Molecular Players in iPSC Technology

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.Molecular Players in iPSC Technology, Volume 12 addresses the molecular players underlying induced pluripotent stem cell (iPSC) generation, maintenance, expansion, and differentiation. The discovery of iPSCs revolutionized biomedical research. iPSC technology involves multiple molecular mechanisms. This volume covers exosomal microRNAs, auxiliary pluripotency-associated genes, inducible caspase-9 suicide gene, cell cycle proteins, ion channels, Notch signaling, kinase signaling, SOCS3/JAK2/STAT3 pathway, NANOG, Krüppel-like factors, H1FOO, and much more in iPSCs. The volume is written for researchers and scientists in stem cell therapy, cellular and molecular biology, and regenerative medicine and is contributed by world-renowned authors in the field.

• Provides overview of the fast-moving field of iPSC technology, regenerative medicine, and therapeutics
• Covers the different key molecular players involved in iPSC formation, maintenance, expansion, and differentiation
• Is contributed by world-renowned experts in the field

• Language: English
• Publisher: Academic Press
• Release date: Aug 29, 2021
• ISBN: 9780323900607

Book preview

Molecular Players in iPSC Technology, Volume 12

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

Contributors

About the editor

Preface

Chapter 1. Engineering exosomal microRNAs in human pluripotent stem cells

Introduction

The role of miRNAs in stem cell properties and disease development

Key signaling pathways regulated by miRNAs in stem cells

The role of exo-miRNAs derived from PSCs and MSCs

Engineering exo-miRNAs produced by stem cells

Conclusions and future directions

Chapter 2. Auxiliary pluripotency-associated genes and their contributions in the generation of induced pluripotent stem cells

Introduction

Esrrb

Sall4

Rex1

Tbx3

Utf1

Zscan4

Nr5a2

Glis1

L-Myc

Zic3

Foxh1

Conclusion

Chapter 3. Improving the safety of iPSC-derived T cell therapy

Introduction

Generation and use of iPSC-derived antigen-specific CTLs

Chimeric antigen receptor T cells generated from iPSCs

Increasing the safety of iPSC

Suicide-gene-based safeguard system

iC9-based safeguard system for iPSC-derived cell therapy

CART cell therapy with iC9 safeguard system

Banking iPSCs for various HLA types

Off-the-shelf T cell therapy and GvHD prevention

Genome-edited T cell therapy and protection from missing-self response of NK cells

Prospects for rejuvenated CTL therapy

Chapter 4. Induced pluripotency and intrinsic reprogramming factors: adult stem cells versus somatic cells

Introduction

Multiple factors/genes responsible for reprogramming/inducing pluripotency-library screening

Signaling pathways that enhance pluripotency during iPSCs generation

Wnt/β-catenin, TGF-β, and hippo signaling pathways

The ubiquitin-proteasome system

Epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition

Methods for induction of pluripotency

Integrative method

Lentiviral vectors

Transfection using linear DNA

Nonintegrative methods

Reprogramming using small molecules

Cell types with intrinsic reprogramming factors and their conversion into iPSC

Possible role and use of epigenetic modifiers in pluripotency induction

Histone H3 Lysine 9 Methylation

Histone H3 Lysine 79 (H3K79) Methylation

Methylation at histone 3 lysine 36 2/3

Histone Deacetylation

Induced pluripotency and gene incorporation—safety and efficacy issues

Cell therapy using induced pluripotent stem cells

Conclusion

Chapter 5. The role of cell cycle in reprogramming toward induced pluripotent stem cells (iPSCs)

Preface

Introduction

G1-phase: main regulators and reprogramming

S-phase/G2 and reprogramming

p53 and other tumor suppressors

Cip/Kip family and reprogramming to pluripotency

Future directions and perspectives

Chapter 6. Signaling pathways regulate cardiovascular lineage commitment of hPSCs

Human pluripotent stem cells

Cardiovascular development in the heart

Cardiac differentiation of hPSCs

Maturation of cardiomyocytes derived from hPSCs

Nonmyocytes derived from cardiac progenitors

HPSCs-derived cardiomyocytes used for disease modeling and drug screening platforms

HPSCs-derived cardiomyocytes for cellular therapy applications

Current challenges and future directions

Chapter 7. Role of ion channels in human induced pluripotent stem cells–derived cardiomyocytes

Introduction

Phases of the cardiac action potential

The ionic basis underlying hiPSC cardiomyocyte action potential

The ionic basis underlying hiPSC cardiomyocyte action potential

Transformation of hiPSC to the cardiac lineage

Strategies to enhance maturation

Summary and future directions

Chapter 8. Notch signaling in induced pluripotent stem cells

Introduction

Notch ligands and receptors

Canonical notch signaling

Noncanonical notch signaling

Receptor- and ligand-dependent notch signaling

Notch signaling interaction with other pathways

Notch signaling in stemness maintenance and differentiation

Notch signaling in induced pluripotent stem cells

Conclusion

Chapter 9. The extracellular signal-regulated kinase signaling pathway in biology of pluripotent stem cells

Introduction

ERK signaling in somatic cell reprogramming

ERK signaling in cardiovascular differentiation of pluripotent stem cells

Conclusions

Chapter 10. SOCS3/JAK2/STAT3 pathway in iPSCs

Introduction

Main text

Conclusions and future perspectives

Chapter 11. Nanog in iPS cells and during reprogramming

Introduction

Nanog discovery, function, and structure

Nanog multilayered regulation

Uncovering Nanog's connection to cell reprogramming

Reprogramming enhancement by Nanog affects efficiency and quality of iPS cells

Nanog is associated to multiple reprogramming roadblocks

Chromatin remodeling linked to Nanog function is relevant during reprogramming

Nanog reactivation is a common feature between reprogramming and some oncogenic processes

Concluding remarks

Chapter 12. The role of Krüppel-like factors in generating induced pluripotent stem cells

Overview of KLFs

Protein structures of KLFs

KLF4 and discovery of iPSC

Substitution of KLF4 by KLF2 and KLF5 in reprogramming

KLFs as members of autoregulated transcriptional network for stem cell identity

Dual role of KLF4 in a stepwise model of reprogramming

Efficiency of reprogramming as determined by stoichiometry

Reprogramming without exogenous KLFs

KLFs contributes to naïve pluripotency

Future direction: KLF interactome and the study of structure–function relationship

Chapter 13. The oocyte-specific linker histone H1FOO plays a key role in establishing high-quality mouse induced pluripotent stem cells

Issues surrounding induced pluripotent stem cells

Oocyte components can promote somatic cell reprogramming

Characteristics of linker histone H1

Oocyte-specific linker histone H1FOO is involved in open chromatin formation

Ectopic expression of H1foo in somatic cells enhances qualified iPSC generation

Transcript expression and methylation characteristics of OSKH-iPSCs

H1foo promotes in vitro and in vivo differentiation potentials of iPSCs

Future directions

Index

Advances in Stem Cell Biology

Series Editor

Alexander Birbrair

Copyright

Academic Press is an imprint of Elsevier

125 London Wall, London EC2Y 5AS, United Kingdom

525 B Street, Suite 1650, San Diego, CA 92101, United States

50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States

The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom

Copyright © 2022 Elsevier Inc. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

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.

Library of Congress Cataloging-in-Publication Data

A catalog record for this book is available from the Library of Congress

British Library Cataloguing-in-Publication Data

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

ISBN: 978-0-323-90059-1

For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci

Acquisitions Editor: Elizabeth Brown

Editorial Project Manager: Billie Jean Fernandez

Production Project Manager: Omer Mukthar

Cover Designer: Mark Rogers

Typeset by TNQ Technologies

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

Poulomi Adhikari

Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India

Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan

Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

Jun Ando,     Department of Hematology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan

Miki Ando

Department of Hematology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan

Division of Stem Cell Therapy, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan

Bipasha Bose,     Stem Cells and Regenerative Medicine Centre, Yenepoya Research Centre, Yenepoya University, Mangalore, Karnataka, India

Malcolm K. Brenner,     Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital and Texas Children's Hospital Houston, Houston, TX, United States

Kirstine Calloe,     Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark

Hee Cheol Cho,     Urowsky-Sahr Scholar in Pediatric Bioengineering, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, United States

Jonathan M. Cordeiro,     Department of Experimental Cardiology, Masonic Medical Research Institute, Utica, NY, United States

Chandrima Dey,     Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India

Camila Vazquez Echegaray

Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Química Biológica, Laboratorio de Regulación Génica en Células Madre, Buenos Aires, Argentina

CONICET - Universidad de Buenos Aires, Instituto de Química Biológica (IQUIBICEN), Buenos Aires, Argentina

Hiroshi Egusa,     Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, Sendai, Miyagi, Japan

Keiichi Fukuda,     Department of Cardiology, Keio University School of Medicine, Tokyo, Japan

Yoshiki Furukawa,     Department of Hematology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan

Yu Gao,     Department of Cardiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

Ranadeep Gogoi,     Department of Biotechnology, National Institute of Pharmaceutical Education and Research Guwahati, Guwahati, Assam, India

Alejandra Sonia Guberman

Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Química Biológica, Laboratorio de Regulación Génica en Células Madre, Buenos Aires, Argentina

CONICET - Universidad de Buenos Aires, Instituto de Química Biológica (IQUIBICEN), Buenos Aires, Argentina

Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Fisiología y Biología Molecular y Celular, Buenos Aires, Argentina

Pengcheng Han,     Urowsky-Sahr Scholar in Pediatric Bioengineering, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, United States

Krishna Kumar Haridhasapavalan,     Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India

Ping He,     Department of Medicine, Renaissance School of Medicine at Stony Brook University, Stony Brook, NY, United States

Michelle Jankova,     Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark

Richard Jeske,     Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL, United States

Saketh Kapoor,     Stem Cells and Regenerative Medicine Centre, Yenepoya Research Centre, Yenepoya University, Mangalore, Karnataka, India

Shintaro Kinoshita,     Department of Hematology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan

Akira Kunitomi,     Department of Fundamental Cell Technology, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan

Yan Li,     Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL, United States

Hiromitsu Nakauchi

Division of Stem Cell Therapy, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, United States

Irina Neganova,     Institute of Cytology, RAS, St-Petersburg, Russia

Thanaphum Osathanon,     Dental Stem Cell Biology Research Unit, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand

Jun Pu,     Department of Cardiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

Khyati Raina,     Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India

Sébastien Sart

Hydrodynamics Laboratory, CNRS UMR7646, Ecole Polytechnique, Palaiseau, France

Laboratory of Physical Microfluidics and Bioengineering, Department of Genome and Genetics, Institut Pasteur, Paris, France

Sudheer P. Shenoy,     Stem Cells and Regenerative Medicine Centre, Yenepoya Research Centre, Yenepoya University, Mangalore, Karnataka, India

Pradeep Kumar Sundaravadivelu,     Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India

S. Sudhagar,     Department of Biotechnology, National Institute of Pharmaceutical Education and Research Guwahati, Guwahati, Assam, India

Madhuri Thool

Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India

Department of Biotechnology, National Institute of Pharmaceutical Education and Research Guwahati, Guwahati, Assam, India

Rajkumar P. Thummer,     Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India

Jacqueline A. Treat,     Department of Experimental Cardiology, Masonic Medical Research Institute, Utica, NY, United States

Vishalini Venkatesan

Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India

Centre for Biotechnology, Anna University, Chennai, Tamil Nadu, India

Universitätsmedizin Göttingen, Göttingen, Germany

Xinxia Wang,     College of Animal Sciences, Zhejiang University, Hangzhou, China

Ruifan Wu,     College of Animal Sciences, Zhejiang University, Hangzhou, China

Vincent W. Yang

Department of Medicine, Renaissance School of Medicine at Stony Brook University, Stony Brook, NY, United States

Department of Physiology and Biophysics, Renaissance School of Medicine at Stony Brook University, Stony Brook, NY, United States

Department of Biomedical Informatics, Renaissance School of Medicine at Stony Brook University, Stony Brook, NY, United States

Lei Ye,     National Heart Research Institute Singapore, National Heart Centre Singapore, Singapore

Xuegang Yuan,     Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL, 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, and, in 2019, he was elected member of the Global Young Academy. 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 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 Molecular Players in iPSC Technology 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 molecular mechanisms involved in the manipulation of iPSCs, enabling us to study the cellular and molecular mechanisms involved in different pathologies. Further insights into these mechanisms will improve the use of iPSC technology for understanding of disease appearance, development, and progression. The authors focus on the modern state-of-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-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 molecular players in 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 key molecular players involved in iPSC technology. Thirteen chapters written by experts in the field summarize the present knowledge about molecular players in iPSC technology.

Yan Li and colleagues from Florida State University introduce exosomal MicroRNAs in iPSCs. Rajkumar P. Thummer and colleagues from Indian Institute of Technology, Guwahati, discuss auxiliary pluripotency-associated genes and their contributions in the generation of iPSCs. Miki Ando and colleagues from Juntendo University School of Medicine bring our attention to the use of inducible caspase-9 suicide gene to improve the safety of iPSC-derived T cell therapy. Sudheer Shenoy and colleagues from Yenepoya Research Center describe induced pluripotency and intrinsic reprogramming factors in adult stem cells versus somatic cells. Irina Neganova from Institute of Cytology updates us with what we know about the role of cell cycle in reprogramming toward iPSCs. Pengcheng Han and Hee Cheol Cho from Emory University School of Medicine compile our understanding of the progress in the signaling pathways that regulate cardiovascular lineage commitment of iPSCs. Jonathan M Cordeiro and colleagues from Masonic Medical Research Institute summarize current knowledge on the role of ion channels in human iPSC-derived cardiomyocytes. Thanaphum Osathanon and Hiroshi Egusa from Chulalongkorn University talk about notch signaling in iPSCs. Lei Ye and colleagues from Heart Research Institute focus on the extracellular signal-regulated kinase signaling pathway in the biology of iPSCs. Wang Xinxia from Zhejiang University addresses the importance of SOCS3/JAK2/STAT3 pathway in iPSCs. Camila Vazquez Echegaray and Alejandra Sonia Guberman from Universidad de Buenos Aires update us on the importance of Nanog in iPSCs. Ping He and Vincent W. Yang from Stony Brook University speak about the role of Krüppel-like factors in generating iPSCs. Finally, Akira Kunitomi and Keiichi Fukuda from Keio University School of Medicine outline the role played by the oocyte-specific linker histone H1FOO in establishing high-quality iPSCs.

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: Engineering exosomal microRNAs in human pluripotent stem cells

Sébastien Sart ¹ , ² , Xuegang Yuan ³ , Richard Jeske ³ , and Yan Li ³       ¹ Hydrodynamics Laboratory, CNRS UMR7646, Ecole Polytechnique, Palaiseau, France      ² Laboratory of Physical Microfluidics and Bioengineering, Department of Genome and Genetics, Institut Pasteur, Paris, France      ³ Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL, United States

Abstract

Stem cells possess unique properties of self-renewal and multilineage differentiation potentials. In addition, stem cells also exhibit important trophic function, by secreting large numbers of regulatory molecules such as cytokines and extracellular vesicles (EVs). Among them, exosomal miRNAs (exo-miRNAs), a type of small noncoding RNAs sorted in EVs, play an important role in the regulation of gene expression in various biological processes through cell–cell communications. As incorporated into EVs, exo-miRNAs can be delivered to recipient cells as cargos to regulate cellular behaviors. This chapter reviews the function of exo-miRNAs in EVs derived from stem cells, including mesenchymal stem cells and pluripotent stem cells, and summarizes current knowledge on the characteristics of miRNA expression at undifferentiated and lineage-primed stages of stem cells. Furthermore, the role of stem-cell-derived exo-miRNAs in regulating the phenotype of somatic and progenitor cells is also analyzed in order to elucidate their participations and the therapeutic function in specific diseases. Lastly, this work summarizes current approaches and progresses in engineering exo-miRNAs and EVs derived by stem cells toward potential clinical applications.

Keywords

Embryonic stem cells; Endosomal sorting complexes required for transport; EV biogenesis; EV cargo Engineering; EV heterogeneity; Exo-miRNA sorting; Exosomes; Extracellular vesicles (EV); Mesenchymal stem cells; microRNA; Mitogen-activated protein kinase; Notch signaling; Pluripotent stem cells; Ultracentrifugation; Wnt signaling

Introduction

The role of miRNAs in stem cell properties and disease development

miRNAs regulate reprogramming, self-renewal, and differentiation of PSCs

miRNAs regulate the differentiation of MSCs

miRNAs are involved in disease development

Key signaling pathways regulated by miRNAs in stem cells

Regulation of the canonical Wnt signaling by miRNAs in stem cells

Regulation of notch signaling by miRNAs in stem cells

Regulation of MAPKs by miRNAs in stem cells

The role of exo-miRNAs derived from PSCs and MSCs

Biological functions of exo-miRNAs derived from PSCs

Biological functions of exo-miRNAs derived from MSCs

Engineering exo-miRNAs produced by stem cells

Isolation and characterization of exo-miRNAs derived from stem cells

Methods to regulate exo-miRNA production in stem cells

Functional consequences of regulating exo-miRNAs production in stem cells

Conclusions and future directions

Acknowledgment

References

Introduction

In the recent years, pluripotent stem cells (PSCs) have emerged as a promising tool for tissue engineering, disease modeling, and drug screening. PSCs possess unique properties of self-renewal and differentiation potential into cellular lineages of the three germ layers (McCauley and Wells, 2017). At different developmental stages, PSCs and their progenies exhibit important trophic function, due to their capability of secreting different types of regulatory molecules (i.e., morphogens, growth factors, extracellular matrix proteins, extracellular vesicles, etc.) (Sart et al., 2014a; Moledina et al., 2012) that can regulate tissue regeneration and homeostasis. PSCs have been derived from the inner cells mass of blastocysts, i.e., embryonic stem cells (ESCs), or through the reprogramming of somatic cells (e.g., dermal fibroblasts, hematopoietic progenitor, etc.). The stable integration of pluripotency-related genes (e.g., OCT-4, KLF-4, SOX-2, c-Myc, etc.) enables differentiated cells to revert to a stage that mimics the behavior of ESCs (Takahashi et al., 2007), which is referred as induced PSCs (iPSCs). Increasing body of evidence has pronounced that this reprogramming of somatic cells into iPSCs represents an invaluable tool to generate patient-specific stem cells and provide a potential unlimited source of PSCs that can be safely used for disease modeling, drug screening, and cell therapy, due to their low risk for immune rejection (Rong et al., 2014).

iPSCs have been recently derived into functional mesenchymal stem cells (iMSCs), a type of mesodermal progenitor cells that can be differentiated into osteoblasts, chondrocytes, and adipocytes. MSCs derived from iPSCs share identical properties with their adult/fetal tissues counterpart (Zhao and Ikeya, 2018). Besides multilineage differentiation potential, MSCs can regulate tissue development and regeneration through paracrine effects, and they are one of the best studied cell populations expressing trophic activities (Sart et al., 2014b). Indeed, MSCs can secrete vast quantities of regulatory molecules, extracellular matrix proteins, as well as extracellular vesicles with functional cargos including growth factors, long noncoding RNAs (lnc-RNA), micro-RNAs (miRNAs), etc., which can modify the surrounding cellular and tissue microenvironment (Fu et al., 2017).

miRNAs are a type of short (about 19–24 nucleotides), generally single-strand, noncoding RNAs that can modulate gene expression by either RNA silencing or posttranscriptional regulations. miRNAs are usually synthetized in the nucleus by RNA polymerase II as long primary miRNAs (pri-miRNAs), which contain a stem loop structure. Pri-miRNAs are then primarily processed into pre-miRNAs by the enzymatic cleavage of the 5′ CAP sequence and the 3′ polyA tail through a complex composed of the Drosha and DGCR8 proteins. The 3′ ends of the pre-miRNAs are then recognized by exportin 5 (EXP5), which mediates their transportation into the cytoplasm. The secondary processing by the RNAse DICER mediates the formation of miRNA duplexes via cleavage of the hairpin loop. The miRNAs are eventually incorporated with Argonaute (Argo) complexes that degrade one of the complementary strands of the duplex (Alberti and Cochella, 2017), leading to the formation of mature RNA-induced silencing complexes (RISCs). RISCs induce the cleavage of perfectly complementary mRNAs or a transcriptional repression for the case of partial complementarity (Fig. 1.1).

miRNAs represent an important role to regulate gene expression of stem cells during differentiation. Moreover, due to the unique repertoire of miRNAs produced by stem cells and their progenies, their delivery by extracellular vesicles (EVs) can mediate cell–cell communication and modify phenotype of the recipient cells. Moreover, changes in microenvironment of stem cells can influence their exo-miRNA profiles, providing a possible engineering strategy for exo-miRNA therapy. Therefore, miRNAs secreted by stem cells have attracted a lot of interests in numerous clinical fields, such as tissue engineering, oncology, immunology, etc.

Based on their biogenesis, miRNAs can be packed and specifically sorted into two major distinctive populations of EVs: microvesicles (MVs) and exosomes. MVs are large EVs (100   nm −¹ μm) generated by direct fission and outward budding at the plasma membrane, through ATPase-dependent signaling, externalization of phosphatidylserine, and cytoskeleton remodeling. MVs are thus expected to retain a large part of cell-derived membrane proteins (e.g., integrins). By contrast, exosomes constitute a different class of EVs that differ in size (50–150   nm) and expression of membrane makers. The biogenesis of exosomes starts with formation of intraluminal vesicles (ILVs) through the invagination of membrane of late endosomes or multivesicular bodies (MVBs), which is mediated by the endosomal sorting complexes required for transport (ESCRT) machinery and its associated proteins (e.g., Alix, Syntenin, and Syndecan). Exosomes are therefore usually characterized by specific endosomal transmembrane proteins such as CD63, CD81, and CD9. The cargos in exosomes (in particular miRNAs) are likely subjected to selective sorting and targeting. The secretion of exosomes is mediated by actin and tubulin, as well as Rab GTPases (in particular Rab27a and Rab27b) that trigger membrane fusion and ultimately release of exosomes to the extracellular space (Fig. 1.2, Hessvik and Llorente, 2018).

Figure 1.1 Mechanisms of formation of miRNAs.Genes of miRNAs are transcribed into primary miRNAs by polymerase II and then transmitted into pre-miRNAs via Drosha/DGCR8 complexes. Pre-miRNAs are released from nucleus to the cytosol via exportin-5 (EXP5) and sliced by DICER into double-stranded miRNAs. With the complex of Argo and DICER, one strand of double-stranded miRNAs is selectively incorporated into RNA-induced silencing complex (RISC) and further inhibits target gene expression.

Both MSCs and PSCs are capable of secreting EVs enriched with miRNAs that play important roles in the process of stem cell fate decisions. The role of EVs derived from PSCs and other derivatives has attracted growing interests recently (Jeske et al., 2019). However, the specific role of exo-miRNAs derived from iPSCs has not been well explored. This article reviews current knowledge on the role of miRNAs in the regulation of stem cell phenotype and disease development. In addition, the progress of recent studies in understanding the role of exo-miRNAs secreted by stem cells to regulate tissue homeostasis is summarized. Finally, by illustrating the role of exo-miRNAs secreted by iMSCs as an example of iPSC derivatives, this chapter sheds light on a broad spectrum of stem-cell-derived exo-miRNAs, including emergent routes, regulation of the production yields, and the diversity of their targets.

Figure 1.2 Mechanisms of formation of extracellular vesicles: microvesicles and exosomes.The origin of microvesicles (MVs) is cytosol and plasma membrane. MVs are budding and fussed from plasma membrane that generally requires cytoskeletal reorganization and interactions with endosomal sorting complexes required for transport (ESCRT) proteins. Cargos in cytosol can be sorted or randomly packed into MVs. Exosomes, however, originate from internal endosomal membrane of late endosomes or multivesicular bodies (MVBs). Complex mechanism is involved in exosome biogenesis including both ESCRT-dependent and independent pathways. Moreover, cargo sorting into exosomes is more selective and specific and is regulated by Syndecan-Syntenin-Alix, lipid rafts, and tetraspanin protein interactions.

The role of miRNAs in stem cell properties and disease development

miRNAs regulate reprogramming, self-renewal, and differentiation of PSCs

ESC-specific cell cycle–regulating (ESCC) family of microRNAs play a pivotal role in overcoming the barriers that limit the reprogramming of somatic cells and further retain the pluripotency of undifferentiated cells (Hao et al., 2017). For instance, miR-93, miR-106b, miR-302, and miR-372 promote the generation of iPSCs by decreasing the activity of transforming growth factor-β signaling and by promoting epithelial-to-mesenchymal transition (EMT) (Li et al., 2011; Subramanyam et al., 2011). In addition, miR-138 promotes reprogramming of embryonic fibroblasts by decreasing p53 expression (Ye et al., 2012), while miR-34 was found to provide a barrier for somatic cell reprogramming (Choi et al., 2011). Alternatively, the miR-290 family enhances the pluripotency of PSCs by repressing nuclear factor kappa B (NF-κB) signaling that regulates EMT (Lüningschrör et al., 2012), while miR-363 regulates the maintenance of an undifferentiated state by inhibiting Notch signaling (de Souza Lima et al., 2019). Moreover, miR-200c-5p inhibits the expression of SIRT2, which in turn decreases oxidative phosphorylation, promotes glycolysis, and reduces the production of reactive oxygen species. This metabolic shift is associated with enhanced expression of stemness genes such as OCT-4, NANOG, and REX-1 within hPSCs (Cha et al., 2017).

About 100 different types of miRNAs have been identified in regulation of the transition between the naïve and the primed stage of PSCs (Wang et al., 2019). In particular, let-7c-5p and miR-134-5p were found to favor an escape from ground state in 2i-medium culture conditions, while naive pluripotency was driven by miR-290/302 (Yan et al., 2017). In addition, the germ-layer specification upon differentiation of iPSCs involves the production of specific miRNAs: miR-483-3p promotes mesoderm formation, while miR-489-3p and miR-1263 promote the differentiation into endoderm derivatives (Ishikawa et al., 2017). Moreover, the directed differentiation of PSCs toward specific lineages involves the expression of particular miRNAs. For instance, overexpression of miR-375 and miR-186 induces pancreatic differentiation of hiPSCs (Lahmy et al., 2014; Shaer et al., 2014), while overexpression of miR-184 induces the differentiation of retinal pigment epithelium cells, by repressing the mammalian target of the rapamycin (mTOR) signaling pathway (Jiang et al., 2016). miR-211 was shown to be involved in the osteoblastic differentiation of PSCs through induction of autophagic events (Ozeki et al., 2017). Alternatively, downregulation of the miR-10a family is involved in neural differentiation by inhibiting Wnt signaling (Kulcenty et al., 2019).

miRNAs regulate the differentiation of MSCs

The phenotype of MSCs is also regulated by differential expression of specific miRNAs (Li, 2018). For instance, miR-193a was found to inhibit osteogenic differentiation by blocking mitogen-activated protein kinase (MAPK) and Wnt signaling (Wang et al., 2018a). In contrast, miR-27 favors osteogenic differentiation by activating Wnt signaling (Wang and Xu, 2010). The miR-320 family is involved in adipogenic differentiation by repressing the expression of RUNX-2 (Hamam et al., 2014). In addition, miR-199a-3p promotes the expression of PPAR-γ, AP-2, and the accumulation of triglyceride in MSCs, through the inhibition of Wnt signaling (Shuai et al., 2019). These results highlight the balance between adipogenic and osteogenic differentiation of MSCs, in which, the differentiation into one lineage inhibits the commitment toward the other lineages (Xu et al., 2016). Finally, miR-218 was found to decrease the expression of osteogenic markers and promote the expression of chondrogenic proteins (e.g., SOX-9 and collagen type II) and glycosoaminoglycans (Chen et al., 2019). Similarly, miR-132 promotes chondrogenesis by increasing the expression level of ADAMTS‐5 (Zhou et al., 2018). In contrast, miR-483 was found to inhibit chondrogenic differentiation, by downregulating the activity of SMAD4 signaling (Anderson and McAlinden, 2017), while miR-30a was shown to promote contrasting effect on chondrogenesis, depending on the MSC tissue source (Tian et al., 2016; Zhang et al., 2019a).

miRNAs are involved in disease development

miRNAs are actively involved in diseases development and can serve as circulating biomarkers for cancers, cardiovascular diseases, and diseases of the lung, kidney, or nervous system (Moldovan et al., 2014). While the abnormal expression of miRNAs can be associated with the emergence of cancers, miRNAs can also serve as tumor suppressors. For example, miR-613 inhibits proliferation and migration and induces apoptosis of various cancer cells (e.g., colorectal-, hepatic-, gastric-, lung-, breast-cancer cells) (Mei et al., 2020). Increased expression of miR-17/92 cluster is associated with hyperproliferation of lung epithelial cells that initiates cancer formation in lungs by targeting myc (Dutta et al., 2019). In addition, a high level of expression of miR-155 has been found to be involved in increased lung cancer development (Shao et al., 2019). miRNAs are also involved in the process of tumor metastasis. For instance, miR-374a participates the process of EMT of breast cancer cells through activation of Wnt signaling (Cai et al., 2013). Alternatively, myocardial infraction exhibits distinct miRNA profile with decreased expression of miR-24, miR-320, and miR-29 and increased expression of miR-320, a proapoptotic miRNA (Wojciechowska et al., 2017; Wang et al., 2016). Similarly, several brain disorders, such as stroke (e.g., miR-210) and spinal cord injury (e.g., miR-33), are also associated with increased expression of miRNAs related to alterations of endothelium integrity and function (Ma et al., 2020).

Key signaling pathways regulated by miRNAs in stem cells

Differential expression of specific miRNAs plays an important role in regulating the phenotype and the differentiation potential of stem cells, by targeting specific signaling pathways (e.g., Wnt, Notch, MAPK, SMAD, etc.). This section discusses the mechanisms of miRNA regulation of stem cell fate decisions via these signaling pathways.

Regulation of the canonical Wnt signaling by miRNAs in stem cells

In the absence of Wnt ligands (e.g., Wnt-3a), glycogen synthase kinase 3 (GSK3) induces the phosphorylation of β-catenin, which is degraded by proteasomes. The binding of Wnt ligands on frizzled transmembrane receptor promotes the inhibition activity of GSK3, which prevents the phosphorylation of β-catenin that accumulates in the cytoplasm. This leads to the translocation of β-catenin into the nucleus and its complexation with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) complex that induces the expression of Wnt-target genes. In MSCs, the activation of Wnt signaling induces osteogenic and chondrogenic differentiation, while inhibiting adipogenic differentiation. For PSCs, however, Wnt signaling plays a dual role: it promotes stem cell self-renewal as well as early stage of lineage commitment (de Jaime-Soguero et al., 2018). A high level of Wnt activation is required to sustain the ground state of PSCs, which is gradually decreased during the transition to primed stage (de Jaime-Soguero et al., 2018). In addition, induction of mesendoderm differentiation requires a significant increase of Wnt activity, which is abrogated during ectodermal differentiation (de Jaime-Soguero et al., 2018).

miRNAs can efficiently trigger the activity of Wnt in MSCs (Jing et al., 2015). For instance, miR-29 can induce the production of β-catenin as well as its degradation in the nucleus, which consequently promotes osteogenic differentiation of MSCs. In contrast, miR-199a-3p was found to decrease Wnt activity in MSCs, by repressing KDM6A (an inducer of WNT3 production). In PSCs, the miR-10a family, in particular hsa-miR-100-5p, was found to repress Wnt signaling probably by promoting the expression of DKK1 (Lu et al., 2017), leading to the neuronal differentiation (Kulcenty et al., 2019).

Regulation of notch signaling by miRNAs in stem cells

The interactions of Notch transmembrane proteins with specific ligands (e.g., Delta, Jagged, etc.) in heterotypic cell–cell interactions can trigger the cleavage of the Notch intracellular domain (NICD). NICD can then translocate to the nucleus where it acts as a transcription factor of Notch target genes (e.g., Hey proteins). In MSCs, the activation of Notch signaling participates in osteogenic differentiation (Cao et al., 2017; Semenova et al., 2020), while its continuous activation abrogates chondrogenic and adipogenic differentiation (Oldershaw and Hardingham, 2010; Song et al., 2015; Tian et al., 2015). For PSCs, Notch activation promotes early commitment and the loss of pluripotency (de Souza Lima et al., 2019) and is involved in gliogenesis (Patterson et al., 2014).

miRNAs have been found to regulate Notch signaling in PSCs. For instance, miR-263 inhibits the expression of Notch receptor leading to better retention of pluripotency (de Souza Lima et al., 2019). Alternatively, let-7 miRNA promotes the differentiation of PSCs toward glial cells, by decreasing Notch signaling and suppressing HES protein expression (Patterson et al., 2014). For MSCs, miR-30a promotes chondrogenesis of rat MSCs by increasing the expression level of the Notch ligand Delta (DLL4) (Tian et al., 2016). In addition, miR-34a-5p regulates Notch that leads to reduced osteogenic differentiation in MSCs treated with dexamethasone.

Regulation of MAPKs by miRNAs in stem cells

The three major classes of MAPKs are extracellular-signal-regulated kinase (ERK) 1/2, Jun N-terminal kinase/p38, and ERK5. MAPKs are serine/threonine protein kinases that are generally activated through the activation of a transmembrane receptor. The phosphorylation cascade results in the activation of transcription factors involved in cell proliferation, apoptosis, and differentiation. In MSCs, MAPKs (in particular ERK) are actively involved in osteogenic and chondrogenic differentiation, while inhibiting adipogenic differentiation (Miao et al., 2018; Ma et al., 2019; Ge et al., 2016). For PSCs, MAPKs play an essential role in inducing their lineage priming, while its inhibition is required to maintain their naïve pluripotent stage (Tsanov et al., 2017).

miRNAs can efficiently trigger MAPKs in MSCs. miR-15b that triggers MAPK in MSCs is required to induce osteogenic differentiation, while its knock-down promotes adipogenesis (Vimalraj and Selvamurugan, 2014). Similarly, miR-143 was found to inhibit MAPK (ERK5) and promote adipogenic differentiation of MSCs (Chen et al., 2014). In PSCs, miR-200 family and miR-290/295 cluster were found to repress MAPK activity, which mediates the suppression of EMT and consequently promotes pluripotency (Xiao et al., 2017). In turn, the time-controlled expression of miR-199 promotes neural differentiation of PSCs by modulating MAPK (PAK4) (Mellios et al., 2018).

The role of exo-miRNAs derived from PSCs and MSCs

miRNAs produced by stem cells can be sorted and incorporated into EVs and delivered to recipient cells for functional output. Functional miRNAs have access to either neighboring cells or distant cells via the circulating EVs. Though other cargos such as proteins and lipids also regulate cellular events and signaling pathways in recipient cells, miRNAs are considered as the major functional compartment in EVs. Traditionally, miRNAs delivered in recipient cells function as negative regulation of targeted mRNA expression. However, recent studies also point out the potentials of exo-miRNAs as binding ligands to the tool-like receptors to further activate immune response (Fabbri et al., 2012). In this section, the studies involved in exo-miRNAs derived from stem cells will be thoroughly reviewed.

Biological functions of exo-miRNAs derived from PSCs

It has been reported that cardiomyocytes derived from ESCs and iPSCs secrete exosomes showing similar repertoire of miRNAs (Lee et al., 2017). However, the information regarding the cargo profile of EVs or the biological function of exo-miRNAs derived from both ESCs and iPSCs remains scarce (Table 1.1). EVs from undifferentiated mouse ESCs have been identified with high enrichment of miR-290-295 cluster, which potentially mediate regeneration of cardiomyocyte and cardiac progenitor cells in postinfarct myocardium (Khan et al., 2015). Moreover, miR-291-3p from undifferentiated ESCs promotes the resistance to cellular senescence of dermal fibroblast (Bae et al., 2019). Similarly, miRNAs (miR-21 and miR-210) in EVs derived from undifferentiated iPSCs inhibit apoptosis of cardiomyocytes, leading to improved therapeutic effects (Wang et al., 2015). Upon differentiation, EVs from linage-specific cells derived iPSCs are enriched with exo-miRNAs that stimulate proliferation and maturation of undifferentiated cells into the phenotype of the secretory cell type (Ye et al., 2019; Quan et al., 2017). Other studies are summarized in Table 1.1.

Table 1.1

AKT, (v-Akt Murine Thymoma Viral Oncogene)/PKB (Protein Kinase-B); ESCs, embryonic stem cells; hATIIC, human alveolar progenitor type II cells; iPSCs, induced pluripotent stem cells; TGF, transforming growth factor; PI3K, phosphoinositide 3-kinase.

Biological functions of exo-miRNAs derived from MSCs

There is considerably more information on the cargo profiles and the biological function of exo-miRNAs secreted by MSCs than PSCs (Table 1.2). Though the miRNA profile in EVs is similar across MSCs isolated from different tissue sources, their relative proportions can slightly differ based on tissue origin (Baglio et al., 2015). Note that the cargo content including proteins and lipids is different across the EVs from different source of hMSCs (Villatoro et al., 2019; Shao et al., 2020). In addition, EV cargo, especially miRNA profile in MSCs, is relatively unstable under in vitro culture due to MSCs' sensitivity to artificial culture environment. Medium composition, oxidative stress, and flow dynamics all result in MSC-EVs with different cargos and biological function (Wei et al., 2016; Liao et al., 2017; Haraszti et al., 2018; Huang et al., 2019). In comparison to the whole pool of miRNAs produced by MSCs or most of other cell types, exosomal miRNAs generally exhibit different profiles, indicating a potential selectively sorting process rather than random incorporation of miRNA into EVs, though the mechanism of miRNAs sorting into EVs is not well understood (Baglio et al., 2015; Huang et al., 2013; Guduric-Fuchs et al., 2012; Baglio et al., 2015). Moreover, miRNAs are found both in mature and in precursor forms in MSCs, which likely influence exo-miRNA profile (Baglio et al., 2015). Primitive MSCs (isolated from tissue sources or derived from iPSCs) secrete numerous exo-miRNAs, which show broad biological functions ranging from the regulation of cancer cell activities, the differentiation, angiogenesis, immune cell activation, etc. (Table 1.2). The broad functional variance of exo-miRNA is likely due to the sources and in vitro culture environment. Similar to differentiated PSCs, differentiated MSCs secrete exo-miRNAs that promote the differentiation into specific lineages. For instance, miRNAs profile is regulated to facilitate adipogenic and osteogenic differentiation of MSCs under induction (Xu et al., 2014; Narayanan et al., 2018). Thus the secreted exo-miRNAs of lineage-specific MSCs can promote respectively adipogenic or osteogenic differentiation (Narayanan et al., 2018; Cui et al., 2016). Consistently, chondrogenically differentiated MSCs secreted exo-miR‐320c that promotes chondrogenesis (i.e., SOX9 expression) (Table 1.2, Sun et al., 2019a).

Engineering exo-miRNAs produced by stem cells

Isolation and characterization of exo-miRNAs derived from stem cells

EVs are usually isolated by ultracentrifugation (× 100,000   g) and sucrose gradients. Consequently, the resulting pellets are usually enriched with exosomes but also with small MVs, which may show different functional properties from exosomes (Hessvik and Llorente, 2018). In fact, most of the isolation strategies result in heterogeneous subpopulation of vesicles including MVs and exosomes (Théry et al., 2018). Therefore, there is an urgent need