Stem Cells in Birth Defects Research and Developmental Toxicology E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 702

Veröffentlichungsjahr: 2018

Beschreibung

This book 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. * Provides important platforms and contemporary accounts of the state of stem cell research in the fields of toxicology and teratology * Considers both in vitro uses of stem cells as platforms for teratology and also stem cellopathies, which are in vivo developmental and degenerative disorders * Helps the pharmaceutical industry and safety and environmental authorities validate the status quo of in vitro toxicity test systems based on human pluripotent stem cells and their derivatives

Leseprobe

Table of Contents

Cover

List of Contributors

Preface

Part I: Introduction and Overview

Chapter 1: The Basics of Stem Cells and Their Utility as Platforms to Model Teratogen Action and Human Developmental and Degenerative Disorders

1.1 Stem Cell Types and Basic Function

1.2 Pluripotency

1.3

In vitro

Uses of Pluripotent Cells

1.4 Adult Stem Cells

In vivo

1.5 Emerging Trends in Stem Cell Culture

1.6 Future Directions

References

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

Chapter 2: Stem Cells and Tissue Engineering Technologies for Advancing Human Teratogen Screening

Abbreviations

2.1 Introduction

2.2 Current DART Regulatory Guidelines and Methods

2.3

In vitro

Animal‐Based Models for Developmental Toxicity Testing

2.4

In vitro

Stem‐Cell‐Based Developmental Toxicity Models

2.5 Conclusion and Future Directions

References

Chapter 3: Use of Embryoid Bodies for the Detection of Teratogens and Analysis of Teratogenic Mechanisms

3.1 Embryoid Body Assays: Background

3.2 Detection of Teratogens Using EBs

3.3 Teratogenic Mechanisms

Acknowledgments

References

Chapter 4: Stem‐Cell‐Based

In vitro

Morphogenesis Models to Investigate Developmental Toxicity of Chemical Exposures

4.1 Introduction

4.2 Stem‐Cell‐Based

In vitro

Morphogenesis Model

4.3 Future Directions: Enhancing Morphogenesis‐Based Assays

4.4 Concluding Remarks

Acknowledgment

References

Chapter 5: Risk Assessment Using Human Pluripotent Stem Cells: Recent Advances in Developmental Toxicity Screens

5.1 Introduction

5.2 Animal Embryo Studies to Evaluate Developmental Toxicity

5.3 Usage of Mouse Embryonic Stem Cells in Developmental Toxicity

5.4 Alternative Endpoint Read‐Out Approaches in the EST

5.5 Novel Methods and Protocols to Replicate Human Development

5.6 Future Applications

Acknowledgments

References

Part III: Human Developmental Pathologies Mediated by Adult Stem Cells

Chapter 6: Modeling the Brain in the Culture Dish: Advancements and Applications of Induced Pluripotent Stem‐Cell‐Derived Neurons

6.1 Introduction

6.2 Methods to Generate Patient‐Derived Neurons

6.3 Neuronal Induction from Fibroblasts and hiPSCs

6.4 Cerebral Organoids: Neural Modeling in Three Dimensions

6.5 Epigenetic Considerations in hiPSC Donor Cell Choice

6.6 Aging Neurons

6.7 Drug Testing Using hiPSCs

6.8 Promises in the Field

6.9 Concluding Remarks

References

Chapter 7: Modeling Genetic and Environment Interactions Relevant to Huntington's and Parkinson's Disease in Human Induced Pluripotent Stem Cells (hiPSCs)‐Derived Neurons

7.1 Gene–Environment Interactions Assessed in hiPSC‐Derived Neurons

7.2 Modeling of Neurological Diseases with hiPSCs

7.3 Cell Viability Assays

7.4 Mitochondria

7.5 Oxidative Stress

7.6 Neurite Length by Immunocytochemistry (ICC)

7.7 Conclusions

References

Chapter 8: Alcohol Effects on Adult Neural Stem Cells – A Novel Mechanism of Neurotoxicity and Recovery in Alcohol Use Disorders

8.1 Introduction

8.2 The “Birth” of the Study of “Neuronal Cell Birth”

8.3 Components of Adult Stem‐Cell‐Driven Neurogenesis

8.4 Alcohol Effects on Adult Neural Stem Cells and Neurogenesis

8.5 Extrinsic Factors Influence the Neurogenic Niche

8.6 Alcohol and the Niche

8.7 Conclusions

References

Chapter 9: Fetal Alcohol Spectrum Disorders: A Stem‐Cellopathy?

9.1 Fetal Alcohol Spectrum Disorders

9.2 Stem Cells

9.3 Endoderm

9.4 Mesoderm

9.5 Ectoderm

9.6 Future Directions

9.7 Conclusion

References

Chapter 10: Toxicological Responses in Keratinocyte Interfollicular Stem Cells

10.1 Epidermal Keratinocyte Stem Cells

10.2 Arsenic

10.3 Dioxin

10.4 Bacterial Toxins

10.5 Conclusions and Prospective Considerations

References

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

Chapter 11: Stem‐Cell Microscale Platforms for Toxicology Screening

11.1 Introduction

11.2 Stem Cell Models for Toxicology Assessment

11.3 Biomimetic Microscale Systems for Drug Screening

11.4 Microtechnologies for Drug Discovery

11.5 Devices for High‐Throughput Toxicology Studies

11.6 Cellular Microarray Platforms

11.7 Microfluidic Platforms

11.8 Conclusions and Future Perspectives

Acknowledgments

References

Chapter 12: HepaRG Cells as a Model for Hepatotoxicity Studies

12.1 Introduction

12.2 Characteristics of HepaRG Cells

12.3 Biotransformation and Detoxification Activities

12.4 Toxicity Studies

12.5 Conclusions and Perspectives

Acknowledgments

References

Index

End User License Agreement

List of Tables

Chapter 02

Table 2.1 Current animal‐based DART testing guidelines and study methodologies.

Table 2.2

In vitro

animal‐based models for developmental toxicity testing.

Table 2.3 Current major

in vitro

stem cell‐based teratogen screening assays.

Chapter 10

Table 10.1 Expressed markers and functional characteristics for recognizing or preferentially isolating interfollicular KCs of differing mitotic reserve.

List of Illustrations

Chapter 01

Figure 1.1

Derivation and use of pluripotent cells for

in vitro

teratology and toxicology applications

. ESCs are derived from the ICM of blastocyst‐stage embryos (mouse or human) and a pluripotent. Alternatively, iPSCs can be produced by factor‐mediated reprogramming of terminally differentiated somatic cells such as fibroblasts. iPSCs are also pluripotent. Pluripotent cells (ESCs or iPSCs) can be differentiated into EBs for modeling of early development and used to detect teratogens or study human genetically specified birth defect mechanisms (provided that iPSCs containing relevant genetic developmental mutations are used). In addition, both ESCs and iPSCs can be subjected to directed differentiation to detect chemically induced impacts on specific cell types. Finally, patient‐specific iPSCs can be used to detect and study individualized pharmacogenomic responses to pharmaceuticals (in terms of efficacy or toxicity).

Figure 1.2

Adult (endogenous) stem cells and developmental toxicity

. An emerging theme is that some human disorders occur due to impacts upon endogenous adult stem cells (“stem cellopathies”). Adult stem cells arise during organogenesis, and if they are compromised during gestation, birth defects and developmental disorders that play out later in life can occur. In addition, adult stem cells are required for tissue homeostasis and organ regeneration, and insults to adult stem cell populations in the adult can also lead to human disorders, impacting a variety of organs. Successive restriction in developmental potency (from totipotency to multipotency) of endogenous stem cells and major landmarks in human development and maturation are shown.

Chapter 02

Figure 2.1

A representative experiment protocol of the ZEDT assay

.

Figure 2.2

Microfabrication technologies to immobilize and perform phenotypic teratogen screening on animal embryos

. (a) Immobilization of chick embryos in a porous microstructure made up of PDMS. (b) Engineered microfluidic systems to dock zebrafish embryos to perform teratogen screening under continuous perfusion culture conditions. (c) A microfabricated embryo‐trapping array to trap multiple embryos within a single microfluidic platform and perform simultaneous high‐throughput phenotypic screening of zebrafish embryos under teratogen treatment. (d) A concentration gradient generator (CGG)‐based microfluidic device to screen the dose‐dependent effect of teratogen on embryo growth on a single microfluidic device, (e) Images displaying the phenotypic dose‐dependent effect of teratogen, valproic acid, on the zebrafish embryo cultured on a microfabricated platform.

Figure 2.3

Cardiac differentiation in the mEST

. (a) Undifferentiated mESCs. (b) EB formation using the hanging drop method. (c) Day 5 EB in suspension culture. (d) EB outgrowth on day 10 of differentiation. The center region was where the beating cardiomyocytes located.

Figure 2.4

The mesoendoderm pattern formation by spatially and temporally controlled hPSC differentiation and migration using the micropatterning technique

. (a) Schematic representation of the μP‐hPSC colony mesoendoderm induction. (b) Phase and fluorescent images of the differentiating μP‐hPSC colonies from day 1 to day 3. T: mesoendoderm marker Brachyury. Scale bar: 200 μm. (c) A montage showing the 3‐day phase imaging on a quarter section of the μP‐hPSC colony. Scale bar, 100 μm.

Source:

Adapted from Xing et al. (2015).

Chapter 03

Figure 3.1

Workflow and considerations for EB analysis of teratogens

. EBs are assembled and differentiated by sequential steps beginning with pluripotent cell culture, aggregation of pluripotent cells, growth and differentiation (during which time suspected teratogens can be added, followed by assays using a variety of endpoints). These steps and technical considerations at each step are shown here.

Figure 3.2

Varieties of EB compromise upon exposure to compounds

. EBs can be affected in a variety of ways upon exposure to compounds, and each of these can be used as quantitative endpoints to determine EB assay outcomes. Effects observed in EBs dosed with compounds include growth retardation, growth arrest, morphological defects, and altered cellular content.

Chapter 04

Figure 4.1

Production and analysis of P19C5 Embryoid Bodies

. (a) Hanging drop culture method to generate embryoid bodies (EBs) with P19C5 stem cells. (Top) Drops (20 μL each) of cell suspension are spotted on the inner surface of the lid of a Petri dish. (Bottom) The lid is flipped and placed over the bottom part of the Petri dish filled with phosphate‐buffered saline to maintain humidity. (b) Time course of morphogenesis of P19C5 EBs. EBs are removed from hanging drops and placed together for photographing. Scale bar = 1 mm. (c) Whole‐mount

in situ

hybridization of Day 4 P19C5 EBs for developmental regulator genes. An arrowhead points to the tight expression of

Wnt3a

. Expressions of

Cdx2

and

Wnt3a

(markers for the posterior end of the normal embryo at E8.5) are restricted to the broader side of the EB, whereas

Meox1

(a marker of somitic mesoderm, which forms more anteriorly) localized to the opposite side of the EB. (d) A schematic diagram, depicting dynamic temporal changes in expression levels of developmental regulator genes. (e) Tracing of the EB circumference using the ImageJ program for morphometric analysis. (Left) Before trace. (Right) After trace with the Polygon Selection tool, showing yellow lines connected with white nodules. (f) Temporal changes in the morphometric parameters of P19C5 EBs. Average values ±95% confidence intervals are shown (

n

= 44).

Figure 4.2

Chemical Exposure of P19C5 Embryoid Bodies

. (a) Images of Day 4 P19C5 EBs that have been cultured with specific chemical exposures: acitretin (0.1 μM), 5‐fluorouracil (1 μM), and ribavirin (1 μM). (b) Images of control Day 4 EBs and those that have been treated with valproic acid (VPA) at 0.6 mM. (c) A model of the molecular mechanisms by which VPA exerts teratogenic actions. HDAC (histone deacetylase), RA (retinoic acid), RAR (retinoic acid receptor), and Ac (acetylated). (d) Time course of morphogenesis of an EB made from human embryonic stem cells (hESCs; H9 line). (e) A group of hESC EBs at Day 4, showing consistency of elongation morphogenesis. Scale bars in (d) and (e)=500 μm. (f) Temporal changes in the morphometric parameters of hESC EBs. Average values ±95% confidence intervals are shown (

n

= 10). (g) Immunohistochemistry for BRACHYURY protein, showing localized expression (arrowhead) in a Day 4 hESC EB.

Chapter 05

Figure 5.1

Schematic depiction of potential uses of stem cells for toxicity assessment

. PSC, pluripotent stem cell; SC, stem cell.

Chapter 06

Figure 6.1

(a) Overexpression of ASCL1, NURR1, and LMX1A generates ~4% pure population of dopaminergic neurons; TuJ1 is a neuronal marker, while TH is a key enzyme in dopaminergic neurons

. (b)

NGN2

overexpression alone rapidly induces iPSCs and ESCs into a glutamatergic fate with near 100% efficiency; cells overexpressing

NGN2

fluoresce green and demonstrate neuronal morphology. (c) Coexpression of two miRNAs and striatal‐enriched transcription factors generate medium spiny neurons from fibroblasts in 1 month; DAPI labels nuclei, while TUBB3 labels neurons and DLX5 labels medium spiny neurons.

Figure 6.2

Illustration of the main steps to derive cerebral organoids from hiPSCs

.

Figure 6.3

Characteristics of aging including cell senescence, inflammation, telomere shortening and metabolic and epigenetic changes can compromise the reprogramming efficiency of aged somatic cells into hiPSCs

. Inhibiting targets that show promise in reprogramming and rejuvenation efficacy may offer additional methodological avenues for hiPSC‐aging.

Figure 6.4

Scientists have ventured into the realm of transplantation‐based therapies using autologous iPSC dopamine neurons derived from the Cynomolgus monkey for a non‐human primate model of Parkinson’s disease

. These neurons provided long‐term functional recovery and survived up to 2 years, reinnervating the host brain. This success has motivated further studies into hiPSC‐based transplantation therapies as viable treatment options for neurodegenerative diseases.

Figure 6.5

As research using hiPSCs advances, scientists hope to address key aspects currently challenging the creation of meaningful patient‐specific in vitro models to study brain disorders

.

Chapter 07

Figure 7.1

hiPSC‐derived mesencephalic dopaminergic (DA) neurons

. Floor plate lineage cells are differentiated from hiPSCs as described (Kumar et al., 2014; Kriks et al., 2011) with a majority expressing lineage‐selective floor plate markers Foxa2 and Lmx1A by day 11 of differentiation. At day 11, the floor plate cells start undergoing final neural maturation and by day 27 of differentiation these neurons express the neuron‐specific marker β3‐tubulin (in red), and the dopamine neuronal marker tyrosine hydroxylase (in green). Cultures were counterstained with Hoechst (in blue), a DNA stain.

Figure 7.2

hiPSC‐derived cortical neurons at day 53 stained with MitoTracker Red CMXRos (orange) and counterstained with Hoechst (blue)

. MitoTracker Red CMXRos is a red fluorescent mitochondrial fixable dye. The accumulation of this stain is dependent on the membrane potential of cells. In this image, there are distinct mitochondrial networks for cortical neurons. With exposure to 300 μM CuSO

4

for 24 h in cortical maintenance media (Shi et al., 2012a), there is fragmentation of the mitochondria and the intricate mitochondrial networks are disrupted.

Figure 7.3

Images of hiPSC‐derived control (a) and toxicant‐treated (d) dopaminergic (DA) neurons stained for β3‐tubulin, tyrosine hydroxylase, and counterstained with Hoechst were acquired using a Molecular Device's ImageXpress Micro XL system

. β3‐tubulin‐positive neurites of control (b) and toxicant‐treated (e) neuronal cultures were quantified using the neurite length module in MetaXpress software. The traced neurites and associated nuclei of control (c) and toxicant exposed (f) neurons are shown as a red overlay.

Chapter 08

Figure 8.1

Sites of constitutive, adult neurogenesis

. Cartoon of a sagittal section of rodent brain showing the two well‐accepted neurogenic niches, the hippocampal dentate gyrus subgranular zone and subventricular zone of the anterior lateral ventricles. Insets show the types of progenitor cells and typical markers used to identify subsets of progenitors as they mature in the subgranular zone (SGZ; left box) and subventricular zone (SVZ; right box). In the SGZ, Type‐1 neural stem cells divide asymmetrically to produce a daughter Type‐2a cell and self‐renew. Type‐2a cells divide symmetrically and can expand the pool in response to a variety of environmental stimuli. Cells become progressively more fate restricted and mature into a granule cell neuron, which is then postmitotic. In the SVZ, Type‐B cells divide asymmetrically to produce daughter transit‐amplifying Type‐C cells, which may divide and also mature into neuroblasts. Neuroblasts then migrate through the rostral migratory stream to the olfactory bulb where cells terminally differentiate into granule cell neurons primarily. (LV, lateral ventricle; RMS, rostral migratory stream; OB, olfactory bulb.)

Chapter 09

Figure 9.1

Alcohol exposure can affect all stages of early stem cell development

. Preconception exposure to alcohol, both maternal and paternal, can affect DNA expression and cellular patterning. After zygote formation, the totipotent morula is formed. The morula further differentiates into two stem cell populations beginning with the blastocyst in which the outer, multipotent, layer goes on to make the placenta, while the inner cell mass of pluripotent stem cells goes on to make the embryo. From there, the inner cell mass grows and a primitive streak forms, through which cellular migration and differentiation occur to form a gastrula containing the three embryonic cell lineages: the endoderm, mesoderm, and ectoderm.

Figure 9.2

The three lineages contained in the gastrula make all of the unique tissues of the body

.

Figure 9.3

Future directions for research in the effects of prenatal alcohol on stem cell biology include the contributions of genetic sex and hormonal milieu, effects on the prenatal formation of resident, lifelong stem cells, and the possibility for stem cell directed therapies including stem cell transplantation

.

Chapter 10

Figure 10.1

Tiered layers of keratinocyte replication and differentiation in the epidermis

. The epidermis is composed of four keratinocyte (KC) strata: basal layer bound by integrin α/β dimers to the basement membrane (BM), located above the dermis; spinous layer composed of postmitotic (PM) cells expressing early differentiation markers such as involucrin and the keratin 1 and 10 pair (K1/10); granular layer expressing late‐differentiation markers such as loricrin and filaggrin; cornified layer composed of enucleated cells (squames) shed to the environment. The most common regeneration modeling posits KC stem cells (SC) that replicate to form transit (also known as transient) amplifying (TA) cells, which undergo limited rounds of replications and ultimately produce PM cells which stratify and differentiate. Delta /Notch1 interactions on adjacent cells (trans interaction) promote the cessation of cell cycling and postmitotic, early differentiation gene expression.

Figure 10.2

Comparison of epidermal KC regeneration models

. Two different models have been described for KC replication and daughter cell progression to PM status. The hierarchal model (a) describes stem cells (SC) having the capability to replicate into either daughter SC or transit (also known as transient) amplifying (TA) cells. TA cells give rise to daughter TA cells for a limited number of cycles and then ultimately PM cells that continue through terminal differentiation (see Figure 10.1). The stochastic model (b) describes the KC replicating population as common progenitor (CP) cells capable of giving rise to daughter CP or PM cells, the latter differentiating as in the hierarchal model. Underlying the CP model is one population of variably mitotically competent KCs able to interconvert between classes of replication (Roshan

et al

.,2016). A balanced replication mode supports KC renewal and replacement under homeostasis versus an expanding replication mode where production of cycling KCs is favored to fill tissue deficits such as during wound healing.

Chapter 11

Figure 11.1

Replica molding using soft lithography

. The process for shaping a pliable polymer material such as PDMS starts by using a microsized frame or model called master mold. The mold is filled with a prepolymer, which is cross‐linked, or cured, and the resulting polymer is peeled off the mold. The master mold can then be recycled to produce multiple copies of the final object.

Figure 11.2

In robotic spotting, a pin or tip controlled by a robotic arm is filled with a solution of interest, and then dispenses the desired material at designated locations of the array surface

. The use of computer‐assisted robotic apparatus enables to customize the resulting microarrays, because it is possible to program the printing of different combinations of materials and dispensing locations.

Figure 11.3

Schematics of the micropillar chip/microwell array system

. The micropillar chip contains cells encapsulated in 3D Matrigel droplets, while the microwell chip contains the compounds to be tested. Stamping of the micropillar chip onto the microwell array allows for testing drug‐induced toxicity. This figure was produced using Servier Medical Art.

Figure 11.4

Schematics of a microfluidic culture device for high‐throughput screening of toxicity effects in stem cells or stem‐cell‐derived tissues

. Typically, a microfluidic platform allows for multiparameter analysis of different concentration gradients due to transport phenomenon properties within microfluidic channels. The microfluidic unit can also be coupled with time‐lapse microscopy and automated operation. This design allows, for example, the tracking of cell proliferation, morphology, and viability. This figure was produced using Servier Medical Art.

Chapter 12

Figure 12.1

Schematic representation of the HepaRG cell line behavior

. Each differentiation process constitutes a maturation program of normal hepatocytes.

Figure 12.2

Morphology of HepaRG cells

. (a) Phase‐contrast microscopic appearance of proliferating bipotent progenitors. (b) Phase‐contrast microscopic appearance of HepaRG cells after differentiation: hepatocyte‐like colonies are surrounded by undifferentiated biliary cells. (c) Immunolabeling of the junctional zonula occludens protein (green); localization of this protein is restricted to bile canaliculi. (d) Fluorescent microscopy showing accumulation into bile canaliculi of carboxydichlorofluorescein diacetate (a fluorescent substrate of MRP2) after 30 min incubation. Bar = 100 μm.

Figure 12.3

Induction of cholestasis, steatosis, and phospholipidosis in HepaRG cells

. (a) Dilatation of bile canaliculi after 2 h treatment with the cholestatic drug flucloxacillin at 4 mM (arrow). (b) Intracytoplasmic accumulation of lipids following Oil Red O staining (red) and unstained vesicles after treatment with 20 μM amiodarone for 24 h. Unstained vesicles corresponding to lamellar bodies are visible in both hepatocyte‐like and biliary‐like cells (arrow). Bar = 100 μm.

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Stem Cells in Birth Defects Research and Developmental Toxicology

Edited by Theodore P. Rasmussen

University of Connecticut Storrs, Connecticut, USA

Copyright

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: Wiley

Cover 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

List of Contributors

Schahram Akbarian

Icahn School of Medicine at Mount Sinai

Department of Neuroscience

New York, NY 10029

USA

Icahn School of Medicine at Mount Sinai

Friedman Brain Institute

New York, NY 10029

USA

Icahn School of Medicine at Mount Sinai

Department of Psychiatry

New York, NY 10029

USA

Brian J. Aneskievich

University of Connecticut

Department of Pharmaceutical Sciences

69 North Eagleville Road, U‐3092 Storrs, CT 06269

USA

Aaron B. Bowman

Vanderbilt University (VU)

Department of Pediatrics and Neurology, Vanderbilt University Medical Center (VUMC)

1161 21st Avenue South, D‐4105 MCN, Nashville, TN 37232

USA

Vanderbilt University (VU)

Department of Biochemistry and Vanderbilt Brain Institute

465 21st Avenue South, 6140 MRB3 Nashville, TN 37232

USA

Kristen Brennand

Icahn School of Medicine at Mount Sinai

Department of Neuroscience

New York, NY 10029

USA

Icahn School of Medicine at Mount Sinai

Friedman Brain Institute

New York, NY 10029

USA

Icahn School of Medicine at Mount Sinai

Department of Psychiatry

New York, NY 10029

USA

Icahn School of Medicine at Mount Sinai

Department of Genetics and Genomics

New York, NY 10029

USA

Kristen Buck

Department of Molecular, Cell and Systems Biology and Stem Cell Center, College of Natural and Agricultural Sciences

University of California Riverside

1113 Biological Sciences Building Riverside, CA, 92521

USA

Joaquim M. S. Cabral

Universidade de Lisboa

Department of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior Técnico

Avenida Rovisco Pais 1049–001, Lisbon

Portugal

Sandhya Chandrasekaran

Icahn School of Medicine at Mount Sinai

Department of Neuroscience

New York, NY 10029

USA

Friedman Brain Institute, Icahn School of Medicine at Mount Sinai

New York, NY 10029

USA

Icahn School of Medicine at Mount Sinai

Department of Psychiatry New York, NY 10029

USA

M. Diana Neely

Vanderbilt University (VU)

Department of Pediatrics and Neurology, Vanderbilt University Medical Center (VUMC)

1161 21st Avenue South, D‐4105 MCN, Nashville, TN 37232

USA

Vanderbilt University (VU)

Department of Biochemistry and Vanderbilt Brain Institute

465 21st Avenue South, 6140 MRB3 Nashville, TN 37232

USA

Tiago G. Fernandes

Universidade de Lisboa

Department of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior Técnico

Avenida Rovisco Pais 1049–001, Lisbon

Portugal

Annette S. Fincher

Texas A&M University

Department of Neuroscience and Experimental Therapeutics Texas A&M University Health Science Center

Bryan, TX 77807

USA

Anthony Flamier

University of Montreal

Maisonneuve‐Rosemont hospital

5415 Boul. l'Assomption, Montréal QC H1T 2M4

Canada

Christiane Guguen‐Guillouzo

Université de Rennes 1, Faculté des Sciences Pharmaceutiques et Biologiques

Inserm UMR 1241, Numecan

2 avenue Prof. Léon Bernard 35043 Rennes Cedex

France

Biopredic International

Parc d’Affaires de la Brétèche, Bât. A4, Saint Grégoire

France

André Guillouzo

Université de Rennes 1, Faculté des Sciences Pharmaceutiques et Biologiques

Inserm UMR 1241, Numecan

2 avenue Prof. Léon Bernard 35043 Rennes Cedex

France

Piyush Joshi

Vanderbilt University (VU)

Department of Pediatrics and Neurology, Vanderbilt University Medical Center (VUMC)

1161 21st Avenue South, D‐4105 MCN, Nashville, TN 37232

USA

Vanderbilt University (VU)

Department of Biochemistry and Vanderbilt Brain Institute

465 21st Avenue South, 6140 MRB3 Nashville, TN 37232

USA

Andrew Klopfer

Texas A&M University

Department of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science Center

Bryan, TX 77807

USA

Soowan Lee

University of Connecticut

Department of Pharmaceutical Sciences

69. North Eagleville Road, Unit 3092, UConn Sch of Pharm Storrs, CT 06269

USA

Amanda H. Mahnke

Texas A&M University

Department of Neuroscience and Experimental Therapeutics, Women's Health in Neuroscience Program, Texas A&M University Health Science Center

Medical Research and Education Building, 8447 State Highway 47 Bryan, TX 77807‐3260

USA

Yusuke Marikawa

University of Hawaii at Manoa

John A. Burns School of Medicine Institute for Biogenesis Research

651 Ilalo Street, Biosciences Building 163A, Honolulu, HI 96813

USA

Rajesh C. Miranda

Texas A&M University

Department of Neuroscience and Experimental Therapeutics Women's Health in Neuroscience Program, Texas A&M University Health Science Center

Medical Research and Education Building, 8447 State Highway 47 Bryan, TX 77807‐3260

USA

Jessica S. Newton

University of Kentucky

Department of Pharmaceutical Sciences

789 S. Limestone, Lee T. Todd Bldg. 473, Lexington, KY 40536

USA

Kimberly Nixon

University of Kentucky

Department of Pharmaceutical Sciences

789 S. Limestone, Lee T. Todd Bldg. 473, Lexington, KY 40536

USA

Rachael A. Olsufka

University of Kentucky

Department of Pharmaceutical Sciences

789 S. Limestone, Lee T. Todd Bldg. 473, Lexington, KY 40536

USA

Hui Peng

University of Kentucky

Department of Pharmaceutical Sciences

789 S. Limestone, Lee T. Todd Bldg. 473, Lexington, KY 40536

USA

Bindu Prabhakar

University of Connecticut

Department of Pharmaceutical Sciences

69. North Eagleville Road, Unit 3092, UConn Sch of Pharm, Storrs CT 06269

USA

Prashanth Rajarajan

Icahn School of Medicine at Mount Sinai

Department of Neuroscience

New York, NY 10029

USA

Icahn School of Medicine at Mount Sinai

Friedman Brain Institute

New York, NY 10029

USA

Icahn School of Medicine at Mount Sinai

Department of Psychiatry

New York, NY 10029

USA

Icahn School of Medicine at Mount Sinai

Department of Genetics and Genomics

New York, NY 10029

USA

Theodore P. Rasmussen

University of Connecticut

Department of Pharmaceutical Sciences

69. North Eagleville Road, Unit 3092, UConn Sch of Pharm, Storrs CT 06269

USA

University of Connecticut Stem Cell Institute

400 Farmington Avenue Farmington, CT 06033

USA

University of Connecticut, Institute for Systems Genomics

Storrs, CT 06269

USA

Geetika Sahni

National University of Singapore

Department of Biomedical Engineering

4, Engineering Drive 3, Block E4 #04‐10, Singapore 117583

Singapore

Nihal A. Salem

Texas A&M University

Department of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science Center

Bryan, TX 77807

USA

Rambon Shamilov

University of Connecticut

Department of Pharmaceutical Sciences

69 North Eagleville Road, U‐3092 Storrs, CT 06269

USA

Yi‐Chin Toh

National University of Singapore

Department of Biomedical Engineering

4, Engineering Drive 3, Block E4 #04‐10, Singapore 117583

Singapore

National University of Singapore

Singapore Biomedical Institute for Global Health Research and Technology

MD6, 14 Medical Drive, #14‐01 Singapore 117599

Singapore

National University of Singapore

NUS Tissue Engineering Program

27 Medical Drive, DSO (Kent Ridge) Building, #04‐01, Singapore 117510

Singapore

National University of Singapore Centre for Life Sciences

Singapore Institute for Neurotechnology

28 medical Drive, Singapore 117456

Singapore

Alexander M. Tseng

Texas A&M University

Department of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science Center

Bryan, TX 77807

USA

Jiangwa Xing

Qinghai University

Department of Basic Medical Sciences, Medical College

Xining, Qinghai, 810016

China

Nicole I. zur Nieden

University of California Riverside

Department of Molecular, Cell and Systems Biology and Stem Cell Center, College of Natural and Agricultural Sciences

1113 Biological Sciences Building Riverside, CA, 92521

USA

Preface

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 progressively restricted as development proceeds through the stages of the totipotent zygote, the pluripotent inner cell mass of the blastocyst, and multipotent primordial 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 development 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 personalized 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 professionals and interested individuals who share interest in human developmental biology and fetal safety. Such individuals include academic researchers who have interest in developmental biology, stem cell research, teratology, toxicology, and human degenerative disorders. In addition, this volume is appropriate for clinicians, including those in the fields of obstetric medicine, genetic 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 testing 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 system 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 neurodegenerative 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 produce 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 interesting 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 bioengineering (Fernandes and Cabral). In stem cell research, it has been difficult to produce hepatocytes that contain full metabolic activities that participate 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. Rasmussen

University of Connecticut

Part IIntroduction and Overview

Chapter 1The Basics of Stem Cells and Their Utility as Platforms to Model Teratogen Action and Human Developmental and Degenerative Disorders

Bindu Prabhakar1, Soowan Lee1, and Theodore P. Rasmussen1,2,3

1University of Connecticut, Department of Pharmaceutical Sciences, 69. North Eagleville Road, Unit 3092, UConn Sch of Pharm, Storrs, CT 06269, USA

2University of Connecticut Stem Cell Institute, 400 Farmington Avenue, Farmington, CT 06033, USA

3University of Connecticut, Institute for Systems Genomics, Storrs, CT 06269, USA

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 embryonic 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 contribute to the embryo proper, and trophectodermal cells, which give rise to the embryonic component of the placenta. Thus, even in the initial stages of mammalian embryogenesis leading to the blastocyst (3.5 days after fertilization in mice and 5.5 days in humans), cells begin with maximal developmental potential (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 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 defining 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, comparable 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 suspended 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, iPSCs have the ability to differentiate into numerous distinct cell types but can be cultured indefinitely under appropriate conditions. In the iPS process, terminally 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 reprogrammed mouse fibroblasts to generate embryonic‐like state by virally expressing 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 factors 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 pluripotent 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 addition, adult stem cells are tissue specific and typically are limited in their differentiation 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 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 differentiation 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 differentiated 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 similar 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 pluripotent state and serves as a framework to establish transcriptional states. Pluripotent cells contain developmentally poised chromatin, which is exquisitely assembled upon the genome with a level of precision that marks key genes for later developmental expression or silencing in appropriate cellular lineages. This is achieved in large measure by the presence of bivalent domains that are positioned throughout the genome on a gene‐by‐gene basis (Bernstein et al., 2006). Bivalent chromatin is unusual in that it contains marks in the histone code that designate specific genes for later expression or silencing, which unfolds in a tissue specific manner over the course of development. Bivalent marks occur on the core nucleosomal histone H3 and consist of the dual trimethylation of lysine residues H3 at positions 4 and 27 within H3 N‐terminal tails (H3K4me3 and H3K27me3). Individually, H3K4me3 specifies active transcription, while H3K27me3 specifies transcriptional silence. However, in pluripotent chromatin, these histone marks co‐occur, resulting in an epigenetic signature that is later resolved during ensuing development, as bivalently marked genes become monovalently marked by the maintenance of either H3K4me3 or H3K24me3 alone, resulting in gene activation or silencing, respectively. Bivalent domain resolution occurs on a tissue‐specific basis. For example, a neurally expressed gene is bivalently marked in the ICM, and in ESCs and iPSCs, but in a terminally differentiated neuron, the resolved mark consists of H3K4me3 while H3K27 becomes unmethylated. The same gene in a nonneuronal differentiated cell (a liver cell for instance) becomes unmethylated at H3K4 and retains H3K27me3, resulting in gene silencing.

Once pluripotent cells are coaxed to differentiate in vitro, they exit the pluripotent state and undergo a process analogous to gastrulation where they proceed along endodermal, ectodermal, and mesodermal lineages. In practice, differentiation methods have been developed that employ two general approaches: (i) undirected differentiation, which is induced by the removal of pluripotency‐signaling factors (LIF or bFGF) leading to the stochastic production on mixtures of endodermal ectodermal and mesodermal cells and (ii) the removal of pluripotency‐supporting factors combined with the addition of specific germ layer inducing signaling molecules. An example of the latter is the removal of bFGF from hESC or hiPSC culture, combined with the addition of Activin A, which induces differentiation to a relatively uniform population of endodermal progenitor cells (D'amour et al., 2005). These can subsequently be exposed to custom series of growth factors, media, and conditions to yield a desired type of endodermal cell, such as hepatocytes for example.

1.2.2 Undirected Differentiation of Pluripotent Cells to Embryoid Bodies

Undirected (spontaneous) differentiation of pluripotent cells is a poor choice if the goal is to produce a uniform population of differentiated cells of a single cellular identity. However, undirected differentiation is highly useful for the production of embryoid bodies (EBs), which are useful for the detection of potential teratogens (compounds that can cause birth defects). This is because EBs serve as a reasonable model for early postimplantation embryogenesis. EBs are initiated from starter cultures consisting of cohesive aggregates of undifferentiated pluripotent cells. Especially in the case of hESCs and hiPSCs, aggregates are crucial since human pluripotent cells form junctions with neighboring cells mediated by E‐cadherins and other cell adhesion proteins. These contacts are preserved in aggregates. Aggregates consisting of dozens to hundreds of cells are transferred to medium lacking pluripotency‐signaling growth factors (i.e. LIF of bFGF is removed). Cells within the aggregate then begin to spontaneously differentiate into cell of all three germ layers, and after several days, a variety of cell types of endodermal, ectodermal, and mesodermal origin form. Many of these cells differentiate quite far in developmental terms, and mature EBs typically contain abundant neurons and cardiomyocytes, which begin to spontaneously contract leading to observable rhythmic contractions in EBs (often called “beating heart” EBs at this stage). Classic methods of EB production start with aggregates of varying cell number made simply by the partial disaggregation of ESC cultures. These are then plated in nonadherent cell culture wells or flasks and as differentiation proceeds, a collection of variously sized EBs form. Individual aggregates have also been cultured in hanging drop cultures, which allows the individual culture of EBs of random size. Recently, it has become possible to mass‐produce human EBs of uniform from pluripotent cells. This is achieved by disaggregating hESC colonies to single cells (which is now possible with ROCK inhibitor, which can compensate for the disruption of E‐cadherins cell–cell interactions), followed by aggregation of a chosen number of cells into EBs. This allows cohorts of EBs of similar size to differentiate synchronously, and these can be cultured individually in multiwell formats (Flamier et al., 2017). This improvement now makes human EB systems suitable for the testing of a large number of suspected environmental and pharmaceutical teratogens.

1.2.3 Directed Differentiation of Pluripotent Cells

A great deal of research effort has been devoted to the directed differentiation of pluripotent cells to specific final cell types. This effort has been driven in large measure by the desire to eventually utilize such cells for cell replacement (stem cell) therapies. However, a useful outcome of this work is that it is now possible to use these cells as platforms for toxicological assays. Primary human cell culture in which cultures are derived from postmortem human organs and biopsies are difficult at best, and directed stem cell differentiation procedures can reproducibly produce such cells. Thus, it is now possible to test compounds for toxicity on relevant cell types from the appropriate species. For instance, hepatotoxicity, neural toxicity, and cardiotoxicity can now be tested on human hepatocytes, neurons, and cardiomyocytes of stem cell origin – a vast improvement over the methods in use only a decade ago (and even now) in which compounds were routinely tested on abnormal immortalized human cell lines and often on cells of irrelevant identity and/or species origin.

1.3 In vitro Uses of Pluripotent Cells

Classic approaches to toxicological and teratological assays have made heavy use of rodents (mice and rats) as well as rabbits. Though animal testing has the advantage of being an in vivo system, species‐specific differences between animal models and humans are often large, resulting in frequent false‐positive and false‐negative errors. Furthermore, animal testing of compounds is laborious, costly, and time‐consuming and requires the use of a large number of animals. Now, it is possible to use pluripotent cells (ESCs and iPSCs) as platforms to investigate both toxicological effects and teratogenic effects as an alternative to animal approaches (Figure 1.1). These approaches supplement existing animal approaches and provide a platform that utilizes relevant cells, and in the case of human ESCs and iPSCs, from the appropriate species. Furthermore, the advent of human iPS platforms now allows the testing of pharmaceuticals for their impact upon specific human populations with genetically altered drug metabolism.

Figure 1.1Derivation and use of pluripotent cells for in vitro teratology and toxicology applications. ESCs are derived from the ICM of blastocyst‐stage embryos (mouse or human) and a pluripotent. Alternatively, iPSCs can be produced by factor‐mediated reprogramming of terminally differentiated somatic cells such as fibroblasts. iPSCs are also pluripotent. Pluripotent cells (ESCs or iPSCs) can be differentiated into EBs for modeling of early development and used to detect teratogens or study human genetically specified birth defect mechanisms (provided that iPSCs containing relevant genetic developmental mutations are used). In addition, both ESCs and iPSCs can be subjected to directed differentiation to detect chemically induced impacts on specific cell types. Finally, patient‐specific iPSCs can be used to detect and study individualized pharmacogenomic responses to pharmaceuticals (in terms of efficacy or toxicity).

1.3.1 Pluripotent Cells for Toxicology

Animal research to assess toxicological effects of compounds in vivo comes with a substantial logistical load, but in general, toxicological assays can be conducted on adult mice followed by sacrifice to assess consequences of compound exposure for tissue histology combined with assessment of biomarkers of toxicity such as release of liver enzymes into peripheral blood. The U.S. Environmental Protection Agency (EPA) ToxCast program has identified thousands of compounds that have been deemed of importance for toxicological assessment (Dix et al., 2007), but the low throughput nature of animal testing makes the complete assessment of ToxCast compounds in animals a huge endeavor, which will require decades of testing and the use of a large, nearly prohibitive number of rodents to complete this task. In addition, rodents developmental biology differs from that of humans, and false‐positive and false‐negative results due to species differences are frequent. Though the use of animals will likely not be supplanted, testing in cell culture platforms is of obvious interest for reasons of expediency and the ability to use human cells of relevant identity.

In pharmaceutical drug development, the most common types of toxicity that result in failure of candidate drugs are cardiotoxicity and hepatotoxicity. Cardiotoxicity is a common pharmaceutical side effect, and cardiomyocytes derived from stem cells serve as a platform for predictive toxicology. For example, doxorubicin can induce cardiotoxic effects in human subjects. The cardiotoxicity of doxorubicin is evident in in vitro stem cell systems in which stem‐cell‐derived cardiomyocytes are used (Farokhpour et al., 2009; Singla, 2015; Maillet et al., 2016). In fact, patients exhibit idiosyncratic cardiotoxicity in response to doxorubicin and patient‐specific susceptibility to doxorubicin can be detected in cardiomyocytes derived from patient‐specific iPSCs (Burridge et al., 2016). Hepatotoxicity is also a common pharmacological side effect, and similar stem cell systems to detect drug‐induced hepatotoxicity have also been developed (Greenhough et al., 2010; Takayama and Mizuguchi, 2017).

1.3.2 Pluripotent Cells for Teratology

In the United States, approximately 3% of live‐born infants are affected by recognizable birth defects according to the Centers for Disease Control and Prevention (CDC). In addition, spontaneous abortion is frequent, and many of these may be caused by teratogenic exposure. Potentially teratogenic compounds exist in the environment, in food and drinking water, and in pharmaceuticals, and these may contribute to birth defects of nongenetic origin. Often, teratogenic pharmaceuticals have become known only after release of a drug to the public. In the late 1950s and early 1960s, over 10 000 birth defects occurred after thalidomide use by pregnant women (Franks