iPSCs from Diverse Species

The series Advances in Stem Cell Biology is a timely and expansive collection of comprehensive information and new discoveries in the field of stem cell biology.

iPSCs from Diverse Species, Volume 2 addresses how induced pluripotent stem cells (iPSCs) can be derived from different species.

The volume teaches the reader about modern state-of-the-art methodologies to derive iPSCs from distinct species. This volume will cover how to derive iPSCs from species like nonhuman primates, horses, dogs, pigs, rats, rabbits, and others. It also discusses the importance of iPSCs in species conservation. Detailed description on methods used to obtain iPSCs from humans and other species expands the knowledge and understanding of stem cell biology and provides a potent tool to model diseases.

The volume is written for researchers and scientists in stem cell biology, and regenerative medicine and is contributed by world-renowned authors in the field.

• Provides overview of the fast-moving field of iPSC technology
• Covers iPCSs from the following species: humans, monkeys, horses, dogs, pigs, rats, rabbits, and more
• Consists of contributions from stem cell leaders around the world

• Language: English
• Publisher: Academic Press
• Release date: Sep 29, 2020
• ISBN: 9780323851855

Book preview

iPSCs from Diverse Species, Volume 2

Edited by

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

Copyright

Dedication

Contributors

About the editor

Preface

Chapter 1. Human iPSCs and their uses in developmental toxicology

Developmental toxicology: A brief history

iPSCs in developmental toxicology

iPSCs use and study developmental toxicology

Endodermal lineage differentiation for hepatic developmental toxicology

Ectodermal lineage differentiation for neuronal developmental toxicology

Mesodermal lineage differentiation for cardiomyocyte developmental toxicology

Alternative applications of iPSCs

Challenges surrounding iPSCs

Conclusions

Chapter 2. Induced pluripotent stem cells from nonhuman primates

Introduction

Advances in the range of NHP species that have been subjected to reprogramming

Advances in genetic modification of NHP iPSCs

Advances in cell transplantation in NHP models

Summary

Chapter 3. Equine induced pluripotent stem cells

Introduction

Derivation of equine iPSCs

Characterization of equine iPSCs

Directed differentiation of equine iPSCs

Cell therapy with equine iPSCs

Equine iPSCs for disease modeling

Future directions

Chapter 4. Canine induced pluripotent stem cells: an in vitro approach to validate the dog as a large animal model for Alzheimer’s disease

Introduction

Canine cognitive dysfunction—a natural model for Alzheimer’s disease

Generation of induced pluripotent stem cells from geriatric dogs

Generation of disease-affected target cell types from canine iPSCs

Role of signaling pathways in neural development

Neural induction

Neuronal differentiation

Conclusions

Chapter 5. Porcine iPSCs

Introduction

The porcine model

Historical perspective of piPSCs generation

Characterization

Culture strategies

Future trends

Glossary

Chapter 6. Bovine iPSC and applications in precise genome engineering

Introduction

Pluripotent stem cells

Isolation of ESC from Bos taurus species

Conclusion

Chapter 7. Induced pluripotent stem cells from buffalo

Introduction

Factors and delivery approaches for cellular reprogramming

Derivation of iPS cells from buffalo

Culture conditions of buffalo iPS cells

Characterization of buffalo iPS cells

Differentiation of buffalo iPS cells

Future prospectives

Chapter 8. Establishment of induced pluripotent stem cells from prairie vole-derived fibroblast

Introduction

Optimization of the reprogramming conditions for prairie vole iPS cells with prairie vole immortalized cells

Establishment of prairie vole iPS cells from primary fibroblasts

Evaluation of iPS cells from prairie vole primary cells

Conclusions

A commentary on the likely future trends or directions

Chapter 9. Rabbit induced pluripotent stem cells: the challenges

Rabbit as a disease model

Pluripotent stem cells

Regulating pathways of pluripotency: lessons learned from the rabbit ESC model

Technological aspects of reprogramming cells into rabbit iPSCs

Prospects and conclusions

Chapter 10. Naked mole rat iPSCs and their noncanonical features: a novel tool for aging research

Introduction

Generation of NMR induced pluripotent stem cells

Characterization of NMR iPSCs

Noncanonic features of NMR iPSCs

Commentary on possible future research directions

Chapter 11. Induced pluripotent stem cells in species conservation: advantages, applications, and the road ahead

Introduction

iPSCs: what makes them so attractive for species conservation?

Applications of iPSCs for species conservation

The long road ahead: the need for further research into iPSCs

Future perspectives

Index

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.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

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

ISBN: 978-0-12-822228-7

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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)

Contributors

Mette Berendt,     Department of Veterinary Clinical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark

Vilceu Bordignon,     Department of Animal Science, McGill University, Montreal, QC, Canada

Abinaya Chandrasekaran,     Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark

Rkia Dardari,     Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada

Laís Vicari de Figueiredo Pessôa,     Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, São Paulo, Brazil

Naomi Dicks,     Department of Animal Science, McGill University, Montreal, QC, Canada

Andras Dinnyes

BioTalentum Ltd, Gödöllő, Hungary

Szent Istvan University, Molecular Animal Biotechnology Laboratory, Gödöllő, Hungary

College of Life Sciences, Sichuan University, Chengdu, China

Ina Dobrinski,     Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada

Clayton Edenfield,     Department of Environmental Health Science, College of Public Health, University of Georgia, Athens, GA, United States

Kristine Freude,     Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark

Tomokazu Fukuda

Wildlife Genome Collaborative Research Group, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan

Graduate School of Science and Engineering, Iwate University, Morioka, Japan

Soft-Path Engineering Research Center (SPERC), Iwate University, Morioka, Iwate, Japan

Vadim N. Gladyshev,     Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States

Debbie Guest,     Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire, United Kingdom

Peter J. Hornsby,     Department of Cellular and Integrative Physiology, and Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, TX, United States

Poul Hyttel,     Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark

Maryam Ahmadi Jeyhoonabadi,     Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada

Masafumi Katayama

Center for Environmental Biology and Ecosystem, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan

Wildlife Genome Collaborative Research Group, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan

Dharmendra Kumar,     Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, Haryana, India

Pradeep Kumar,     Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, Haryana, India

Nathalia de Lima e Martins Lara,     Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada

Sang-Goo Lee,     Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States

Jun Liu,     Stem Cells and Genome Editing, Genomics and Cellular Sciences, Agriculture Victoria Research, Bundoora, VIC, Australia

Luis F. Malaver-Ortega,     Monash Functional Genomics Platform, Monash University, Clayton, VIC, Australia

Gabriela F. Mastromonaco,     Reproductive Sciences Unit, Toronto Zoo, Toronto, ON, Canada

Aleksei E. Mikhalchenko,     Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, Portland, OR, United States

Praopilas Phakdeedindan,     Department of Animal Husbandry, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand

Naresh L. Selokar,     Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, Haryana, India

Jacob Siracusa,     Department of Environmental Health Science, College of Public Health, University of Georgia, Athens, GA, United States

Huseyin Sumer,     Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC, Australia

Theerawat Tharasanit,     Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand

Barbara Blicher Thomsen,     Department of Veterinary Clinical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark

Paul J. Verma,     South Australian Research & Development Institute (SARDI), Turretfield Research Centre, Rosedale, SA, Australia

Ruoning Wang,     College of Nursing, University of New Mexico, Albuquerque, NM, United States

Franklin D. West,     Regenerative Bioscience Center, Neuroscience Program, Biomedical and Health Sciences Institute, Department of Animal and Dairy Science, College of Agricultural and Environmental Sciences, University of Georgia, Athens, GA, United States

P.S. Yadav,     Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, Haryana, India

Xiaozhong Yu,     College of Nursing, University of New Mexico, Albuquerque, NM, 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 (ABC), and in 2019, he was elected member of the Global Young Academy (GYA). 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

Alexander Birbrair ¹ , ² ,      ¹ Department of Pathology, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil,      ² Department of Radiology, Columbia University Medical Center, New York, NY, United States

This book's initial title was iPSCs: Recent Advances. Nevertheless, because of the ongoing strong interest in this theme, we were capable 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 from Diverse Species 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 the derivation of iPSCs from different species in vitro, enabling us to study the cellular and molecular mechanisms involved in different pathologies in various species. 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, the present book is an attempt to describe the most recent developments in the area of iPSCs biology 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 how we derive iPSCs from distinct species. Eleven chapters written by experts in the field summarize the present knowledge about iPSCs derived from different species.

Xiaozhong Yu and colleagues from University of Georgia introduce the derivation of human iPSCs. Peter J. Hornsby from University of Texas Health Science Center discusses iPSCs from nonhuman primates. Debbie Guest from Animal Health Trust describes iPSCs formed from horses. Kristine Freude and colleagues from University of Copenhagen compile our understanding of iPSCs derived from dogs. Ina Dobrinski and colleagues from University of Calgary update us with what we know about porcine iPSCs. Paul J Verma and colleagues from Turretfield Research Centre summarize current knowledge on bovine iPSCs and their applications in precise genome engineering. Dharmendra Kumara and colleagues from Central Institute for Research on Buffaloes address the importance of iPSCs from buffalo. Masafumi Katayama and Tomokazu Fukuda from National Institute for Environmental Studies focus on the establishment of iPSCs from prairie vole–derived fibroblasts. Andras Dinnyes and colleagues from Chulalongkorn University give an overview of iPSCs derived from rabbit. Vadim N. Gladyshev and colleagues from Harvard Medical School present naked mole rat iPSCs and their noncanonical features, as a novel tool for aging research. Finally, Gabriela F. Mastromonaco and colleagues from Toronto Zoo update us on the use of iPSCs in species conservation.

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: Human iPSCs and their uses in developmental toxicology

Clayton Edenfield ¹ , Jacob Siracusa ¹ , Ruoning Wang ² , and Xiaozhong Yu ² , ∗       ¹ Department of Environmental Health Science, College of Public Health, University of Georgia, Athens, GA, United States      ² College of Nursing, University of New Mexico, Albuquerque, NM, United States

∗ Correponding author. 

Abstract

Developmental toxicology is an interdisciplinary field that focuses on xenobiotic induced alterations at all stages of prenatal development. Human induced pluripotent stem cells (hiPSCs) have provided a human and patient-specific physiological model of a disease state, or induced toxicity can be observed rapidly and predictably while providing insights into mechanisms of developmental signals. hiPSCs model all major embryonic lineages as well as toxicologically relevant cell types. With recent advancements in coculturing, 3D culturing, and organoid formation, hiPSCs are primed to be a standard model for developmental toxicology.

Keywords

Animal induced pluripotent stem cells; Cardiomyocytes; Developmental toxicology; Hepatocytes; Human induced pluripotent stem cells; In vitro; Neuronal cells; Stem cells

Acknowledgments

Developmental toxicology: A brief history

iPSCs in developmental toxicology

iPSCs use and study developmental toxicology

Endodermal lineage differentiation for hepatic developmental toxicology

Ectodermal lineage differentiation for neuronal developmental toxicology

Mesodermal lineage differentiation for cardiomyocyte developmental toxicology

Alternative applications of iPSCs

Drug discovery/screening

Personalized toxicology through disease-specific modeling

Regenerative medicine

Animal iPSCs in veterinary medicine, modeling diseases, and species conservation

Challenges surrounding iPSCs

Conclusions

References

Developmental toxicology: A brief history

Developmental toxicology is an interdisciplinary field investigating the effects on a developing organism that may result from exposure to a xenobiotic before conception, throughout prenatal development, or even postnatally to the point of sexual maturation (EPA Guidelines for Developmental Toxicity Risk Assessment, 1991). Death of the developing organism, structural abnormalities, altered growth, and functional alterations are the four significant manifestations of developmental toxicity. These adverse manifestations may arise at any point in the life span of the organism. Epidemiological studies often have the power to evaluate the four developmental effects. At the same time, the first three are observed in laboratory animals using conventional developmental toxicity assays (also known as teratogenicity or Segment II testing) (EPA Guidelines for Developmental Toxicity Risk Assessment, 1991). These traditional models consist of a multigenerational or continuous breeding study. Most frequently, developmental toxicity expresses one or more of the possible endpoints that are used in the evaluation of potential agents to induce abnormal development. Generally, developmental toxicity occurs in a dose-dependent manner; however, there are many critical windows of susceptibility in development that may affect how toxic a xenobiotic is.

In 1966, Edwin Goldenthal scripted a letter to pharmaceutical companies highlighting the methods used in drug evaluation. His three-segment protocol for adverse reproductive effects is still used today (Scialli et al., 2018). The three-segment protocol consists of fertility and embryonic developmental component, embryo–fetal development component, and finally, a pre- and postnatal exposure study. While the three-segment approach study design has remained the same, recent modifications have arisen. Current testing protocols often include the use of two different species of animals and the administration of a test compound at a concentration relevant to human exposure data. In vivo models test prenatal exposures to assess malformations, structural variations, resorptions, and fetal growth of litters of the pregnant animals during the windows of susceptibility during organogenesis. Alternatively, in vivo methods dose both the males and females prior to mating and conceptus to evaluate the developmental endpoints after implantation, or only dosing pregnant dams and allowing the young to be delivered and raised to record offspring viability and functional/structural characteristics. However, in vivo developmental toxicity assays require a substantial amount of resources, including time, compounds, labor, and animal use. Therefore, the implementation of the three   R’s (reduction, refinement, and replacement) has led to broad interest in alternative methods, especially in vitro assays, to identify compounds with the potential to induce developmental toxicity.

Conventional in vitro developmental toxicity models include: the use of rodent embryo and zebrafish embryo cultures to evaluate the mechanisms of teratogenic potentials of compounds on diverse and well-documented organisms, and most recently the use of embryonic stem cells (ESCs) (reviewed in Luz and Tokar, 2018). Unfortunately, these traditional in vitro models lack the organ and organ relevant physiology that in vivo models excel in. Therefore, the use of human-derived cell lines examines a more representative in vitro model to human exposure, which has two significant subfamilies: primary cell lines and human pluripotent stem cells (hPSCs). Primary cell lines are obtained directly from a patient, and therefore, only certain cell types are available in minute quantities. Additionally, primary cells will be fixed at specific stages of development and require some form of transformation to induce proliferation in cell culture conditions. On the other hand, hPSCs have several qualities that make them better suited to investigate human developmental processes, as these cells have the potential to differentiate into any cell lineage using Yamanaka transforming factors (OCT4, SOX2, KLF4, and c-MYC). hPSCs aid in cell type investigation with identical genotypes as well as in the elucidation of how cells, embryos, and even organs develop. Furthermore, hPSCs have an unlimited self-renewal potential to provide an abundance of cellular material and the ability to examine varying specific developmental stages.

hPSCs split into two major subfamilies: ESCs and induced pluripotent stem cells (iPSCs), with the advantage of iPSCs not carrying the ethical barriers that surround ESCs. The development of iPSCs has provided a unique look into cell developmental mechanisms that traditional in vivo and in vitro models failed to deliver. More recently, iPSCs appear in high-throughput screening (HTS) and high-content analysis (HCA) assays, which allow for the rapid, unbiased characterization of a compound’s pharmaceutical and toxicological profiles. The major downside surrounding iPSCs come with the added cost of having to derive new cell lineages for each experiment, adding further risk of mutagenesis and oncogene activation and the potential of reprogramming alterations that remain in the cells’ epigenetic memory. Additionally, they fail to develop fully into their adult somatic cell counterparts. Technical improvements in differentiation protocols and three-dimensional modeling have shown promise in propagating maturation. The introduction of iPSCs has led to the burgeoning of developmental toxicology as a relevant human, and patient-specific physiological model of a disease state or induced toxicity can be observed rapidly and predictably while providing insights into mechanisms of developmental signals.

iPSCs in developmental toxicology

Currently, there are no golden standards for human in vitro developmental modeling; however, regulatory agencies recommend the use of human iPSCs (hiPSCs) to investigate the developmental processes, as the use of hiPSCs allows for investigators to examine developmental signaling pathways from the single-cell level throughout its specified maturation from an iPSC to a more representative adult somatic cell.

Recall that the four manifestations of developmental toxicology are: (1) death of the developing organisms, (2) structural abnormalities, (3) altered growth, and (4) functional alterations. While these manifestations traditionally are used at the organism level, they are useful to the single-cell level. The relationship between these two levels is simultaneously simple and complex. For instance, depending on the window of susceptibility and exposure, a xenobiotic can initiate cell death, thus resulting in the death of the developing organism. However, for a more nuanced manifestation, such as a structural abnormality, the relationship may not be as obvious. An example of this is limb development, where at the cell level, there may not be apparent abnormalities in cell morphology. Alterations at the cellular level may influence higher-order structures, such as tissue and muscular development, resulting in adverse outcomes such as stunted limb development.

Therefore, researchers need to be able to elucidate the mechanisms of the more apparent manifestations, such as cell death. For instance, iPSC-derived cardiomyocytes (iPSC-CMs) would be beneficial to investigate the effects of nicotine replacement therapies (NRT) and bupropion as remedies for smoking cessation. These methods have demonstrated adverse effects in offspring cardiomyocytes (Gopalakrishnan et al., 2017). These iPSC-CMs allow investigators to examine developmental processes throughout compound administration and iPSC-CM maturation. As mentioned previously, if NRT or bupropion induced cardiomyocyte cell death, more than likely organ/organismal fatality will occur. Furthermore, investigators would be able to determine the dose–response effects of NRTs or bupropion in a human in vitro model without the necessary translation from previously published in vivo or other in vitro systems. As the determination of a dose–response relationship is a principal component of toxicological research, it is vital to remember that environmentally relevant exposures may induce manifestations of developmental toxicology before cell death.

Traditionally, cell death is an endpoint in toxicological studies, while manifestations of structural abnormalities, altered growth, and functional alterations may result in cell death, they are essential to study as endpoints themselves. That is a xenobiotic that induces structural abnormalities before causing cell death, and which may be due to the exposure route or a nonlethal dose. For example, iPSC-derived hepatocytes are beneficial to study the effects of compounds such as icariin, a compound that induced off-target toxicity in the liver by affecting expression levels of F-actin thus perturbing the cellular scaffolding (Wang et al., 2015). F-actin analysis would allow the researcher to determine differences in cell cytoskeleton parameters compared to control at the single-cell level and correlate these alterations to structural abnormalities at a higher developmental level.

Cellular size and cell proliferation are the characterizations for growth. For the most part, cellular size correlates with structural alterations. For instance, if a cell cytoskeleton is perturbed, then the cell may not exhibit normal morphology and, therefore, could alter the size of the cell compared to the control. Most often, cell size is often overlooked as toxicants or gene mutations seldomly impair cell size in the majority of the exposed populations. Nevertheless, cell size is closely related to zygotic genome activation, as decreased cell size is known to induce premature zygotic genome activation. Additionally, reduced cell size affects the protein gradients inside individual cells changing cell polarity and the future fate of asymmetric or symmetric division. On the other hand, if a cell expands beyond its limits, necessary materials are not able to cross the membrane efficiently enough to accommodate its increased cellular volume. Therefore, cells must divide into smaller