Recent Advances in iPSC-Derived Cell Types

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.

Recent Advances in iPSC-derived Cell Types, Volume 4 addresses how different cell types can be derived from induced pluripotent stem cells.

Somatic cells can be reprogrammed into Induced pluripotent stem cells by the expression of specific transcription factors. These cells are transforming biomedical research in the last 15 years. The volume teaches readers about current advances in the field. This book describes the use of induced pluripotent stem cells to form different cell types which can be used in cell therapy as well as to model several diseases in vitro, enabling us to study the cellular and molecular mechanisms involved in different pathologies. In recent years, remarkable progress has been made in the obtention of induced pluripotent stem cells and their differentiation into several cellular populations, tissues and organs using state-of-art techniques. This volume will cover what we know so far about the use of iPSCs to derive different cell types, such as: erythroid cells, mucosal-associated invariant T cells, megakaryocytes, cerebral cortical neurons, inner ear cell types, airway epithelial cells, male germ cells, trophoblasts, cardiomyocytes, ßpancreatic cells, and more.

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

• Provides overview of the fast-moving field of induced pluripotent stem cell technology, regenerative medicine and therapeutics
• Covers the following cell types derived from iPSCs: erythroid cells, mucosal-associated invariant T cells, megakaryocytes, cerebral cortical neurons, inner ear cell types, airway epithelial cells, male germ cells, trophoblasts, cardiomyocytes, ßpancreatic cells, and more
• Contributions from stem cell leaders around the world

• Language: English
• Publisher: Academic Press
• Release date: Feb 21, 2021
• ISBN: 9780128224540

Book preview

Recent Advances in iPSC-Derived Cell Types, Volume 4

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

Copyright

Dedication

Contributors

About the editor

Preface

Chapter 1. iPSC-derived erythroid cells

Hematopoiesis and erythropoiesis in vivo

Globin expression and the globin switch

Hematopoietic differentiation of iPSCs

Erythroid differentiation of iPSC-derived HSCs

The globin expression profile of iPSC-derived erythroid cells

Identifying the enucleation defect of iPSC-derived erythroid cells

Improving the performance of iPSC-derived erythroid cells

Use of iPSCs to create models of RBC diseases

Streamlining the generation of erythroid cells from iPSCs

Karyotype of iPSCs

Future perspectives

Chapter 2. iPSC-derived mucosal-associated invariant T cells

Introduction

Perspective

Conclusions

Chapter 3. Advances in stem cell biology: induced pluripotent stem cells—novel concepts iPSC-derived megakaryocytes

Introduction

Platelets and transfusions

Megakaryocytes

Megakaryocyte production in vitro

Megakaryocytes from hematopoietic stem cells

Megakaryocytes from pluripotent stem cells

Producing platelets from megakaryocytes

Conclusion

Chapter 4. From human pluripotent stem cells to cerebral cortical neurons

Introduction

Cortical neurons in vivo: diversity and developmental mechanisms

Human cortical neurons in vitro: pluripotent stem cell-derived models

Chapter 5. Differentiation of inner ear cell types from human-induced pluripotent stem cells for the therapeutic application in sensorineural hearing loss

Human auditory system

Etiological factors for hearing loss and current therapeutic options

Cell types in the inner ear

Stem cells as a source of inner ear cell types

Differentiation of the OPCs

Cell fate of iPSC-derived OPCs after transplantation

Differentiation of inner ear HCs from stem cells

Differentiation of SGNs

Differentiation of outer sulcus cells

iPSC-derived otic organoids

Conclusion and future concepts

Chapter 6. Application of iPS cell-derived airway epithelial cells for cell-based therapy and disease models

Introduction

How to obtain airway epithelial cells from iPS cells

Application of iPS cell-derived airway epithelial cells for cell-based therapy and disease models

Future directions

Chapter 7. Male germ cell derivation from PSCs

Introduction

Mammalian germ cell development

The mechanisms of PGC fate specification

Male germ cell derivation from mouse PSCs

Male germ cell derivation from human PSCs

Male germ cell derivation from PSCs in nonhuman primates

Perspectives

Chapter 8. Induction of trophoblast differentiation in human-induced pluripotent stem cells by cell adhesion restriction

Introduction

Adhesion restriction triggers self-organization and differentiation of human iPS cells

Establishing human trophoblast stem cells from mesh-derived hiPSC cysts

Conclusion

Chapter 9. Induced pluripotent stem cell-derived cardiomyocytes: generation and enrichment protocols, immature and mature structure and function

Introduction

Procedures for differentiating iPSC into cardiomyocytes, selection and enrichment

Functional properties of immature iPSC-CMS

Experimental procedures for promoting maturation of immature induced pluripotent stem cell-derived cardiomyocytes

Concluding remarks

Chapter 10. A demanding path from iPSCs toward pancreatic β- and α-cells

Introduction

The pancreatic niche

iPSCs as a potential source of pancreatic cells

From iPSCs toward β-cells

From PSCs toward α-cells differentiation

Potential role of iPCs-derived β- and α-cells in diabetes treatment

Challenges and future directions

Index

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 © 2021 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: 978-0-12-822230-0

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 Fernande

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, 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 father, Lev Birbrair, and my beloved mom, Marina Sobolevsky, of blessed memory (July 28, 1959–June 3, 2020)

Contributors

Polina Baskin,     Department of Physiology, Biophysics and Systems Biology, Ruth and Bruce Rappaport Faculty of Medicine and Research Institute, Technion – Israel Institute of Technology, Haifa, Israel

Ofer Binah,     Department of Physiology, Biophysics and Systems Biology, Ruth and Bruce Rappaport Faculty of Medicine and Research Institute, Technion – Israel Institute of Technology, Haifa, Israel

Deborah E. Daniels,     School of Biochemistry, University of Bristol, Bristol, United Kingdom

Mor Davidor,     Department of Physiology, Biophysics and Systems Biology, Ruth and Bruce Rappaport Faculty of Medicine and Research Institute, Technion – Israel Institute of Technology, Haifa, Israel

Roxana Deleanu,     Medical University of Innsbruck Institute for Neuroscience, Innsbruck, Austria

Irit Dolgopyat,     Department of Physiology, Biophysics and Systems Biology, Ruth and Bruce Rappaport Faculty of Medicine and Research Institute, Technion – Israel Institute of Technology, Haifa, Israel

Daniel C.J. Ferguson,     School of Biochemistry, University of Bristol, Bristol, United Kingdom

Domingos Ferreira,     Faculdade de Farmácia, University of Porto, Porto, Portugal

Helena Florindo,     Faculdade de Farmácia, University of Lisbon, Lisbon, Portugal

Jan Frayne,     School of Biochemistry, University of Bristol, Bristol, United Kingdom

Cédric Ghevaert

Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, Cambridge, Cambridgeshire, United Kingdom

Department of Haematology, University of Cambridge and NHS Blood and Transplant, Cambridge, Cambridgeshire, United Kingdom

Akihiro Hazama,     Department of Cellular and Integrative Physiology, School of Medicine, Fukushima Medical University, Fukushima, Japan

Yi-Chao Hsu,     Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan

Fatemeh Kermani,     Institute of Physiology and Pathophysiology, Division of Cardiovascular Physiology, University of Heidelberg, Heidelberg, Germany

Osamu Kurosawa,     The Compass to Healthy Life Research Complex Program, RIKEN Institute, Kobe, Hyogo, Japan

Moyra Lawrence

Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, Cambridge, Cambridgeshire, United Kingdom

Department of Haematology, University of Cambridge and NHS Blood and Transplant, Cambridge, Cambridgeshire, United Kingdom

Zhousi Li,     The Compass to Healthy Life Research Complex Program, RIKEN Institute, Kobe, Hyogo, Japan

Dongli Liang,     Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China

Katherine A. MacInnes,     School of Biochemistry, University of Bristol, Bristol, United Kingdom

Joana Moreira Marques

i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

Faculdade de Farmácia, University of Porto, Porto, Portugal

Rute Nunes,     i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

Kennedy Omondi Okeyo,     Institute for Frontier Life and Medical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan

Danielle Regev,     Department of Physiology, Biophysics and Systems Biology, Ruth and Bruce Rappaport Faculty of Medicine and Research Institute, Technion – Israel Institute of Technology, Haifa, Israel

Bruno Sarmento

i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde & Instituto Universitário de Ciências da Saúde, Gandra, Portugal

Chie Sugimoto,     Host Defense Division, Research Center for Advanced Medical Science, Dokkyo Medical University, Tochigi, Japan

Chia-Ling Tsai,     Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan

Nina D. Ullrich,     Institute of Physiology and Pathophysiology, Division of Cardiovascular Physiology, University of Heidelberg, Heidelberg, Germany

Hiroshi Wakao,     Host Defense Division, Research Center for Advanced Medical Science, Dokkyo Medical University, Tochigi, Japan

Yuan Wang,     Department of Animal Sciences, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI, United States

Susumu Yoshie,     Department of Cellular and Integrative Physiology, School of Medicine, Fukushima Medical University, Fukushima, Japan

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 the biology of induced pluripotent stem cells (iPSCs) from different perspectives. Therefore, the book was subdivided into several volumes.

This volume "Recent Advances in iPSC-derived Cell Types" 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 use of iPSCs to different cell types 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-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, this 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 what we know so far about the derivation of distinct cell types from iPSCs. Ten chapters written by experts in the field summarize the present knowledge about iPSC-derived cell types.

Jan Frayne and colleagues from the University of Bristol discuss the derivation of erythroid cells from iPSCs. Hiroshi Wakao and Chie Sugimoto from Dokkyo Medical University describe iPSC-derived mucosal-associated invariant T cells. Moyra Lawrence and Cédric Ghevaert from the University of Cambridge compile our understanding of iPSC-derived megakaryocytes. Roxana Deleanu from Medical University of Innsbruck updates us with what we know about iPSC-derived cerebral cortical neurons. Yi-Chao Hsu and Chia-Ling Tsai from Mackay Medical College summarize current knowledge on differentiation of inner ear cell types from iPSCs. Susumu Yoshie and Akihiro Hazama from Fukushima Medical University address the importance of iPSC-derived airway epithelial cells. Dongli Liang and Yuan Wang from Michigan State University talk about male germ cell derivation from iPSCs. Kennedy Omondi and colleagues from Kyoto University focus on trophoblast differentiation from human iPSCs. Ofer Binah and colleagues from Technion – Israel Institute of Technology give an overview of iPSC-derived cardiomyocytes. Finally, Bruno Sarmento and colleagues from the University of Porto present a demanding path from iPSCs toward β- and α-pancreatic cells.

It is hoped that the chapters 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: iPSC-derived erythroid cells

Daniel C.J. Fergusona, Katherine A. MacInnesa, Deborah E. Daniels, and Jan Frayne     School of Biochemistry, University of Bristol, Bristol, United Kingdom

Abstract

The generation of erythroid cells from iPSCs has the potential to open avenues of opportunity in blood cell technologies. These include fundamental research, large-scale manufacture of blood products for transfusion medicine, and the creation of disease model systems for the development of novel therapeutics. Before erythroid differentiation, iPSC needs to be under a process of hematopoietic differentiation to obtain the necessary progenitor cells. The optimal system for this is not yet determined although steps have been made to both simplify and increase efficiency. Similarly, erythroid culture systems vary, but in most cases erythroid differentiation is achieved although expansion rates are low and, in particular, enucleation rates are poor and highly variable between studies, the mechanistic details for which are still largely unresolved. Notwithstanding, significant improvements have been made in recent years with adjustments to culture systems and components. Furthermore, iPSC-derived erythroid cells express predominantly fetal and embryonic, with little adult globin although as above approaches to resolve, including coculture and genetic manipulation are promising. Finally, albeit within the described caveats, iPSC technology provides an important approach to creating much needed human model cellular systems for red blood cell diseases, for both study and as drug testing platforms. As improvements to the erythroid potential of iPSC lines continue, the existing potential and application of this technology for such approaches will be revealed.

Keywords

Adult globin; Cellular disease models; Diamond Blackfan anemia; Embryonic globin; Enucleation; Erythroid; Erythroid progenitor; Erythropoiesis; Fetal globin; Hematopoietic stem cell (HSC); Hematopopoietic; Hemoglobin; Sickle cell disease; Thalassemia

Hematopoiesis and erythropoiesis in vivo

Globin expression and the globin switch

Hematopoietic differentiation of iPSCs

Erythroid differentiation of iPSC-derived HSCs

The globin expression profile of iPSC-derived erythroid cells

Identifying the enucleation defect of iPSC-derived erythroid cells

Improving the performance of iPSC-derived erythroid cells

Use of iPSCs to create models of RBC diseases

Hemoglobinopathies

Diamond Blackfan anemia

Congenital dyserythropoietic anemia type IV

Streamlining the generation of erythroid cells from iPSCs

Karyotype of iPSCs

Future perspectives

References

Red blood cells (RBCs) cultured in vitro from human stem cell sources represent an attractive alternative to donated blood, particularly for patients with rare or hard-to-match blood group phenotypes, who represent an unmet clinical need. These stem cell-derived erythroid cultures seek to replicate normal erythropoiesis to produce cells that are functionally identical to those from a donor source (Anstee et al., 2012). In recent years, groups have established efficient erythroid culture systems to support the terminal differentiation of adult peripheral blood (PB) and umbilical cord blood (CB) CD34+ stem cells in vitro (Griffiths et al., 2012a; Kupzig et al., 2017). However, although such CD34+ cells undergo efficient erythroid differentiation, with enucleation rates of 60%–95% (Griffiths et al., 2012a; Kupzig et al., 2017; Timmins et al., 2011), the resultant erythroid cells have a restricted expansion potential that limits the yield of reticulocytes from any culture (Anstee et al., 2012), requiring repeat collection of stem cells from donors. The generation of erythroid cells from induced pluripotent stem cells (iPSCs) has the potential to overcome this hurdle. Theoretically, iPSCs expand indefinitely, providing an inexhaustible supply of progenitors for transfer to erythroid culture systems. In principle, banks of iPSCs generated from donors with desired blood group phenotypes can be established, or a novel universal donor line created by gene editing technologies (Hawksworth et al., 2018). iPSCs could provide a sustainable source of erythroid cells for therapeutic and diagnostic purposes.

Studying the molecular defects underlying red blood cell disorders is impeded by the paucity of suitable and adequate quantities of material from patients. Limited expansion of CD34+ cells isolated from patient PB and the complications associated with repeated collections from anemic patients mean that most red cell disease mechanisms have been studied in mouse models. However, fundamental differences between mouse and human erythropoiesis are increasingly apparent (An et al., 2014). Therefore, the generation of iPSCs from such patients has the potential to provide human model systems of these diseases. Such models could be used for the evaluation of therapeutic agents.

In this chapter, we summarize the work that aims to recapitulate human hematopoietic and erythroid differentiation from iPSCs, both as tools for studying normal and pathological erythropoiesis and as potential therapeutic products.

Hematopoiesis and erythropoiesis in vivo

During mammalian embryogenesis, hematopoiesis occurs in three successive waves, each giving rise to distinct hematopoietic cell types (Hansen et al., 2019; Ivanovs et al., 2017; Georgomanoli and Papapetrou, 2019). Primitive hematopoiesis occurs in yolk sac hemangioblasts, derived from the posterior primitive streak region of the mesoderm in the gastrulating embryo, producing primitive RBCs, megakaryocytes, and macrophages. Primitive hematopoietic progenitors are identifiable by their lack of a c-KIThi population co-expressing CD41¹⁰. Distinct from those that arise later in development, primitive RBCs are large and enucleate after entering the circulation (Palis et al., 1999). Second, erythromyeloid progenitors (EMPs), originating from the yolk sac hemogenic endothelium, migrate and transiently seed the fetal liver, producing definitive RBCs, megakaryocytes, and myeloid cells. As EMPs emerge from hemogenic endothelium, they co-express both endothelial and hematopoietic markers (Swiers et al., 2013), including c-KIT, CD41, VEC, CD45, and CD16/32. EMPs are rapidly replaced by definitive hematopoietic stem cells (HSC) from the aorta-gonad-mesonephros region of the embryo (Ivanovs et al., 2017), which produce definitive RBCs, megakaryocytes, and B-cells. HSCs initially expand in the fetal liver through cycles of self-renewal and subsequently migrate to the bone marrow. Colonization of the bone marrow by HSCs begins at 11–12 weeks of development in humans and continues until after birth (Palis, 2014), supplying lifelong hematopoiesis. Mesenchymal stromal cells and endothelial cells within the perivascular niche of the bone marrow core support HSCs by providing cytokines and cell-cell interactions required for differentiation (Morrison and Scadden, 2014). HSCs also co-express endothelial and hematopoietic markers, but they can be separated from EMPs by their lack of CD16/32 and the expression of Sca1 (McGrath et al., 2015).

We should note that our understanding of embryogenesis is derived predominantly from murine studies, because of ethical concerns and the inaccessibility of human embryos. Therefore, there are significant knowledge gaps in the development of human hematopoiesis.

RBCs (also known as erythrocytes) are the principal means by which oxygen is transported to, and carbon dioxide is removed from, body tissues. In adult humans, approximately 20–30 million circulating RBCs are required to fulfill this role, making up 84% of the cells in the body (Sender et al., 2016). RBCs are highly adapted for their role as oxygen carriers, containing no organelles to maximize their capacity for the oxygen-binding protein hemoglobin, with 270 million molecules in each cell (D’Alessandro et al., 2017). Highly efficient gas exchange is afforded by the characteristic biconcave shape of RBCs, which endows them with a large surface area. RBC function is further specialized by a highly flexible plasma membrane and cytoskeleton, enabling extensive and reversible deformation for passage through narrow capillaries (Diez-Silva et al., 2010). Although RBCs have a relatively long circulatory life span of ∼120 days (Franco, 2012), continuous turnover causes new cells to be produced at a rate of over 2 million per second (Palis, 2014). To meet this demand, HSCs continuously generate RBCs via a differentiation process known as erythropoiesis.

The first committed progenitors of the erythroid lineage to form from HSCs undergoing erythropoiesis are the burst-forming unit-erythroid (BFU-E) and the colony-forming unit-erythroid (CFU-E). These cells subsequently develop into the earliest morphologically recognizable erythroid cells, proerythroblasts, which sequentially differentiate into basophilic, polychromatic, and orthochromatic erythroblasts (Fig. 1.1). Orthochromatic erythroblasts undergo enucleation to form a reticulocyte by expelling the plasma membrane-bound nucleus, called the pyrenocyte. Reticulocytes are released from the bone marrow into the circulatory system and undergo further maturation (further loss of plasma membrane and cytoplasmic organelles) to form RBCs within 1–2 days (Nandakumar et al., 2016). In the native hematopoietic tissue, erythropoiesis occurs in a specialized niche known as the erythroblastic island where maturing erythroblasts bind to one or more central macrophages in concentric rings (Manwani and Bieker, 2008; Mohandas and Chasis, 2010). These macrophages are thought to support the proliferation and terminal differentiation of erythroid cells by directly transferring iron to developing erythroblasts (Leimberg et al., 2008), promoting growth through survival signals (Hanspal and Hanspal, 1994; Rhodes et al., 2008) and phagocytosis of the pyrenocyte post-enucleation (Toda et al., 2014). A core network of erythroid transcription factors, including key factors GATA1, KLF1 (EKLF or erythroid Krüppel-like factor), and TAL1 (T-cell acute lymphocytic leukemia-1), is crucial for many features of erythropoiesis, including cell cycle regulation, cytoskeletal arrangement, heme synthesis, iron transport and globin expression (reviewed by Nandakumar et al., 2016).

Figure 1.1  Schematic of surface marker expression during erythroid differentiation of primary CD34 + cells.

Globin expression and the globin switch

The expression of hemoglobin is a tightly regulated process and essential to RBC function. The hemoglobin protein is a tetramer of two subunits of an α-type globin and two subunits of a β-type globin. These subunits are produced from two distinct gene clusters: the α-locus encodes embryonic ζ-globin and two adult α-globins, while the β-locus encodes embryonic ε-globin, two fetal γ-globins and the adult δ- and β-globins. There are also additional minor globin genes.

Hemoglobin switching takes place at several points during human ontogeny when the globin subunit expression changes from an embryonic to fetal isoform, and then subsequently to the adult isoform. These switches roughly coincide with the changes in hematopoiesis highlighted earlier. RBCs produced during yolk sac hematopoiesis express embryonic hemoglobin (Hbemb; ζ2ε2 or α2ε2); those generated by EMPs express fetal HbF (α2γ2); and those arising from bone marrow HSCs express predominantly HbA (α2β2), with a small and variable amount of HbF (Hansen et al., 2019; Cumano and Godin, 2007). Many researchers have an interest in this fetal–adult hemoglobin switch because of the potential of HbF to ameliorate the symptoms of β-hemoglobinopathies.

The transcription factors LRF (ZBTB7A) and BCL11A (Masuda et al., 2016), the latter in cooperation with SOX6 (Xu et al., 2010), have an important role in the fetal–adult hemoglobin switch. These factors suppress γ-globin production, switching erythroid cells to an adult β-globin expression profile. KLF1 increases β-globin production by upregulating BCL11A and facilitating the interaction of the β-globin promoter with the locus control region, an enhancer upstream of the β-locus (Zhou et al., 2010; Esteghamat et al., 2013; Siatecka and Bieker, 2011). The subtleties of the switch in globin expression patterns during human development can be read in more detail in the following reviews (Stamatoyannopoulos, 2010; Sankaran et al., 2010).

Hematopoietic differentiation of iPSCs

Before differentiation to erythroid cells, iPSCs must first undergo hematopoietic differentiation to generate erythroid progenitor cells. However, capacity for generating hematopoietic stem and progenitor cells in vitro is limited, with low cell yield and purity, no doubt caused by an incomplete understanding of the required differentiation cues (Olivier et al., 2016; Hansen et al., 2018). Moreover, understanding of human hematopoiesis and that of iPSCs continues to develop. A consensus has not yet been reached regarding the optimal culture conditions, nor the most appropriate markers to identify and isolate desired progeny.

Hematopoiesis of iPSCs mimics early embryonic development, as iPSCs closely correspond to the inner-cell mass (embryoblast) of the blastocyst stage of embryogenesis (Hansen et al., 2019). The type of hematopoiesis appears to change temporally, in a manner that reflects development in the embryo (Kardel and Eaves, 2012). Erythroid cells arising from progenitors early in culture express both embryonic and fetal globins, while predominantly fetal globin-expressing erythroid cells arise as the culture continues (Kardel and Eaves, 2012). Broadly, hematopoiesis of iPSCs is carried out via two spatially distinct approaches, two-dimensional monolayers on a feeder cell layer or on matrix-coated dishes, or three-dimensional cultures of spontaneous or forced aggregation of iPSCs to form embryoid bodies (EBs). Both approaches attempt to recreate aspects of embryogenesis and have been combined with supplementation of culture media with growth factors and morphogens that have been implicated in the mesoderm’s induction (e.g., BMP4, activin A, and bFGF) (Razaq et al., 2017; Ruiz and Larochelle, 2018) and promote hematopoiesis (e.g., BMP4, WNT, bFGF, and VEGF) (Razaq et al., 2017; Ruiz and Larochelle, 2018; Pick et al., 2007).

The three-dimensional properties of early embryogenesis have been recreated via EB formation (Stover and Schwartz, 2011; Ng et al., 2005; Braham et al., 2019), whereby suspension of iPSCs in low- or nonbinding culture dishes results in spontaneous aggregation (Ng et al., 2005). Successful differentiation of the EBs depends on their quality, which can vary substantially between protocols (Van Winkle et al., 2012), dictated primarily by EB size, which affects morphology and viability. Centrally localized cells within large EBs suffer from the decreased transfer of nutrients (e.g., oxygen, glucose) and cytokine signaling, which can lead to differentiation to undesirable lineages and decreased survival (Van Winkle et al., 2012). Mechanical separation can also create EBs (i.e., by scraping or cutting) from iPSC colonies, but this process leads to the formation of nonuniform EBs. Alternatively, more control over the size of EBs has been obtained by dissociating iPSCs into single-cell suspensions before EB formation. Typically, single-cell suspensions of iPSCs exhibit low viability, undergoing dissociation-induced apoptosis (anoikis) (Amit et al., 2000). Inhibition of Rho-associated coiled-coil kinase (ROCK) has been used to improve the survival of single iPSC cultures (Watanabe et al., 2007; Chambers et al., 2009; Ishizaki, 2000), increasing EB yield from suspension cultures. Hanging drop techniques, which force EB formation in suspended droplets of media, allow limited control over EB size and are currently not suitable for large-scale EB formation (Dang et al., 2002). EB formation has been physically restricted using sub-millimeter micro-well plates (Spelke et al., 2011; Ungrin et al., 2008), providing accurate control over EB size and producing homogeneous EBs in a reproducible manner. Spin EBs have been formed