iPSCs - State of the Science

iPSCs - State of the Science, Volume Sixteen, the latest release in the Advances in Stem Cell Biology series, is an expansive collection of information and new discoveries in the field. This volume addresses the importance of induced pluripotent stems cells and how can they be derived from different sources. It addresses advances in research in induced pluripotent stem cells from alternate sources, such as spermatogonial stem cells, ovarian tissue, cancer cells, and many other sources. It is written for researchers and scientists in stem cell therapy, cell biology, regenerative medicine and organ transplantation, and is contributed by world-renowned authors.

• Provides an overview of the fast-moving field of stem cell biology and function, regenerative medicine and therapeutics
• Covers iPSCs derived from amniotic fluid, oral tissue derived iPSCs, muse cells, postmortem tissue, and much more
• Contributed by world-renowned experts in the field

• Language: English
• Publisher: Academic Press
• Release date: Jan 9, 2022
• ISBN: 9780323856454

Book preview

iPSCs - State of the Science

Volume 16

Editor

Alexander Birbrair

Federal University of Minas Gerais, Department of Pathology, Belo Horizonte, Minas Gerais, Brazil

Columbia University Medical Center, Department of Radiology, New York, NY, United States

Table of Contents

Cover image

Title page

Advances in Stem Cell Biology

Copyright

Dedication

List of contributors

About the editor

Preface

Chapter 1. Revealing nervous and cardiac system interactions by iPSC-Based platforms

Development and physiology of the cardiovascular system

Innervations of the heart

Cardiac–nervous system interactions: relevance to cardiac disorders

Cardiac and peripheral nervous system studies before pluripotent stem cells

Induced pluripotent stem cells (iPSCs)

New approaches to explore cardiac–nervous system interactions

Future perspectives

Limitations

Chapter 2. Human iPSC models of cardiac electrophysiology and arrhythmia

Abbreviations

Introduction

Disorders of impulse formation

Disorders of impulse conduction

Design considerations for in vitro models of arrhythmia

Expanded applications of human-induced pluripotent stem cell–derived cardiomyocyte models

Summary

Chapter 3. Human iPSCs for modeling of hepatobiliary development and drug discovery

Introduction

Key signals in induced pluripotent stem cell hepatobiliary differentiation

Modeling hepatotoxicity and liver diseases

The present and future of drug discovery

Chapter 4. The application of iPSC-derived kidney organoids and genome editing in kidney disease modeling

Introduction

Kidney

Kidney organoids

Kidney disease modeling

Genome editing technologies

Genetic kidney disease model with genome editing

Challenges of kidney disease modeling with genome editing

Chapter 5. Toward the in vitro understanding of iPSC nucleoskeletal and cytoskeletal biology, and their relevance for organoid development

Introduction

Chapter 6. Induced pluripotent stem cell models for mitochondrial disorders

Introduction

Mitochondrial genetics

Mitochondrial biochemistry

Mitochondrial diseases

Human stem cell models

Metabolic switching and cell fate decisions during early development

Differentiation potential and therapeutic potential for human-induced pluripotent stem cells

Concluding remarks

Chapter 7. Induced pluripotent stem cells for studying genetic autonomic disorders

Overview

Introduction

Autonomic nervous system disorders modeled with human pluripotent stem cells

Future directions and technical challenges

Chapter 8. Applications of iPSCs in Gaucher Disease and other rare sphingolipidoses

Summary

Introduction: from multisystemic lysosomal diseases to induced pluripotent cells

Overview of induced pluripotent stem cells as models of Gaucher disease

Overview of induced pluripotent stem cells as regenerative medicine tools

Future trends and directions of induced pluripotent stem cells in Gaucher disease and beyond

Conclusion

Chapter 9. Application of CRISPR/Cas system in iPSC-based disease model of hereditary deafness

Introduction

Induced pluripotent stem cells model of hereditary deafness

Application of CRISPR/Cas9 in induced pluripotent stem cells for hearing loss disease modeling

Future perspectives

Chapter 10. Progress and possibilities for patient-derived iPSCs and genetically engineered stem cells in cancer modeling and targeted therapies

Introduction

Cancer modeling with patient-derived induced pluripotent stem cells

Engineered disease trait pluripotent stem cell models

Applications of pluripotent stem cells

Conclusion

Index

Advances in Stem Cell Biology

Series Editor

Alexander Birbrair

Copyright

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Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

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A catalogue record for this book is available from the British Library

ISBN: 978-0-323-85767-3

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Publisher: Stacy Masucci

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Editorial Project Manager: Billie Jean Fernandez

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Dedication

This book is dedicated to my mother, Marina Sobolevsky, of blessed memory, who passed away during the creation of this volume. Professor of Mathematics at the State University of Ceará (UECE), she was loved by her colleagues and students, whom she inspired by her unique manner of teaching. All success in my career and personal life I owe to her.

My beloved mom Marina Sobolevsky of blessed memory (July 28, 1959–June 3, 2020)

List of contributors

Olga Amaral

Human Genetics Department, Unit of Research and Development, CSPGF, National Health Institute Doctor Ricardo Jorge (INSA, IP), Porto, Portugal

ICETA, CECA, University of Porto, Porto, Portugal

Enrico Bertini,     Genetics and Rare Diseases Research Division, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy

José Bragança

Department of Biomedical Sciences and Medicine and Centre for Biomedical Research (CBMR), University of Algarve, Campus of Gambelas, Faro, Portugal

ABC—Algarve Biomedical Centre, Faro, Portugal

Esra Cagavi

Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul, Turkey

Department of Medical Biology, School of Medicine, Istanbul Medipol University, Istanbul, Turkey

Medical Biology and Genetics Graduate Program, Health Sciences Institute, Istanbul Medipol University, Istanbul, Turkey

Claudia Compagnucci,     Genetics and Rare Diseases Research Division, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy

Ana Duarte

Human Genetics Department, Unit of Research and Development, CSPGF, National Health Institute Doctor Ricardo Jorge (INSA, IP), Porto, Portugal

ICETA, CECA, University of Porto, Porto, Portugal

Julian A. Gingold,     Department of Obstetrics & Gynecology and Women's Health, Einstein/Montefiore Medical Center, Bronx, NY, United States

Subhajit Giri,     Developmental Neurobiology Unit, GIGA-Stem Cells, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), CHU Sart Tilman, University of Liège, Liège, Belgium

Mo-Fan Huang

Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, United States

MD Anderson Cancer Center, UTHealth Graduate School of Biomedical Sciences, The University of Texas, Houston, TX, United States

Shilpa Iyer

Department of Biological Sciences, J. William Fulbright College of Arts and Sciences, University of Arkansas, Fayetteville, AR, United States

Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, United States

Yoon-Young Jang

Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, United States

Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, United States

Dung-Fang Lee

Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, United States

MD Anderson Cancer Center, UTHealth Graduate School of Biomedical Sciences, The University of Texas, Houston, TX, United States

Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, TX, United States

Center for Precision Health, School of Biomedical Informatics, The University of Texas Health Science Center at Houston, Houston, TX, United States

Justin Lowenthal

Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, United States

Medical Scientist Training Program, Johns Hopkins University School of Medicine, Baltimore, MD, United States

Brigitte Malgrange,     Developmental Neurobiology Unit, GIGA-Stem Cells, Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), CHU Sart Tilman, University of Liège, Liège, Belgium

Fibi Meshrkey

Department of Biological Sciences, J. William Fulbright College of Arts and Sciences, University of Arkansas, Fayetteville, AR, United States

Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, United States

Ryuji Morizane

Nephrology Division, Massachusetts General Hospital, Boston, MA, United States

Department of Medicine, Harvard Medical School, Boston, MA, United States

The Wyss Institute, Harvard University, Cambridge, MA, United States

Harvard Stem Cell Institute, Cambridge, MA, United States

Ozlem Mutlu Burnaz,     Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul, Turkey

Lon Kai Pang

Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, United States

Rice University, Houston, TX, United States

Mezthly Pena

Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, United States

Rice University, Houston, TX, United States

Raj R. Rao

Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, United States

Department of Biomedical Engineering, College of Engineering, University of Arkansas, Fayetteville, AR, United States

Diogo Ribeiro

Human Genetics Department, Unit of Research and Development, CSPGF, National Health Institute Doctor Ricardo Jorge (INSA, IP), Porto, Portugal

ICETA, CECA, University of Porto, Porto, Portugal

Renato Santos,     Human Genetics Department, Unit of Research and Development, CSPGF, National Health Institute Doctor Ricardo Jorge (INSA, IP), Porto, Portugal

Marco Tartaglia,     Genetics and Rare Diseases Research Division, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy

Gordon F. Tomaselli,     Office of the Dean, Albert Einstein College of Medicine, New York, NY, United States

Tamara Traitteur

Nephrology Division, Massachusetts General Hospital, Boston, MA, United States

Department of Medicine, Harvard Medical School, Boston, MA, United States

Eidgenössische Technische Hochschule Zürich, Zurich, Switzerland

The Wyss Institute, Harvard University, Cambridge, MA, United States

Leslie Tung,     Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, United States

Heidi Ulrichs,     Center for Molecular Medicine, University of Georgia, Athens, GA, United States

Yichen Wang,     Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, United States

Franklin D. West,     Regenerative Bioscience Center, The University of Georgia, Athens, GA, United States

Hsueh Fu Wu

Center for Molecular Medicine, University of Georgia, Athens, GA, United States

Department of Biochemistry and Molecular Biology, Franklin College of Arts and Sciences, University of Georgia, Athens, GA, United States

Brenda Yang,     Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, United States

Nadja Zeltner

Center for Molecular Medicine, University of Georgia, Athens, GA, United States

Department of Biochemistry and Molecular Biology, Franklin College of Arts and Sciences, University of Georgia, Athens, GA, United States

Department of Cellular Biology, Franklin College of Arts and Sciences, University of Georgia, Athens, GA, United States

Chengcheng Zhang

Nephrology Division, Massachusetts General Hospital, Boston, MA, United States

Department of Medicine, Harvard Medical School, Boston, MA, United States

Ruiying Zhao,     Department of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, United States

About the editor

Alexander Birbrair

Dr. Alexander Birbrair received his bachelor's biomedical degree from Santa Cruz State University in Brazil. He completed his PhD in Neuroscience, in the field of stem cell biology, at the Wake Forest School of Medicine under the mentorship of Osvaldo Delbono. Then, he joined as a postdoc in stem cell biology at Paul Frenette's laboratory at Albert Einstein School of Medicine in New York. In 2016, he was appointed faculty at Federal University of Minas Gerais in Brazil, where he started his own lab. His laboratory is interested in understanding how the cellular components of different tissues function and control disease progression. His group explores the roles of specific cell populations in the tissue microenvironment by using state-of-the-art techniques. His research is funded by the Serrapilheira Institute, CNPq, CAPES, and FAPEMIG. In 2018, Alexander was elected affiliate member of the Brazilian Academy of Sciences, in 2019, he was elected member of the Global Young Academy (GYA), and in 2021 he was elected affiliate member of The World Academy of Sciences (TWAS). He is the Founding Editor and Editor-in-Chief of Current Tissue Microenvironment Reports, and Associate Editor of Molecular Biotechnology. Alexander also serves in the editorial board of several other international journals: Stem Cell Reviews and Reports, Stem Cell Research, Stem Cells and Development, and Histology and Histopathology.

Preface

This book's initial title was iPSCs: Recent Advances. Nevertheless, because of the ongoing strong interest in this theme, we were able to collect more chapters than would fit in one single volume, covering induced pluripotent stem cells (iPSCs) biology from different perspectives. Therefore, the book was subdivided into several volumes.

This volume iPSCs—State of the Science offers contributions by known scientists and clinicians in the multidisciplinary areas of biological and medical research. The chapters bring up-to-date comprehensive overviews of current advances in the field. This book describes recent advances in the use of iPSCs to model several diseases in vitro, enabling us to study the cellular and molecular mechanisms involved in different pathologies. Further insights into these mechanisms will have important implications for our understanding of disease appearance, development, and progression. The authors focus on the modern state-of-the-art methodologies and the leading-edge concepts in the field of stem cell biology. In recent years, remarkable progress has been made in the obtention of iPSCs and their differentiation into several cell types, tissues, and organs using state-of-the-art techniques. These advantages facilitated identification of key targets and definition of the molecular basis of several disorders. Thus, this book is an attempt to describe the most recent developments in the area of iPSCs technology which is one of the rising hot topics in the field of molecular and cellular biology today. Here, we present a selected collection of detailed chapters on what we know so far about iPSC technology. Ten chapters written by experts in the field summarize the present knowledge about iPSC technology.

Ozlem Mutlu Burnaz and Esra Cagavi from Istanbul Medipol University discuss nervous and cardiac system interactions by iPSC-based platforms. Leslie Tung and colleagues from Johns Hopkins University School of Medicine look at human iPSC models of cardiac electrophysiology and arrhythmia. Yichen Wang and Yoon-Young Jang from Johns Hopkins University School of Medicine describe human iPSCs for modeling of hepatobiliary development and drug discovery. Ryuji Morizane and colleagues from Harvard Medical School compile our understanding of the application of iPSC-derived kidney organoids and genome editing in kidney disease modeling. Claudia Compagnucci and colleagues from IRCCS update us with what we know about in vitro understanding of iPSC nucleoskeletal and cytoskeletal biology, and their relevance for organoid development. Shilpa Iyer and colleagues from University of Arkansas summarize current knowledge on the use of iPSCs to model mitochondrial disorders. Nadja Zeltner and colleagues from University of Georgia talk about iPSCs for studying genetic autonomic disorders. Olga Amaral and colleagues from University of Porto addresses the importance of iPSCs in Gaucher disease and other rare sphingolipidoses. Subhajit Giri and Brigitte Malgrange from University of Liège focus on the application of CRISPR/Cas system in iPSC-based disease model of hereditary deafness. Finally, Dung-Fang Lee and colleagues from The University of Texas Health Science Center at Houston update us with the progress and possibilities for patient-derived iPSCs and genetically engineered stem cells in cancer modeling and targeted therapies.

It is hoped that the articles published in this book will become a source of reference and inspiration for future research ideas. I would like to express my deep gratitude to my wife, Veranika Ushakova, and Ms. Billie Jean Fernandez and Ms. Elisabeth Brown from Elsevier, who helped at every step of the execution of this project.

Alexander Birbrair

Editor

Chapter 1: Revealing nervous and cardiac system interactions by iPSC-Based platforms

Ozlem Mutlu Burnaz ¹ , and Esra Cagavi ¹ , ² , ³       ¹ Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul, Turkey      ² Department of Medical Biology, School of Medicine, Istanbul Medipol University, Istanbul, Turkey      ³ Medical Biology and Genetics Graduate Program, Health Sciences Institute, Istanbul Medipol University, Istanbul, Turkey

Abstract

Cardiac function is regulated by the autonomic nervous system. In exchange, the sensorial information from the heart is relayed to the brain via sensory neurons as a crucial modulatory feedback mechanism. Cardiovascular and neurological diseases constitute the majority of deaths globally. The high morbidity associated with cardiac and neurological disorders is mostly due to the limited number of targeted therapeutics. Moreover, many new drug candidates are withdrawn from clinical use due to cardiotoxicity or neurotoxicity. Previously, experimental animal models, biopsy materials, or immortalized cell lines were the basis of disease studies and drug screens. However, the differences between these models and human physiology particularly in neural and cardiac functions resulted in limited clinical success. To overcome the complications related to the organism mismatch and cell source, human induced pluripotent stem cells (hiPSCs) provide ways to investigate molecular mechanisms in embryonic, adult, and diseased states. hiPSC-derived cardiac cells and various neuron subtypes could replicate complex interactions in the physiologically relevant organoids or multiorgan microdevices. Using these novel technical developments, recent models of neuronal regulation of heart tissue started to provide unique insights in systemic interactions and molecular basis to develop more precise therapeutic approaches. In this chapter, a brief historical perspective and recent advances in iPSC-based models of cardiac and nervous system interactions are reviewed.

Keywords

Cardiac differentiation; Cardiac innervations; Cardiology; Cardiovascular disease; In vitro disease models; iPSC; Microfluidics; Nervous system; Neural differentiation; Organ-on-a-chip; Organoids; Sensory neurons; Tissue engineering

Development and physiology of the cardiovascular system

Innervations of the heart

Cardiac–nervous system interactions: relevance to cardiac disorders

Cardiac and peripheral nervous system studies before pluripotent stem cells

Induced pluripotent stem cells (iPSCs)

Modeling the cardiovascular system with iPSCs

Modeling the peripheral nervous system with iPSCs

New approaches to explore cardiac–nervous system interactions

Organ-on-chips

Organoids

Tissue engineering

Future perspectives

Limitations

Acknowledgements

References

Development and physiology of the cardiovascular system

The heart is the central component of the circulation system and is composed of four chambers working coherently to supply the blood flow throughout the body (Kurokawa and George, 2016). The cardiac tissue is composed of endocardium, myocardium, and epicardium, from which the myocardium is responsible to generate contractile function. Cardiomyocytes (CMs) together with the support of fibroblasts, endothelial cells, smooth muscle cells, immune cells, and perivascular cells construct the myocardium (Wang et al., 2018). The human heart has various CM populations categorized based on the localization and function, including atrial, ventricular, or nodal CMs, Purkinje cells, and the cells of the His bundle. The CM subpopulations differ by the distinct transcriptional profiles providing unique molecular signatures, electrophysiological excitability, and contractile properties (Litviňuková et al., 2020). Adult CMs have a rodlike shape and intercalated discs giving the cardiac muscle fibers a spiral alignment to provide the force required to pump the blood. Gap junctions formed specifically by connexin-43 (Cx43) proteins enable synchronized contractions and rapid signal transmission in the cardiac tissue.

Heart tissue is highly vascularized with coronary arteries to meet the high requirement for oxygen and nutrients for the myocardium. Vascular endothelial cells secrete growth factors and signaling modulators such as inotropic factor endothelin-1, neuregulin-1, and nitric oxide, which regulate cardiac development, contractility, and blood pressure in the adult (Kurokawa and George, 2016). Vascular smooth muscle cells, resident cardiac lymphocytes, and macrophages contribute to the structure and function of the heart (Wang et al., 2018). Cardiac fibroblasts support the cardiac niche by secreting extracellular matrix (ECM) and remodeling enzymes, signaling molecules, growth factors, cytokines and could couple with CMs to modulate electrophysiological properties (Fan et al., 2012). ECM in the cardiac tissue predominantly contains periostin, fibronectin, fibrillin, elastin, and collagen types I, III, and VI in a stage-specific combination (Kurokawa and George, 2016). From these, periostin was shown to induce proliferation in the developing myocardium (Kühn et al., 2007), whereas collagens provide strength to the cardiac tissue (Marijianowski et al., 1994).

The heart is the first organ developed in the vertebrate embryo. Cardiac development begins with cardiac stem cells forming the cardiogenic plate and heart tube, which are characterized by the expression of Nkx-2.5, Isl-1, and GATA-4 transcription factors. In the third week of human embryonic development, the heart tube loops, and synchronous contractions begin. Predominantly sonic hedgehog (Shh), activin, nodal, Ptx2, lefty, and retinoic acid expression together with other morphogens contribute to the left–right asymmetry and branching of the heart (Olson and Srivastava, 1996). Mechanical forces and chemical signals originating from neural crest cells participate in chamber formation (Kirby and Waldo, 1995). Ventricular growth is dependent on signaling between myocardium and endocardium. Especially, neuregulin expressed in endocardium is essential to regulate ERBB2 and ERBB4 receptor signaling for the ventricular trabeculae formation (Gassmann et al., 1995; Meyer and Birchmeier, 1995). As of note, endothelial plexus also develops simultaneously to provide circulatory networks, in which the cardiac jelly, contractions, and sheer pressure by the blood flow stimulate angiogenesis and vasculogenesis (Moorman et al., 2003; Lindsey et al., 2014). The complex nature of the cardiovascular system including various cell types and the unique ECM contribute to the steady or diseased states while shaping the developmental organization. The regulation of cardiac activity by nerve innervations adds to the complexity yet to be explored.

Innervations of the heart

The regulation of the cardiac tissue by the nervous system is maintained by efferent and afferent neurons in the intracardiac nervous system, extracardiac-intrathoracic ganglia, spinal cord, brain stem, and central nervous system (CNS) elements such as insular cortex, hypothalamus, and the amygdala. The vagal and sympathetic fibers stem from medulla oblongata via the preganglionic neurons descending to the heart. The cardiac tissue is innervated by the autonomic sympathetic, parasympathetic, and sensory neurons to regulate the function and homeostasis of the heart (Hasan, 2013). From these neuron populations, sympathetic neurons innervating the cardiac tissue reside mainly in the stellate ganglia and have been extensively studied. They project to the pacemaker cells, the atria, and the ventricles. Sympathetic neurons are characterized by the expression of PHOX2B, ASCL1, TH, and DBH gene expression and signal through catecholamines by targeting primarily the β1 and β2 adrenergic receptors on the cardiac cells. The sympathetic neural activity increases heart rate (chronotropic), conduction velocity (dromotropic), and myocardial contraction (inotropic), whereas the parasympathetic division decreases these cardiac outputs. The parasympathetic neurons stem from the cardiac ganglia, which are located on the atrial epicardium and project to the sinoatrial and atrioventricular nodes. Parasympathetic neuron somas reside in the cardiac ganglia and convey signals through muscarinic receptors on the heart by using acetylcholine.

Functionally, the sensory neurons transmit signals such as pain and pressure from the heart to the CNS via either upper dorsal root ganglia (DRG) and thoracic dorsal horn or vagal fibers and nodose ganglia (Akgul Caglar et al., 2020; Kukanova and Mravec, 2006 ). The nodose ganglion is reported to relay mostly chemical information related to the heart and DRG is thought to transmit chemical and mechanical information related to the cardiac muscle and circulatory system (Armour, 2007). The sensory neurons in these ganglia are classified in three groups: nociceptive neurons that transmit pain signals, mechanoreceptive neurons that transmit pressure and tension, and proprioceptive neurons that transmit location information. Nociceptive, mechanoreceptive, and proprioceptive neurons are distinguished by TrkA, TrkB, and TrkC receptor expression, respectively (Viventi and Dottori, 2018).

The activity of the sympathetic and parasympathetic neurons is regulated based on the signals acquired via the sensory neurons (Wang and Ma, 2000). In one report, the blockade of TRPV1 expressing sensory neurons with resiniferatoxin was shown to decrease the sympathetic tone, recovery time, and cardiac contractility in experimental animal models (Wang et al., 2014). The interactions between sensory neurons and CMs are largely unknown at the cellular and molecular level.

Developmentally, sympathetic and sensory neurons originate from the trunk neural crest cells, whereas parasympathetic neurons originate from cardiac neural crest cells as summarized in Fig. 1.1 (Kimura et al., 2012). Both neural crest populations migrate toward the developing heart and participate in the formation of the aortic arches, cardiac ganglia, truncal cushion, interventricular septum, and semilunar valve development (Olson and Srivastava, 1996). Neurotrophic factors and chemorepellents secreted from the cardiac tissue have significant roles in the development, survival, migration, and proliferation of the innervating neurons. Numerous reports have demonstrated the effects of neural growth factor (NGF) secreted from CMs binding to the TrkA receptor on neurons stimulating survival and axonal growth in rodents (Hassankhani et al., 1995; Kimura et al., 2007, 2010). Importantly, the deficiency of either NGF or TrkA proteins in transgenic animal models showed a significant reduction in the numbers of sympathetic neurons of the superior cervical ganglia (Crowley et al., 1994; Fagan et al., 1996), whereas overexpression resulted in hyperinnervation in transgenic mice (Hassankhani et al., 1995). Interestingly, inhibition of NGF or TrkA receptors was shown to drive neonatal rat CMs into apoptosis indicating the dual survival effect of NGF on CMs and innervated neurons (Caporali et al., 2008). Moreover, NGF enhances synaptic transmission, which was shown by in vitro cocultures of neonatal rat CMs with superior cervical ganglion sympathetic neurons (Lockhart et al., 1997). In addition, sensory neurons release substance P and calcitonin gene–related peptide (CGRP) both for restoration and regeneration of the cardiac tissue (Fig. 1.1) (Liu et al., 2008).

Figure 1.1 Developmental organization of sympathetic, parasympathetic, and sensory neurons innervating the heart. Reciprocal interactions of cardiac and nerve cells cause mature phenotypes. Major factors and receptors involved in the maturation process are indicated.

In addition to the role of NGF in sympathetic and sensory neurons, other members of the neurotrophic family, glial-derived neurotrophic factor (GDNF), and neurturin support the parasympathetic development and innervations. Deficiencies in GDNF, neurturin, or their receptors (GFRa2, GFRa1, and RET) resulted in reduced cholinergic innervations in the heart, although sympathetic patterning remained intact (Fig. 1.1) (Hiltunen et al., 2000). Neurotrophin-3 (NT-3) is another important factor of peripheral neuron survival and was shown to stimulate growth and axonal elongations both in vitro and in vivo (Kimpinski et al., 1997; Gordon, 2009; Fornaro et al., 2020). Particularly, neurons were shown to grow toward the NT-3-expressing endothelial cells of the blood vessels (Hsieh et al., 2006). NT-3 shares common receptors with NGF, TrkA, and p75 (Francis et al., 1999). The positive regulation provided by neurotrophic factors is balanced by a chemorepellent, Sema3a, whose deficiency resulted in disrupted sympathetic patterning and malformation in stellate ganglia (Tang et al., 2004; Ieda et al., 2007). Collectively, cardiac and nervous system cells give feedback to each other by physical connections and releasing chemical factors, and these interactions are important regulators of homeostasis in the cardiovascular system.

Cardiac–nervous system interactions: relevance to cardiac disorders

Several experimental and clinical studies indicated that the disruption or disorganization of the cardiac innervations contribute to various cardiac disorders including sudden cardiac death, congenital heart failure, atrial fibrillation, and arrhythmia (Hasan et al., 2013; Shen and Zipes, 2014). As a part of the postmyocardial infarction (MI) pathology, cardiac innervation density was demonstrated to become heterogeneous due to the Wallerian degeneration, neurilemmal cell proliferation, and unguided axonal regeneration, (Chen et al., 2007).

In the congestive heart failure, increased sympathetic tone (Cohn et al., 1984), insufficient norepinephrine reuptake (Eisenhofer et al., 1996), sympathetic neuron loss (Himura et al., 1993) as well as decreased parasympathetic tone, reduced vagal transmission, and altered muscarinic receptor density were reported (Olshansky et al., 2008). Moreover, adenosine, capsaicin, bradykinin, ATP, ROS, and serotonin secreted from CMs were speculated to regulate sensory neuron behavior in damaged myocardium (Kukanova and Mravec, 2006) and to cause ventricular arrhythmia via sympathoexcitatory reflex (Rajendran, 2016). Information obtained from these studies led to the therapeutic approach that stimulation of the vagal or spinal cord segments could decrease the symptoms of heart failure (De Ferrari et al., 2011; Schwartz and De Ferrari, 2011; Tse et al., 2015). Furthermore, overexpression of Sema3a was demonstrated to result in increased β-adrenergic density, reduced innervations, prolonged action potential duration, and ventricular tachyarrhythmia (Tang et al., 2004; Ieda et al., 2007). For these cases, neurochemical stimulation is an option with adrenergic receptor blockers to control symptoms of heart failure (Chatterjee et al., 2013) and cholinesterase inhibitor—pyridostigmine—to reduce ventricular arrhythmias (Behling et al., 2003). Therefore, revealing the molecular mechanisms behind cardiac diseases by exploring cardiac–nervous system interactions could provide unique opportunities to develop novel pharmacological or physiological approaches to modulate disease symptoms.

Cardiac and peripheral nervous system studies before pluripotent stem cells

The most established strategy to investigate cardiac–nervous system interactions has been the use of experimental animal models to replicate disease conditions and to evaluate the effects of candidate drugs. However, the results obtained from in vivo animal models and transgenic strategies had limitations due to the complexity of contributing factors and differences in physiological, immunological, or species-specific mechanisms (Hoekstra et al., 2012). The physiological differences in contraction rates, electrophysiological properties, and pharmacological responses between animal models and human result in partial correlation to human disorders (Karakikes et al., 2015). Immortalized cell lines, human cadaveric tissues, and primary cell cultures from biopsies have been the other commonly used in vitro approaches with drawbacks besides their merits. Immortalized cell lines solve the problems related to limited cell source, but phenotype and gene expression patterns after immortalization tend to change the physiological properties of the cells. Primary cell cultures isolated from the heart or neural structures as well as human cadaveric tissue allowed investigation of the human material, but reaching those tissues often required ethically inconvenient invasive surgeries and only a small amount of tissue can be recovered that would not create a reproducible cell source for multiple experiments. Moreover, human or animal-derived tissues are hard to maintain in culture and are reported to change phenotype and go through cell senescence shortly after culturing (Jastrzebska et al., 2016).

The functional relationship between the cardiac and autonomic nervous system has been studied to a certain extent in experimental animal models; however, the signaling mechanisms at the cellular or molecular level have not been deciphered extensively. Attempts to coculture sympathetic, parasympathetic, and sensory neurons with CMs were reported to investigate the molecular and functional properties in animal-derived tissues. In one pioneering study, explant cultures of chick sympathetic neurons and rat CMs were evaluated to show neurotransmitter release, neuronal calcium ion currents, and voltage-clamped calcium currents to be reduced by three- to five fold following stimulation (Wakade et al., 1995). It should be noted that interactions between CMs and neuron cell bodies may have interfered with the results since neurons communicate through their axons in normal physiological conditions. Also, the isolated neuron cultures were impure, which may have complicated the interpretation of their findings. The advances in pluripotent stem cells provided the possibility to investigate the cardiac interactions with the innervating neurons with human cells, eliminating the challenges regarding the cell type, origin, and reproducibility.

Induced pluripotent stem cells (iPSCs)

The human iPSCs (hiPSCs) are defined by the unlimited renewal capacity and ability to differentiate into the majority of cell types in the human body. In 2007, human somatic cells were reprogrammed into iPSCs with the viral transfer of Oct3/4, Sox-2, c-Myc, and Klf-4 genes, known as Yamanaka factors (Takahashi et al., 2007). Disease modeling, drug screening, toxicity studies, and personalized cell therapy are some of the applications where iPSCs have been utilized. The use of iPSCs offers many advantages including refined screening applications with relevant human cell types, controlled microenvironment, and modeling hereditary or acquired diseases compared with experimental in vivo models (Wang et al., 2018). These superiorities give hopes for developing more effective therapeutic strategies in the future.

The efficiency of differentiation protocols continues to improve, and functionally validated iPSC-derived mature CMs and neuron subtypes of human origin attract more attention due to wider applicability and reproducibility (Sharma et al., 2020). Recently, several clinical trials have been conducted and are planned by using personalized tissues generated by hiPSCs to develop new cellular therapies for the regeneration