Stem Cells in Birth Defects Research and Developmental Toxicology

Stem Cells in Birth Defects Research and Developmental Toxicology

Edited by Theodore P. RasmussenUniversity of ConnecticutStorrs, Connecticut, USA

This edition first published 2018© 2018 John Wiley & Sons, Inc.

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Library of Congress Cataloging-in-Publication Data:Names: Rasmussen, Theodore P., 1962- editor.Title: Stem cells in birth defects research and developmental toxicology /

[edited by] Theodore P. Rasmussen.Description: First edition. | Hoboken, NJ : Wiley, 2018. | Includes

bibliographical references and index. | Identifiers: LCCN 2017056789 (print) | LCCN 2017059841 (ebook) | ISBN

9781119283225 (pdf) | ISBN 9781119283232 (epub) | ISBN 9781119283218 (hardback)

Subjects: | MESH: Stem Cell Research | Pluripotent Stem Cells–drug effects | Fetal Research | Teratogens–analysis | Neurodevelopmental Disorders | Toxicity Tests

Classification: LCC QH588.S83 (ebook) | LCC QH588.S83 (print) | NLM QU 325 | DDC 616.02/774–dc23

LC record available at https://lccn.loc.gov/2017056789

Cover design by: WileyCover images: Immunofluorescent staining of a human iPS cell colony during early stages of iPS stem cell line derivation. Green staining identifies the expression of the pluripotency-associated transcription factor SOX2, red staining identifies LIN28, and blue staining (DAPI) allows visualization of cell nuclei. Image courtesy of A. Flamier. (Background image) © Pinghung Chen/EyeEm/Gettyimages

Set in 10/12pt WarnockPro by SPi Global, Chennai, India

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v

List of Contributors xiiiPreface xix

Part I Introduction and Overview 1

1 The Basics of Stem Cells and Their Utility as Platforms to Model Teratogen Action and Human Developmental and Degenerative Disorders 3Bindu Prabhakar, Soowan Lee, and Theodore P. Rasmussen

1.1 Stem Cell Types and Basic Function 31.2 Pluripotency 61.2.1 Poised Chromatin of the Pluripotent Epigenome 61.2.2 Undirected Differentiation of Pluripotent Cells to Embryoid

Bodies 71.2.3 Directed Differentiation of Pluripotent Cells 81.3 In vitro Uses of Pluripotent Cells 91.3.1 Pluripotent Cells for Toxicology 91.3.2 Pluripotent Cells for Teratology 111.3.3 Limitations of Pluripotent Stem Cells 121.4 Adult Stem Cells In vivo 131.5 Emerging Trends in Stem Cell Culture 141.5.1 Use of Coculture 151.5.2 Organoids 161.5.3 Microfluidics 171.5.4 Other Cell Types with Stem-Cell-Like Properties 181.6 Future Directions 181.6.1 iPSCs, Pharmacogenomics, and Predictive Teratology 181.6.2 Stem Cell Systems for Environmental Toxicology 19 References 20

Contents

Contentsvi

Part II Using Pluripotent Cells for the Detection and Analysis of Teratogens 25

2 Stem Cells and Tissue Engineering Technologies for Advancing Human Teratogen Screening 27Jiangwa Xing, Geetika Sahni, and Yi-Chin Toh

Abbreviations 272.1 Introduction 282.2 Current DART Regulatory Guidelines and Methods 292.2.1 Governing Bodies 292.2.2 Terminologies and Definitions 292.2.3 Testing Methodologies 302.2.4 Limitations of Animal-Based DART Testing 322.3 In vitro Animal-Based Models for Developmental

Toxicity Testing 332.3.1 Current In vitro Animal-Based Models for Developmental Toxicity

Testing 332.3.2 The MM Assay 352.3.3 The WEC Assay 352.3.4 The ZEDT Assay 362.3.5 New Engineering and Microfabrication Technologies for Model

Improvement 382.4 In vitro Stem-Cell-Based Developmental Toxicity Models 422.4.1 Embryonic Stem Cell Test (EST) 422.4.2 ReproGlo Reporter Assay 452.4.3 Metabolite Biomarker Assay Using hESCs 462.4.4 Mesoendoderm Biomarker-Based Human Pluripotent Stem

Cell Test (hPST) 472.4.5 The Micropatterned Human Pluripotent Stem Cell Test

(μP-hPST) 482.5 Conclusion and Future Directions 50 References 51

3 Use of Embryoid Bodies for the Detection of Teratogens and Analysis of Teratogenic Mechanisms 59Anthony Flamier

3.1 Embryoid Body Assays: Background 593.1.1 Teratogens and Teratogenesis 593.1.2 Classic Protocols for Teratogen Assays 603.1.3 Pluripotent Stem Cell Technology and its Applications for Teratogen

Detection 623.2 Detection of Teratogens Using EBs 63

Contents vii

3.2.1 Formation of Embryoid Bodies for Teratogen Assays 633.2.2 Cytotoxicity versus Teratogenicity 653.2.3 EB Treatments 653.3 Teratogenic Mechanisms 653.3.1 EB Growth and Morphogenesis 653.3.2 Molecular Analysis 663.3.3 Alternative Analyses 67 Acknowledgments 67 References 67

4 Stem-Cell-Based In vitro Morphogenesis Models to Investigate Developmental Toxicity of Chemical Exposures 71Yusuke Marikawa

4.1 Introduction 714.2 Stem-Cell-Based In vitro Morphogenesis Model 734.2.1 Mouse P19C5 EB as an In vitro Gastrulation Model 734.2.2 Quantitative Evaluation of Morphogenetic Impact 774.2.3 Detection of Developmentally Toxic Exposures Using Morphometric

Analyses 784.2.4 Investigations into the Molecular Mechanisms of Teratogen Actions

Using P19C5 EBs 814.3 Future Directions: Enhancing Morphogenesis-Based

Assays 834.3.1 Analyses of Changes in Gene Expression Relevant

for Teratogenesis 834.3.2 Detection of Proteratogens Using Metabolic Systems 844.3.3 Representation of Additional Developmental Regulator Signals 844.3.4 Recapitulation of Human Embryogenesis Using Human Embryonic

Stem Cells 854.4 Concluding Remarks 85 Acknowledgment 86 References 86

5 Risk Assessment Using Human Pluripotent Stem Cells: Recent Advances in Developmental Toxicity Screens 91Kristen Buck and Nicole I. zur Nieden

5.1 Introduction 915.2 Animal Embryo Studies to Evaluate Developmental Toxicity 915.3 Usage of Mouse Embryonic Stem Cells in Developmental

Toxicity 945.4 Alternative Endpoint Read-Out Approaches in the EST 965.4.1 Simple and Complex Methods – Trends Are Ever Changing 96

Contentsviii

5.4.2 Genomics, Transcriptomics, Proteomics, and Metabolomics 985.5 Novel Methods and Protocols to Replicate Human

Development 995.5.1 Human Embryonic Stem Cells 1005.5.2 Multipotent Stem Cells and Beyond 1035.6 Future Applications 105 Acknowledgments 105 References 106

Part III Human Developmental Pathologies Mediated by Adult Stem Cells 119

6 Modeling the Brain in the Culture Dish: Advancements and Applications of Induced Pluripotent Stem-Cell-Derived Neurons 121Sandhya Chandrasekaran, Prashanth Rajarajan, Schahram Akbarian, and Kristen Brennand

6.1 Introduction 1216.2 Methods to Generate Patient-Derived Neurons 1226.2.1 Directed Differentiation of Neurons from Pluripotent

Stem Cells 1226.2.2 Dopaminergic Neurons 1236.2.3 Glutamatergic Neurons 1236.2.4 GABAergic Interneurons 1246.2.5 Striatal Neurons 1256.2.6 Other Neurons (Serotonergic and Motor) 1266.2.7 Limitations of Directed Differentiation 1276.3 Neuronal Induction from Fibroblasts and hiPSCs 1276.3.1 Induced Neurons (iNeurons) 1286.3.2 Dopaminergic iNeurons 1296.3.3 Glutamatergic iNeurons 1306.3.4 Induced GABAergic Interneurons 1306.3.5 Induced Medium Spiny Neurons 1316.3.6 Serotonergic iNeurons 1316.3.7 Induced Motor Neurons 1316.3.8 Limitations of Neuronal Induction 1326.4 Cerebral Organoids: Neural Modeling in Three Dimensions 1326.4.1 Current Methods for Deriving Cerebral Organoids 1326.4.2 Applications of Cerebral Organoids: Disease Modeling 1346.4.3 Limitations in the Use of Cerebral Organoids 1356.5 Epigenetic Considerations in hiPSC Donor Cell Choice 136

Contents ix

6.6 Aging Neurons 1376.6.1 Techniques to Age hiPSCs 1376.6.2 Aging and Dedifferentiation 1386.6.3 Future Directions 1396.7 Drug Testing Using hiPSCs 1406.7.1 Facilitating Clinical Trials 1406.7.2 Titrating Drug Dosage 1406.7.3 Evaluating Chemotherapies 1416.7.4 Steering Personalized Medicine 1416.7.5 Forging Neural Networks 1426.8 Promises in the Field 1426.8.1 High-Throughput Automation 1426.8.2 Neural Tissue Engineering Using hiPSCs 1426.8.3 hiPSC-Based Transplantation Therapies 1436.8.4 Advances Using Gene-Editing Technologies 1446.9 Concluding Remarks 145 References 146

7 Modeling Genetic and Environment Interactions Relevant to Huntington’s and Parkinson’s Disease in Human Induced Pluripotent Stem Cells (hiPSCs)-Derived Neurons 159Piyush Joshi, M. Diana Neely, and Aaron B. Bowman

7.1 Gene–Environment Interactions Assessed in hiPSC-Derived Neurons 159

7.2 Modeling of Neurological Diseases with hiPSCs 1607.3 Cell Viability Assays 1627.4 Mitochondria 1637.5 Oxidative Stress 1647.6 Neurite Length by Immunocytochemistry (ICC) 1647.7 Conclusions 166 References 167

8 Alcohol Effects on Adult Neural Stem Cells – A Novel Mechanism of Neurotoxicity and Recovery in Alcohol Use Disorders 173Rachael A. Olsufka, Hui Peng, Jessica S. Newton, and Kimberly Nixon

8.1 Introduction 1738.2 The “Birth” of the Study of “Neuronal Cell Birth” 1758.3 Components of Adult Stem-Cell-Driven Neurogenesis 1808.3.1 Permissive Sites of Adult Neurogenesis in Brain 1808.3.2 Stem Cells Versus Progenitors 1828.3.3 Proliferation 1848.3.4 Differentiation and Migration 187

Contentsx

8.3.5 Cell Survival and Integration 1888.4 Alcohol Effects on Adult Neural Stem Cells

and Neurogenesis 1898.4.1 Proliferation 1898.4.2 Differentiation and Migration 1938.4.3 Survival and Integration 1948.5 Extrinsic Factors Influence the Neurogenic Niche 1968.6 Alcohol and the Niche 1988.7 Conclusions 200 References 201

9 Fetal Alcohol Spectrum Disorders: A Stem-Cellopathy? 223Amanda H. Mahnke, Nihal A. Salem, Alexander M. Tseng , Annette S. Fincher, Andrew Klopfer , and Rajesh C. Miranda

9.1 Fetal Alcohol Spectrum Disorders 2239.2 Stem Cells 2259.2.1 Totipotent Stem Cells 2279.2.2 Placental Stem Cells – Trophoblast 2309.2.3 Embryonic Stem Cells and Induced Pluripotent Stem Cells 2319.3 Endoderm 2349.3.1 Liver 2349.4 Mesoderm 2359.4.1 Cardiac Development 2359.4.2 Kidney 2379.5 Ectoderm 2389.5.1 Neuroectoderm Development 2389.5.2 Neural Crest 2399.5.3 Neural Tube Development 2409.6 Future Directions 2439.6.1 Fetal Origin of Adult Stem Cells 2439.6.2 Sex Differences 2449.6.3 Stem Cell Therapy 2459.7 Conclusion 245 References 246

10 Toxicological Responses in Keratinocyte Interfollicular Stem Cells 261Rambon Shamilov and Brian J. Aneskievich

10.1 Epidermal Keratinocyte Stem Cells 26110.2 Arsenic 26710.3 Dioxin 26910.4 Bacterial Toxins 273

Contents xi

10.5 Conclusions and Prospective Considerations 274 References 275

Part IV Recent Innovations in Stem Cell Bioassay and Platform Development 285

11 Stem-Cell Microscale Platforms for Toxicology Screening 287Tiago G. Fernandes and Joaquim M. S. Cabral

11.1 Introduction 28711.2 Stem Cell Models for Toxicology Assessment 28811.3 Biomimetic Microscale Systems for Drug Screening 29011.3.1 Design and Microfabrication: Soft Lithography and Replica

Molding 29011.3.2 Microcontact Printing and Surface Patterning 29211.3.3 Robotic Spotting and Printing 29211.4 Microtechnologies for Drug Discovery 29311.5 Devices for High-Throughput Toxicology Studies 29411.6 Cellular Microarray Platforms 29511.7 Microfluidic Platforms 29811.8 Conclusions and Future Perspectives 301 Acknowledgments 301 References 302

12 HepaRG Cells as a Model for Hepatotoxicity Studies 309André Guillouzo and Christiane Guguen-Guillouzo

12.1 Introduction 30912.2 Characteristics of HepaRG Cells 31012.2.1 A Bipotent Human Liver Cell Line 31012.2.2 HepaRG Hepatocytes Express Liver-Specific Functions 31412.2.3 Long-Term Functional Stability of HepaRG Hepatocytes 31512.3 Biotransformation and Detoxification Activities 31612.3.1 Drug Metabolism Capacity 31612.3.2 Biokinetics and Intrinsic Clearances 31812.3.3 Applications 31912.4 Toxicity Studies 32012.4.1 Hepatotoxicity Screening 32012.4.2 Cellular Cytotoxicity 32212.4.3 Genotoxicity and Carcinogenicity Screening 32412.4.4 Identification of Target Genes 32512.4.5 Cholestasis 32612.4.6 Steatosis 327

ftoc.indd 11 3/23/2018 7:50:16 PM

Contentsxii

12.4.7 Phospholipidosis 32812.5 Conclusions and Perspectives 328 Acknowledgments 329 References 330

Index 341

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xiii

Schahram AkbarianIcahn School of Medicine at Mount SinaiDepartment of NeuroscienceNew York, NY 10029USA

Icahn School of Medicine at Mount SinaiFriedman Brain InstituteNew York, NY 10029USA

Icahn School of Medicine at Mount SinaiDepartment of PsychiatryNew York, NY 10029USA

Brian J. AneskievichUniversity of ConnecticutDepartment of Pharmaceutical Sciences69 North Eagleville Road, U‐3092 Storrs, CT 06269USA

Aaron B. BowmanVanderbilt University (VU)Department of Pediatrics and Neurology, Vanderbilt University Medical Center (VUMC)1161 21st Avenue South, D‐4105 MCN, Nashville, TN 37232USA

Vanderbilt University (VU)Department of Biochemistry and Vanderbilt Brain Institute465 21st Avenue South, 6140 MRB3 Nashville, TN 37232USA

Kristen BrennandIcahn School of Medicine at Mount SinaiDepartment of NeuroscienceNew York, NY 10029USA

Icahn School of Medicine at Mount SinaiFriedman Brain InstituteNew York, NY 10029USA

List of Contributors

List of Contributorsxiv

Icahn School of Medicine at Mount SinaiDepartment of PsychiatryNew York, NY 10029USA

Icahn School of Medicine at Mount SinaiDepartment of Genetics and GenomicsNew York, NY 10029USA

Kristen BuckDepartment of Molecular, Cell and Systems Biology and Stem Cell Center, College of Natural and Agricultural SciencesUniversity of California Riverside1113 Biological Sciences Building Riverside, CA, 92521USA

Joaquim M. S. CabralUniversidade de LisboaDepartment of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior TécnicoAvenida Rovisco Pais 1049–001, LisbonPortugal

Sandhya ChandrasekaranIcahn School of Medicine at Mount SinaiDepartment of NeuroscienceNew York, NY 10029USA

Friedman Brain Institute, Icahn School of Medicine at Mount SinaiNew York, NY 10029USA

Icahn School of Medicine at Mount SinaiDepartment of Psychiatry New York, NY 10029USA

M. Diana NeelyVanderbilt University (VU)Department of Pediatrics and Neurology, Vanderbilt University Medical Center (VUMC)1161 21st Avenue South, D‐4105 MCN, Nashville, TN 37232USA

Vanderbilt University (VU)Department of Biochemistry and Vanderbilt Brain Institute465 21st Avenue South, 6140 MRB3 Nashville, TN 37232USA

Tiago G. FernandesUniversidade de LisboaDepartment of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior TécnicoAvenida Rovisco Pais 1049–001, LisbonPortugal

Annette S. FincherTexas A&M UniversityDepartment of Neuroscience and Experimental Therapeutics Texas A&M University Health Science CenterBryan, TX 77807USA

List of Contributors xv

Anthony FlamierUniversity of MontrealMaisonneuve‐Rosemont hospital5415 Boul. l’Assomption, Montréal QC H1T 2M4Canada

Christiane Guguen‐GuillouzoUniversité de Rennes 1, Faculté des Sciences Pharmaceutiques et BiologiquesInserm UMR 1241, Numecan2 avenue Prof. Léon Bernard 35043 Rennes CedexFrance

Biopredic InternationalParc d’Affaires de la Brétèche, Bât. A4, Saint GrégoireFrance

André GuillouzoUniversité de Rennes 1, Faculté des Sciences Pharmaceutiques et BiologiquesInserm UMR 1241, Numecan2 avenue Prof. Léon Bernard 35043 Rennes CedexFrance

Piyush JoshiVanderbilt University (VU)Department of Pediatrics and Neurology, Vanderbilt University Medical Center (VUMC)1161 21st Avenue South, D‐4105 MCN, Nashville, TN 37232USA

Vanderbilt University (VU)Department of Biochemistry and Vanderbilt Brain Institute465 21st Avenue South, 6140 MRB3 Nashville, TN 37232USA

Andrew KlopferTexas A&M UniversityDepartment of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science CenterBryan, TX 77807USA

Soowan LeeUniversity of ConnecticutDepartment of Pharmaceutical Sciences69. North Eagleville Road, Unit 3092, UConn Sch of Pharm Storrs, CT 06269USA

Amanda H. MahnkeTexas A&M UniversityDepartment of Neuroscience and Experimental Therapeutics, Women’s Health in Neuroscience Program, Texas A&M University Health Science CenterMedical Research and Education Building, 8447 State Highway 47 Bryan, TX 77807‐3260USA

Yusuke MarikawaUniversity of Hawaii at ManoaJohn A. Burns School of Medicine Institute for Biogenesis Research651 Ilalo Street, Biosciences Building 163A, Honolulu, HI 96813USA

List of Contributorsxvi

Rajesh C. MirandaTexas A&M UniversityDepartment of Neuroscience and Experimental Therapeutics Women’s Health in Neuroscience Program, Texas A&M University Health Science CenterMedical Research and Education Building, 8447 State Highway 47 Bryan, TX 77807‐3260USA

Jessica S. NewtonUniversity of KentuckyDepartment of Pharmaceutical Sciences789 S. Limestone, Lee T. Todd Bldg. 473, Lexington, KY 40536USA

Kimberly NixonUniversity of KentuckyDepartment of Pharmaceutical Sciences789 S. Limestone, Lee T. Todd Bldg. 473, Lexington, KY 40536USA

Rachael A. OlsufkaUniversity of KentuckyDepartment of Pharmaceutical Sciences789 S. Limestone, Lee T. Todd Bldg. 473, Lexington, KY 40536USA

Hui PengUniversity of KentuckyDepartment of Pharmaceutical Sciences789 S. Limestone, Lee T. Todd Bldg. 473, Lexington, KY 40536USA

Bindu PrabhakarUniversity of ConnecticutDepartment of Pharmaceutical Sciences69. North Eagleville Road, Unit 3092, UConn Sch of Pharm, Storrs CT 06269USA

Prashanth RajarajanIcahn School of Medicine at Mount SinaiDepartment of NeuroscienceNew York, NY 10029USA

Icahn School of Medicine at Mount SinaiFriedman Brain InstituteNew York, NY 10029USA

Icahn School of Medicine at Mount SinaiDepartment of PsychiatryNew York, NY 10029USA

Icahn School of Medicine at Mount SinaiDepartment of Genetics and GenomicsNew York, NY 10029USA

Theodore P. RasmussenUniversity of ConnecticutDepartment of Pharmaceutical Sciences69. North Eagleville Road, Unit 3092, UConn Sch of Pharm, Storrs CT 06269USA

List of Contributors xvii

University of Connecticut Stem Cell Institute400 Farmington Avenue Farmington, CT 06033USA

University of Connecticut, Institute for Systems GenomicsStorrs, CT 06269USA

Geetika SahniNational University of SingaporeDepartment of Biomedical Engineering4, Engineering Drive 3, Block E4 #04‐10, Singapore 117583Singapore

Nihal A. SalemTexas A&M UniversityDepartment of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science CenterBryan, TX 77807USA

Rambon ShamilovUniversity of ConnecticutDepartment of Pharmaceutical Sciences69 North Eagleville Road, U‐3092 Storrs, CT 06269USA

Yi‐Chin TohNational University of SingaporeDepartment of Biomedical Engineering4, Engineering Drive 3, Block E4 #04‐10, Singapore 117583Singapore

National University of SingaporeSingapore Biomedical Institute for Global Health Research and TechnologyMD6, 14 Medical Drive, #14‐01 Singapore 117599Singapore

National University of SingaporeNUS Tissue Engineering Program27 Medical Drive, DSO (Kent Ridge) Building, #04‐01, Singapore 117510Singapore

National University of Singapore Centre for Life SciencesSingapore Institute for Neurotechnology28 medical Drive, Singapore 117456Singapore

Alexander M. TsengTexas A&M UniversityDepartment of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science CenterBryan, TX 77807USA

Jiangwa XingQinghai UniversityDepartment of Basic Medical Sciences, Medical CollegeXining, Qinghai, 810016China

Nicole I. zur NiedenUniversity of California RiversideDepartment of Molecular, Cell and Systems Biology and Stem Cell Center, College of Natural and Agricultural Sciences1113 Biological Sciences Building Riverside, CA, 92521USA

xix

Were it not for the action of stem cells, all multicellular organisms with body plans organized into distinct tissues and organs would not be possible. Human development relies entirely upon the action of stem cells, which are progres-sively restricted as development proceeds through the stages of the totipotent zygote, the pluripotent inner cell mass of the blastocyst, and multipotent pri-mordial germ layers of the gastrula, ultimately leading to fetal organogenesis and growth. Even in adult life, resident adult stem cells participate in organ renewal, and their dysfunction leads to organ degeneration and aging. The ability to culture and differentiate pluripotent stem cells in vitro has provided a wealth of resources for the investigation of basic mechanisms of human devel-opment at cellular and molecular levels. Indeed, stem‐cell‐based models now exist that can detect compounds that can cause birth defects (teratogens), and these models can also be used to explore mechanisms whereby teratogens exert their effects. Furthermore, the use of induced pluripotent stem cells has led to numerous “disease‐in‐a dish” models of human genetic disorders. In addition, induced pluripotent stem cells can also serve as platforms for person-alized medicine since individual patient genomes are retained in these cells.

This volume Stem Cells in Birth Defects Research and Developmental Toxicology contains material contributed by forward‐looking scientists who work at the interface of stem cell research and applied science with the aim to improve human fetal safety and the understanding of human developmental and degenerative disorders. This volume is unique in that it considers both in vitro uses of stem cells as platforms for teratology and also “stem cellopathies,” which are human developmental and degenerative disorders that are caused by harmful impacts to resident adult stem cells in vivo.

This volume harbors a wealth of information of interest to a variety of pro-fessionals and interested individuals who share interest in human developmen-tal biology and fetal safety. Such individuals include academic researchers who have interest in developmental biology, stem cell research, teratology, toxicol-ogy, and human degenerative disorders. In addition, this volume is appropriate for clinicians, including those in the fields of obstetric medicine, genetic

Preface

Prefacexx

counseling, and environmental safety. Also, those who are interested in neurodegenerative diseases and alcohol abuse will find novel content. Finally, those interested in safety assessment of developmental pharmaceuticals will find useful content on expedient cell culture approaches that can supplement the use of animal teratogen testing, leading to faster assessments of compound safety and a reduction in the use of animals for pharmaceutical assessment.

The book is organized into four parts: (i) an introduction, which provides an overview of basic stem cell function and applications, (ii) a section on the use of stem cells for in vitro assays to detect suspected teratogens, (iii) a section on human “stem cellopathies,” and (iv) a section on current technical innovation in stem cell bioassays.

Part II considers in vitro stem cell platforms for the detection and assessment of teratogenic compounds. The initial chapter in Part II (Xing et al.) contains an excellent overview of existing regulatory agencies and approved animal test-ing procedures, followed by a discussion of stem cell approaches that are of higher throughput than animal assays, thus leading to the ability to assess the large number of compounds of suspected teratogenicity, which cannot be quickly assessed by animal use alone. This section also includes a summary of embryoid body approaches (Flamier), and a unique chapter that outlines a sys-tem using embryoid bodies that can undergo exquisite morphological changes that recapitulate early postimplantation development (Marikawa). The chapter concludes with content on the embryonic stem cell test (EST) for teratogen detection (Buck and zur Nieden).

Part III is devoted to human degenerative disorders mediated by impacts on adult stem cells (“stem cellopathies”). This section is heavily focused on neuro-degenerative disorders and contains two chapters on the modeling of human degenerative disorders in vitro using pluripotent stem cells: One chapter (Chandrasekaran et al.) provides a comprehensive account of strategies to pro-duce a range of neural cell types by directed differentiation, and the second (Joshi et  al.) describes the use of pluripotent cells to model Parkinson’s and Huntington’s diseases in vitro and considers the involvement of heavy metals and pesticides in neurodegeneration. The final three chapters in Part III focus on chemical impacts upon endogenous fetal and adult stem cells in humans. Chapters 8 and 9 are focused on the effects of ethanol exposure. Chapter 8 (Olsufka et al.) considers the impacts of alcohol use on adult neural stem cells and adult neurogenesis. Chapter 9 provides a novel perspective on fetal alcohol spectrum disorder (FASD) as a “stem cellopathy” syndrome. Finally, an inter-esting chapter by Shamilov and Aneskievich is focused on replacement of cells of the skin and disorders of the skin that are caused by chemical insults to dermal stem and progenitor cells.

Part IV focuses on technological development. Recent advances in stem cell research and developmental biology are yielding increasingly innovative and sophisticated stem cell culture approaches, which can now be combined with

Preface xxi

bioengineering (Fernandes and Cabral). In stem cell research, it has been difficult to produce hepatocytes that contain full metabolic activities that par-ticipate in toxicological responses, but a cell line with stem‐cell‐like properties (HepaRG cells) that have better metabolic activity is described (Guillouzo and Guguen‐Guillouzo).

Storrs, Connecticut, 2018 Theodore P. RasmussenUniversity of Connecticut

1

Part I

Introduction and Overview

Stem Cells in Birth Defects Research and Developmental Toxicology,First Edition. Edited by Theodore P. Rasmussen.© 2018 John Wiley & Sons, Inc. Published 2018 by John Wiley & Sons, Inc.

3

1

1.1 Stem Cell Types and Basic Function

All organisms with body plans organized into specialized organs containing tissue-specific cell types rely upon the action of stem cells in order to produce their adult body plans over the course of development. In placental mammals, the process of development commences upon fertilization of the egg to yield the zygote, a single cell with a fixed genome from which all subsequent cells arise over the course of development. The zygote is imbued with the property of totipotency; i.e. it has the potential to give rise to all cells of the conceptus, including cells of the developing embryo proper as well as cells of the embry-onic component of the placenta. After a short series of rapid cleavage‐stage cell divisions, the morula forms, in which all cells are still totipotent. Morula cells then execute the first asymmetric cell division, resulting in daughter cells that are a component of either the inner cell mass (ICM), which subsequently con-tribute to the embryo proper, and trophectodermal cells, which give rise to the embryonic component of the placenta. Thus, even in the initial stages of mam-malian embryogenesis leading to the blastocyst (3.5 days after fertilization in mice and 5.5 days in humans), cells begin with maximal developmental poten-tial (totipotency), which is then restricted during a single‐key asymmetric cell division culminating in the production of pluripotent cells of the ICM and multipotent cells of the trophectoderm. This most basic example illustrates two key features of stem cells in vivo: (i) the capacity for self‐renewal without loss of developmental potentiality and (ii) the capacity to execute carefully

The Basics of Stem Cells and Their Utility as Platforms to Model Teratogen Action and Human Developmental and Degenerative DisordersBindu Prabhakar1, Soowan Lee1, and Theodore P. Rasmussen1,2,3

1 University of Connecticut, Department of Pharmaceutical Sciences, 69. North Eagleville Road, Unit 3092, UConn Sch of Pharm, Storrs, CT, 06269, USA2 University of Connecticut Stem Cell Institute, 400 Farmington Avenue, Farmington, CT 06033, USA3 University of Connecticut, Institute for Systems Genomics, Storrs, CT 06269, USA

1 The Basics of Stem Cells and Their Utility as Platforms to Model Teratogen Action4

regulated asymmetric cell divisions resulting in two differing types of cells each with distinct developmental trajectories.

These two features (self‐renewal and asymmetric cell division) are the defin-ing features of all stem cells, in both the developing embryo and adult tissues. However, in postimplantation development asymmetric cell divisions of stem cells are of two types: (i) Asymmetric cell divisions that yield two new cell types, both of which have lineage‐restricted developmental potential and (ii) asymmetric cell divisions that yield a replacement daughter stem cell similar to the parental cell and a differentiated cell that is committed to differentiation. This second type of stem cell division is common in adult tissues and organs and is responsible for the establishment and maintenance of a pool of resident adult stem cells that serve to renew and replenish the organ with new cells that replace those lost to aging, degeneration, and injury.

Embryonic stem cells (ESCs) are pluripotent cells that are derived from the ICM of blastocyst‐stage embryos and are becoming increasingly employed in cell culture systems that are designed to assess compounds and conditions that affect development and cellular function. ESCs were first developed in 1981 from isolated mouse blastocysts (Evans and Kaufman, 1981). However, compa-rable human ESCs were not derived until 1998 (Thomson et al., 1998). The cells of the ICM are pluripotent, having the capacity to differentiate into the approximately 200 distinct cell types present in the adult mammal body plan, and this level of pluripotency is retained by ESCs. The ICM persists in the mammalian embryo for little more than a day in vivo, but culture conditions for ESC maintenance have been optimized such that this pluripotent state can be maintained indefinitely during ESC culture. This is because key signaling factors such as leukemia inhibitory factor (LIF, for mouse ESCs) and basic fibroblast growth factor (bFGF, for human ESCs) have been discovered, and these maintain the pluripotent state of ESCs indefinitely during culture in vitro. In addition, ESCs are conditionally immortal in culture since they can divide indefinitely without loss of pluripotency. Thus, ESCs exist in a state of sus-pended stasis with regard to their pluripotentiality, which is only a transient state in vivo. Upon removal of LIF or bFGF, ESCs spontaneously differentiate in vitro to form cells of endodermal, ectodermal, and mesodermal lineages, reminiscent of the process of gastrulation in their in vivo counterparts. Upon differentiation, conditional immortality is lost and the differentiated cells eventually senesce. In the last decade, widespread advances have been realized that allow the directed differentiation of ESCs to specific terminal cells types derived from each of the three principal germ layers, but the initial steps of each of these individual directed differentiation procedures commence with the removal of factors that support pluripotency, leading to the formation of definitive endodermal, ectodermal, or mesodermal cells.

In addition to ESCs, it is now possible to produce induced pluripotent stem cells (iPSCs) from terminally differentiated somatic cells. Similarly to ESCs,

1.1 Stem Cell Types and Basic Function 5

iPSCs have the ability to differentiate into numerous distinct cell types but can be cultured indefinitely under appropriate conditions. In the iPS process, ter-minally differentiated cells are reprogrammed to a pluripotent state by the forced expression a key set of powerful transcription factors that normally function in the ICM. In 2006, Takahashi and Yamanaka successfully repro-grammed mouse fibroblasts to generate embryonic‐like state by virally express-ing four core pluripotency reprogramming factors: Oct3/4, Sox2, Klf4, and c‐Myc (Takahashi and Yamanaka, 2006). The resulting iPSCs are functionally equivalent to ESCs. In the following year, they showed that the same four fac-tors could be used to produce iPSCs by the reprogramming of adult human fibroblasts (Takahashi et al., 2007). Two of these transcription factors (OCT4 and SOX2), together with NANOG, are especially important as they regulate the expression of hundreds of genes in pluripotent cells. When OCT4 and SOX2 are expressed in fibroblasts or other differentiated cells, they “boot up” the expression of a large set of embryonic genes, leading to the reprogramming of somatic cells to pluripotent cells. Since the same transcription factors can successfully reprogram both mouse and human fibroblasts to pluripotency, this finding shows that two divergent mammalian species (human and mouse) contain conserved sets of transcription factors that govern the pluripotent state. Subsequently, iPS approaches have been used to produce iPSCs from other species including rhesus monkey (Liu et  al., 2008), pig (Esteban et  al., 2009), and rat (Coppiello et  al., 2017, Hamanaka et  al., 2011, Li et  al., 2009, Zhou et al., 2011). In addition, many types of somatic cells can be reprogrammed including nucleated cord blood cells (Haase et al., 2009) epithelial cells from the urinary tract (Zhou et al., 2011), and many others. iPS technology allows the facile production of pluripotent cells from fibroblasts or nucleated peripheral blood cells, which bypasses the need for embryos for the derivation of pluripo-tent cells. Importantly, iPS technology works well on human cells, and human iPSCs retain the entire genome of the human somatic cell donor, thus allowing the creation of patient‐specific iPSC lines. This approach opens the door to personalized medicine approaches in which individual patient genomes (with an idiosyncratic set of patient‐specific genetic variations) can be used to test for individualized responses to drug activation and susceptibility to toxicological effects caused by genetically altered drug metabolism.

Adult stem cell populations exist in most if not all adult organs and serve to replenish cells lost to aging and damage. However, adult stem cells have in general proven to be much more difficult to isolate and culture in vitro (with the notable exceptions of hematopoietic and mesenchymal stem cells). In addi-tion, adult stem cells are tissue specific and typically are limited in their dif-ferentiation abilities, as they can usually only produce the terminally differentiated cell types present within a specific organ. Thus, adult stem cells are considered multipotent. In summary, developmental potentiality in vivo becomes progressively restricted, transiting from totipotent (the zygote and

1 The Basics of Stem Cells and Their Utility as Platforms to Model Teratogen Action6

morula) to pluripotent (the ICM) to multipotent (adult stem cells), and finally unipotent (terminally differentiated cells), which often become postmitotic as a final stage.

1.2 Pluripotency

Pluripotency is a developmental biology term that describes the breadth of developmental potential of cells of the blastocyst inner cells mass. ESCs and iPSCs have a highly similar level of pluripotency. This is exemplified by the finding that individual mouse ESCs and iPSCs can completely contribute to embryogenesis in vivo after transfer into tetraploid host blastocysts, which are then implanted into pseudopregnant surrogate female mice (Kang et al., 2009). In this embryological method, the host tetraploid blastocyst (which is made artificially tetraploid) supports the engrafted ESCs or iPSCs resulting in the fetal development and live birth of pups entirely derived from ESCs or iPSCs. To date, this achievement provides the best proof that ESCs and iPSCs have pluripotency comparable to that of normal embryonic ICM cells. In addition, a vast body of work from the field of stem cell research has shown that directed differentiation approaches can be devised that guide pluripotent cell differen-tiation in vitro to a wide variety of terminal cell types.

In the case of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), proof of pluripotency in vivo is not possible due to medical ethics, but hESCs and hiPSCs have been successfully differenti-ated to a wide variety of terminal cellular types, perhaps even more numerous than has been achieved with mouse ESCs. In addition, numerous “omics” reports have shown that the transcriptome of hESCs and hiPSCs are very simi-lar to native human ICM cells that the epigenome of hESCs and hiPSCs are also very similar to native human ICM. Finally, detailed and comprehensive comparisons between human and mouse pluripotent cells have shown a high degree of similarity in terms of their relative transcriptomes and epigenomes, though notable differences exist, probably due to divergent evolution resulting in species‐specific idiosyncrasies. Overall, the transcriptional and epigenomic state of mouse and human ICM cells, ESCs, and iPSCs share a high degree of concordance, and key pathways that collude to maintain the pluripotent state are shared by all these cells.

1.2.1 Poised Chromatin of the Pluripotent Epigenome

The configuration of the epigenomes is key for the maintenance of the pluripo-tent state and serves as a framework to establish transcriptional states. Pluripotent cells contain developmentally poised chromatin, which is exqui-sitely assembled upon the genome with a level of precision that marks key