movie analysis set-up ……….……..90

Fig. 3C.4 Characteristics of the field potential activity in the BioCell MEA set-up ………...92

Fig. 3C.5 Effects of isoprenaline on beating frequency and field potential frequency ………..93

Fig. 3C.6 Quantification by movie analysis of beating areas ……….94

Fig. AI.1 Characteristics of ESCs ………..130

Fig. AI.2 Correlations between electrocardiograms, action potentials and field potentials ………135

Fig. AI.3 Potential characteristics of cardiotoxicity assays based on hESCs ……….135

Fig. AI.4 Potential effects of hESCs on the numbers of drugs passing the drug development phases….141 Fig. AI.5 An in vitro test is the combination of a in vitro assay and a prediction model ………146

Fig. AI.6 The meaning of reliable and relevant as addressed in a validation study ……….…147

Fig. AII.1 Characteristics of ESCs ………..157

Fig. AII.2 Potential characteristics of cardiotoxicity assays based on hESCs ……….158

Fig. AII.3 The EST prediction model ………..162

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List of Tables

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List of Tables

Table 1.1 Characteristics of mouse and human ESCs ………..…..….16

Table 1.2 The 17 identified signalling pathways of importance for normal development ……….23

Table 1.3 OECD test guidelines for reproductive/developmental toxicity testing ..………....26

Table 1.4 The 3-study design of the ICH S5(R2) test guideline .………..…...26

Table 1.5 OECD test guidelines for repeated dose toxicity testing ………..….37

Table 1.6 ICH guidelines relating to cardiotoxicity ..………...37

Table 3A.1 EST results for CrIII, CrVI and MeHg ...………...51

Table 3B.1 Human ESC neuronal differentiation media tested ...72

Table 4.1 Markers used in the present thesis ……….…....106

Table AI.1 ICH guidelines relating to non-clinical safety assessments of pharmaceuticals …….……...132

Table AI.2 Characteristics of hESCs, primary and immortalised cells ..……….……...141

Table AI.3 ECVAMs modular approach for test validation ..……….………....147

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1. Introduction

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1. Introduction

1.1. Embryonic Stem Cells

1.1.1. Characteristics of mouse and human embryonic stem cells

Embryonic stem cell (ESC) lines are isolated either from the blastomeres of the morula or from the inner cell mass of the pre-implantation blastocyst as shown in Fig. AI.1 and AII.1 (Klimanskaya et al., 2006; Thomson et al., 1998). In human embryonic development the morula cleavage stage occurs 3 days after fertilization (Carlson, 2004). The morula consists of approximately 16 compactly arranged identical cells named blastomeres, which can give rise to ESC lines (Carlson, 2004; Klimanskaya et al., 2006). At day 4-5 of gestation, the blastocyst consists of approximately 58 to 250 cells and pre-implantation is initiated (Carlson, 2004; NIH, 2001). The blastocyst is a hollow sphere of cells containing an outer layer of trophoblast cells surrounding a small inner group of inner cell mass cells. The inner cell mass gives rise to the embryo proper plus several extra-embryonic structures, while the trophoblast gives raise only to extra-embryonic tissue, including the placenta (Carlson, 2004). ESCs isolated from pre- implantation blastocysts are derived from the inner cell mass cells (NIH, 2001; Thomson et al., 1998). The first mouse ESC (mESC) lines were isolated in 1981, while human ESC (hESC) lines emerged two decades later (Evans and Kaufman, 1981; Martin, 1981; Thomson et al., 1998).

In contrast to most other cell lines, ESCs are genetically normal (diploid). Moreover, ESCs are considered pluripotent, as they are capable of both self-renewal without senescence and multi-lineage differentiation, i.e. they can give rise to differentiated cell lineages of all three germ layers, endoderm, mesoderm and ectoderm, as well as germ cells (Fig. AI.1 and AII.1) (Aflatoonian and Moore, 2006; Thomson et al., 1998). In fact, many studies have dealt with the establishment of protocols for spontaneous or directed in vitro differentiation of ESCs into cells with morphological and functional characteristics resembling those of cell types of the adult body. Some examples are differentiation into pancreatic cells (Lumelsky et al., 2001), a variety of neuronal subtypes and glial cells (Okabe et al., 1996; Pomp et al., 2005; Zhang et al., 2001), hepatocytes (Hay et al., 2007; Soderdahl et al., 2007), chondrocytes (Kramer et al., 2000), osteoblasts (Bielby et al., 2004), endothelial and haematopoietic cells (Wang, 2006), insulin- producing β-cells (Assady et al., 2001), skeletal muscle cells (Prelle et al., 2000) and cardiomyocytes (Boheler et al., 2005; Capi and Gepstein, 2006; Reppel et al., 2004). Many additional somatic-like cell types have been derived (NIH, 2001). The following two sections will give details on the characteristics of cardiomyocyte-like and neuron-like cells derived from

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1. Introduction

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ESCs, since the present thesis uses cardiac differentiation of mESCs and neuronal differentiation of mouse and human ESCs.

ESCs have an intrinsic potential to differentiate, thus their pluripotency must be ensured in long term culturing. Teratoma formation, expression of the transcription factor Pouf1, high alkaline phosphatase levels and expression of specific surface markers are among the common markers used to confirm pluripotency (Table 1.1) (Loring and Rao, 2006). A teratoma is a tumour composed of tissues from the three embryonic germ layers (NIH, 2001). Teratomas can be experimentally induced in animals by injecting mESCs or hESCs, as means to determine the ability of the ESCs to differentiate into tissues of the three germ layers, i.e. as a control for pluripotency (NIH, 2001). Both undifferentiated mESCs and hESCs express the transcription factor Pou5f1 and high levels of alkaline phosphatase, but differ in their expression of some surface markers (Table 1.1). Human ESCs express the stage specific antigens SSEA-3, SSEA-4 and the glycoproteins TRA-1-60 and TRA-1-81 (NIH, 2001). None of these are detectable in undifferentiated mESCs, which in contrast express e.g. SSEA-1 (NIH, 2001).

Undifferentiated mESCs can grow as single cells in monolayer and can therefore be cultured using simple standard cell culture techniques including enzymatic dissociation and growth on cell culture plastics (Table 1.1). Originally, mESCs were grown on feeder cells in serum containing medium, but the early experimental work proved that leukemia inhibitory factor (LIF) was the active agent produced by the feeder cells that was needed to sustain pluripotency of mESC cultures (Smith et al., 1988; Williams et al., 1988). LIF is a polyfunctional glycoprotein cytokine inducibly produced by many tissues. LIF acts on responding cells by binding to a heterodimeric membrane receptor (Metcalf, 2003). Today, the use of LIF in combination with serum containing medium is the standard culture condition used in many laboratories to maintain pluripotent mESCs.

In contrast to mESCs, undifferentiated hESCs grow in colonies and generally have a very low survival rate as single cells. Human ESC colonies are commonly split by manual cutting and grown on mitotic inactivated primary mouse embryonic fibroblast feeder cells (Fig. AI.1).

Hence, the passaging of hESCs is rather laborious and delicate. Recent publications report on methods for propagation of hESCs by enzymatic dissociation, and inactivation of feeder cells by irradiation (Ameen et al., 2008; Pouton and Haynes, 2007). In addition, the possibility to grow hESCs on human feeder cells, in feeder-free conditions and in chemically defined medium have been demonstrated (Ameen et al., 2008; Pouton and Haynes, 2007). None of these approaches are currently universally accepted for culturing of hESCs in the undifferentiated state (see Annex I for a detailed discussion on the need for hESC maintenance and differentiation quality criteria).

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1. Introduction

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mESCs hESCs

Karyotype Diploid (XY and XX) Diploid (XY and XX)

Teratoma formation in vivo Yes Yes

SSEA-1 Yes No

SSEA-3 No Yes

SSEA-4 No Yes

TRA-1-60 No Yes

TRA-1-81 No Yes

Alkaline phosphatase Yes Yes

Pou5f1 Yes Yes

Growth characteristics in vitro Grow as single cells in monolayer Grow in colonies Factors aiding in self-renewal Feeder cells + serum

LIF can substitute for feeder cells

Feeder cells + serum

bFGF + serum replacement can substitute for serum

Table 1.1 Mouse and human ESCs have many characteristics in common, but also differ with respect to their properties. The table gives an overview and is by no means complete. Modified from NIH (2001).

1.1.2. Cardiac differentiation

Several research groups have demonstrated that mouse and human ESCs can differentiate in vitro into cardiomyocyte-like cells. Successful cardiac differentiation of ESCs is readily identifiable, since the cardiomyocytes contract spontaneously. The number of spontaneously beating areas and the rate of contraction within each beating area change with continued differentiation (Boheler et al., 2005). Beating of mESC derived cardiomyocytes may continue from several days to >1 month (Boheler et al., 2005). The fully differentiated cardiomyocytes may stop contracting but can be maintained for many weeks (Boheler et al., 2005). ESC derived cardiomyocytes have structural and functional properties resembling those of their primary counterparts, including cardiac ion channels, action potentials, sarcomeric proteins, development of rhythmically beating cell clusters and responsiveness to β-adrenergic and muscarinic pharmacological modulation (Boheler et al., 2005; Capi and Gepstein, 2006; Reppel et al., 2004;

Wobus et al., 1991). In particular, the demonstration of human ether-a-go-go related gene (hERG) conducted rapid delayed rectifier potassium currents in hESC derived cardiomyocytes is important for their applicability in drug safety pharmacology (He et al., 2003; Sartiani et al., 2007). The reason is that pharmacological inhibition of hERG plays a major role in induction of delayed ventricular repolarisation, which may result in the life-threatening form of ventricular tachycardia “torsades de pointes” (see section 1.3.3). Evidence for the in vivo functionality of hESC derived cardiomyocytes was provided by the demonstration that transplanted hESC derived cardiomyocytes can pace the hearts of swine and guinea pigs with complete atrioventricular block (Kehat et al., 2004; Xue et al., 2005).

The in vivo development of the heart from precardiac mesoderm involves a complex series of morphological and gene expression changes that are reflected by changes in the electrical activity of the differentiating cardiomyocytes. The first observed mechanical activity

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1. Introduction

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occurs in the developing heart soon after the formation of the tubular heart tube and is due to spontaneous action potentials of the pacemaker-like cells (Mikawa and Hurtado, 2007). Several additional electrophysiological activities appear during the development of the embryonic heart into the adult heart, including the action potentials of the atrium, ventricles, sinoatrial node, atrioventricular node, His bundle and Purkinje fibres (Fig. 1.1) (Mikawa and Hurtado, 2007).

The sequence of cardiac differentiation events during embryogenesis, i.e. loss of pluripotency, formation of precardiac mesoderm, appearance of heart progenitor cells and their further maturation, are mirrored during in vitro cardiac differentiation of ESCs (Boheler et al., 2005). Multiple cardiomyocyte subtypes derived from mouse and human ESCs have been identified and characterised (He et al., 2003; Maltsev et al., 1993). Their appearance can be correlated with the length of the in vitro differentiation: early (primary myocardial–like (pacemaker-like) cells), intermediate, and terminal (atrial-, ventricular-, sinoatrial node-, His- and Purkinje-like cells) (Boheler et al., 2005). Hence, the in vitro cardiac differentiation of ESCs seems to mimic the in vivo differentiation occurring during embryonic development.

Fig. 1.1 The left panel shows the electrophysiological activities of the adult heart and the image to the right indicates their location within the heart. SA: Sinoatrial, AV:

Atrioventricular. RV: Right ventricle, LV: Left ventricle, AO:

Aorta. Source: Mikawa and Hurtado (2007).

1.1.3. Neuronal differentiation

Mouse and human ESCs can differentiate in vitro into cells having structural and functional properties resembling those of primary neurons. The ESC derived neuron-like cells produce abundant outgrowth of neurites, which develop into distinct axonal and dentritic structures. In addition, they express neuronal neurotransmitter receptors, voltage- and ligand-gated channels as well as enzymes for neurotransmitter synthesis (Carpenter et al., 2001; Perrier et al., 2004; Yan et al., 2005). They have been shown to have neuron-like functional membrane properties with resting membrane potentials of -30 to -70 mV and evoked, and even spontaneous, fast neuron-

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1. Introduction

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like action potentials (Gottlieb and Huettner, 1999; Perrier et al., 2004; Yan et al., 2005).

Furthermore, ESC derived neuron-like cells have been demonstrated to release neurotransmitters upon membrane depolarisation (Park et al., 2005; Schulz et al., 2004; Yan et al., 2005).

Moreover, functional synaptic connections between ESC derived neurons can be detected as evoked or spontaneous postsynaptic currents (Gottlieb and Huettner, 1999; Yan et al., 2005).

Human ESC derived neural precursors transplanted into neonatal mouse brains incorporated into a variety of brain regions where they differentiated into astrocytes and neurons (Zhang et al., 2001). Grafting of hESC derived dopaminergic cultures into 6-hydroxydopamine-lesioned parkinsonian rats can induce restitution of motor function (Roy et al., 2006; Yang et al., 2008), although a beneficial effect is not always observed (Park et al., 2005; Sonntag et al., 2007). Thus, hESC derived neuronal cells seem to exhibit functionality in vivo.

The formation of the nervous system in vivo is an integrated series of complex developmental steps, which initiates during embryogenesis but continues during postnatal development. Embryonic neuronal development initiates by the formation of the neural plate from the ectodermal germ layer (Carlson, 2004). The neural plate folds to form the neural tube.

The cells of the neural plate and early neural tube are highly mitotic neuroepithelial cells, which are organised in a pseudostratified single cell-layer neuroepithelium (Carlson, 2004). The central nervous system develops from these highly plastic neuroepithelium cells that proliferate, migrate from their places of origin to their final positions, differentiate, acquire regional identities and grow axons with a motile growth cone that is guided to form synapses with postsynaptic partners (Carlson, 2004). Through neural induction, the early central nervous system becomes organised into regions, which develop increasing levels of complexity and finally gain the characteristics of the adult central nervous system. The first reflex movements and brain activities are seen in the sixth week of human embryo development (Carlson, 2004).

Consecutively loss of pluripotency, formation of ectodermal cells and differentiation of neuroepithelial cells are the prerequisite steps towards derivation of neuron-like cells from ESCs (Guan et al., 2001; Yan et al., 2005; Zhang et al., 2001). Moreover, neuronal differentiating ESCs progressively decrease their resting membrane potential, gain neuronal sodium and potassium currents, fire actions potentials of mature types and obtain synaptic currents (Guan et al., 2001; Johnson et al., 2007). Furthermore, neuronal differentiation of ESCs can give rise to the multiple neuronal subtypes of the nervous system, including GABAnergic, glutamatergic, serotonergic, cholinergic and catecholaminergic neurons as well as motorneurons (Guan et al., 2001; Zhang et al., 2007). Overall, these data support the hypothesis that neuronal differentiation of ESCs mimics the in vivo neuronal induction processes.

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1. Introduction

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1.2. Developmental Toxicity 1.2.1. Birth defects

Birth defects are those manifestations of deviant or disruptive development observable at birth or later. The four principal manifestations are malformations, growth retardation, embryo- or foetal-lethality and functional impairment (Schardein, 2000). Malformations are structural abnormalities, which may be single or multiple. Generally, the term teratogenesis refers to a process of inducing malformation, while a teratogen refers to the substance that induces malformations. Growth retardation is abnormally small foetal size. The limit for intrauterine growth retardation in humans is a birth weight of less than 2500g (Schardein, 2000). Death of offspring is another manifestation of birth defects, resulting in resorption or miscarriage depending on the species. Functional impairments are those birth defects affecting behaviour, including sexual maturation, intelligence, social ability and emotionality. Damaged reflex development, motor activity, learning and memory are examples of neurological functional impairments (OECD, 2007).

Major birth defects are abnormalities that are life-threatening, require major surgery or present a significant disability. Such birth defects are observed in 2 to 8% of human infants and foetuses and is an important social and healthcare issue (Eurocat, 2005; NRC, 2000; Queisser- Luft et al., 2002). The causes may be genetic or exposure to environmental factors. Extrinsic causes may include infections, nutritional deficiencies or excesses, natural plant or animal toxicants, radiation, pharmaceuticals, industrial chemicals, cosmetics and food additives (NRC, 2000). Between 1 and 3% of all birth defects are suggested to be attributable to chemicals and drugs, but the figure is a rough estimation (NRC, 2000; Schardein, 2000; Webster and Freeman, 2001).

The perception of high risk of chemicals and drugs is strongly related to the thalidomide tragedy. Thalidomide was perceived as a harmless sedative drug that was given to pregnant women in the late 1950s and early 1960s. However, the drug was strongly teratogenic, when given in critical periods of embryo development, and caused more than 7000 children to be born with malformations (Gilbert, 2006). In response to the disaster, rigid regulatory requirements for developmental toxicity testing of pharmaceuticals and industrial chemicals were implemented (Collins, 2006).

1.2.2. Developmental toxicants

Reproductive toxicity is any effect of a substance that interfere with reproductive ability or capacity (UNECE, 2004). Developmental toxicity comprises the part of reproductive toxicity

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1. Introduction

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dealing with the effects on the offspring, i.e. adverse effects induced during pregnancy or as a result of parental exposure, that can be manifested at any point in the life span of the organism (UNECE, 2004). Chemicals or drugs are considered development toxicants when they display developmental toxicity and they have predominant effects on the offspring (Schardein, 2000).

The implication is that a substance producing abnormal development at doses, which also induce maternal toxicity, will be regulated by the toxicity to the adult and hence will not necessarily be classified as developmental toxic.

Most developmental toxicants produce abnormalities only during certain critical periods of gestation (Carlson, 2004; Schardein, 2000). In line, some stages of development are more susceptible to toxicants than others (Fig. 1.2). Exposure of the embryo to a toxicant during cleavage and the formation of the epiblast and hypoblast are unlikely to result in congenital abnormalities because it either damages so few cells that the embryo can recover or it damages so many cells that the embryo dies. The period with maximal susceptibility to developmental harm is between the third and eighth week (the embryonic period), because most organogenesis occurs in this interval and interference with the processes may lead to gross malformations. Most organs are well established after the eighth week of gestation, making it unlikely that major malformations will be induced. Abnormalities arising during the third to the ninth month of gestation, the foetal period, tend to be functional or to be disturbances in the growth of established body parts. It should be considered that some developmentally harmful agents might cause their effects at the molecular level at an early stage of development, although the effects may not be recognized before later, perhaps postnatally. Other agents may destruct already established structures. A recent literature analysis of the standardised toxicological endpoints assessed in current in vivo developmental toxicity testing of chemicals revealed the frequency of endpoints with statistically significant findings for 202 developmental toxicants (Fig. 1.3) (Bremer et al., 2007). The data showed the most common manifestations of developmental toxicity to be post-implantation loss and death, abnormal offspring bodyweight, malformation of the skeleton, external limbs, digits, mouth, jaw and skull as well as malformations of the visceral (pertaining to the internal organs) neurological, urogenital and cardiovascular tissues.

Developmental toxicity is governed by dose-effect relations (Schardein, 2000). Because it is a multicellular phenomenon induction of abnormalities only occurs above a certain threshold of exposure and is not a stochastic phenomenon such as induction of cancer and mutations for which the risk decreases with lower doses but theoretically never disappears. The dose-response curves for developmental toxic effects are probably rather steep, lying within the doses, which will kill the embryo and those without effect.