iPSC Derived Progenitors

iPSC Derived Progenitors, Volume Thirteen in the Advances in Stem Cell Biology series is a timely collection of information and new discoveries in the field of stem cell biology. The book addresses the importance of induced pluripotent stems cells and how can they be differentiated into different progenitors. Progenitors cells are often the first-step to making more differentiating cell types. This volume addresses iPSCs derived from bone, dental pulp, craniofacial, neural stem cells, otic, cardiac, and much more. The volume is written for researchers and scientists in stem cell therapy, cell biology, regenerative medicine, organ transplantation, and is contributed by world-renowned authors.

• Provides an overview of the fast-moving field of stem cell biology and function, regenerative medicine and therapeutics
• Covers how iPSCs can be differentiated into different progenitors
• Contributed by world renowned experts in the field

• Language: English
• Publisher: Academic Press
• Release date: Nov 10, 2021
• ISBN: 9780323900737

Book preview

iPSC Derived Progenitors, Volume 13

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. Induced pluripotent stem cell–derived neural stem cells

Introduction

Development of the nervous system in vivo

Extracellular matrix

In vitro models for neural stem cells and their progeny

Examples for the use of in vitro models

Ethical aspects

Future trends

Chapter 2. Induced pluripotent stem cells-derived craniofacial mesenchymal progenitor cells

Introduction

The neural crest and craniofacial development

Craniofacial pathologies

iPSC-derived craniofacial mesenchymal progenitors

Derivation of NCC-MPCs from iPSCs

Characterization of iPSC-derived NCC-MPCs

Potential applications of NCC-MPCs

Conclusions and perspectives

Chapter 3. Human induced pluripotent stem cell–derived astrocytes progenitors as discovery platforms: opportunities and challenges

Introduction

Human induced pluripotent stem cell–derived astrocytes

Challenges for in vitro modeling

Alternative techniques and new approaches

Modeling normal astrocyte biology with hiPSC-A

Functional characterization of human induced pluripotent stem cells into astrocytes

Modeling astrocyte roles in neurological disorders with human induced pluripotent stem cells into astrocytes

Alexander disease

Transplantation of glial progenitor cells and astrocytes

Conclusion

Chapter 4. Induced pluripotent stem cells-derived mesothelial progenitors; implications in cell-based regenerative medicine

Introduction

The origin of mesothelial cells

Functions of mesothelial cells

Physiological dysfunctions of mesothelial cells

Delivery methods for mesothelial cell therapy

Future trends or directions

Chapter 5. Human induced pluripotent stem cell–derived keratinocyte progenitors

Introduction

Differentiation of keratinocytes from embryonic stem cells

Differentiation of keratinocytes from induced pluripotent stem cells

Alternative methods of keratinocyte differentiation

Direct reprogramming into keratinocytes

Current use of hiPSC-derived keratinocytes and future trends

Chapter 6. Induced pluripotent stem cell–derived bone progenitors

Introduction

Bone-forming cells

Summary

Chapter 7. Induced pluripotent stem cells–derived chondrocyte progenitors

Introduction

New cell sources for cartilage regeneration

Future trends

Conclusions

Chapter 8. Induced pluripotent stem cells–derived dental pulp stem cells: Future application in regenerative medicine

Introduction

Characteristics of dental pulp stem cells

Regenerative potential of SHED/DPSCs-derived iPSCs

Regenerative potential of iPSCs-derived SHED/DPSCs

Advantages in generation of iPSCs from SHED

Other major concerns in tissue engineering and tissue regeneration

Conclusion

Chapter 9. Nephron progenitors in induced pluripotent stem cell–derived kidney organoids

Introduction

Overview of mammalian kidney development

Generation of nephron progenitors from human induced pluripotent stem cells

Disease modeling using induced pluripotent stem cell–derived nephron progenitors

Selective induction of glomerular podocytes from induced pluripotent stem cell–derived nephron progenitors

Propagation of induced pluripotent stem cell–derived nephron progenitors

Combination of nephron progenitors and ureteric buds toward higher-order kidney structures

Challenges ahead

Conclusions

Chapter 10. Interaction of iPSC-derived MSCs with the gastrointestinal tract and microbiome in the management of inflammatory bowel disease

Introduction

Immune mechanisms of gastrointestinal tract inflammation in inflammatory bowel disease

Role of microbiome alterations in inflammatory bowel disease

Need for new approaches

Rationale for use of cellular therapy in managing inflammatory bowel disease

Evidence of MSC and induced pluripotent stem cell–derived MSC efficacy in animal models of inflammatory bowel disease

Impact of induced pluripotent stem cell–derived MSC on the gastrointestinal tract microbiome and role in regulating inflammatory bowel disease severity

Utility of induced pluripotent stem cell–derived MSC for therapeutic use

Properties of induced pluripotent stem cell–derived MSC

Use of induced pluripotent stem cell–derived MSC for treatment of inflammatory bowel disease

Concluding remarks

Chapter 11. Induced pluripotent stem cells–derived hematopoietic progenitors for cellular immunotherapies

Introduction

Induced pluripotent stem cells as a potential source of hematopoietic stem cells and immune cells for immunotherapies

Concluding remarks and future directions

Chapter 12. Induced pluripotent stem cells as the source of cancer stem cells providing novel concepts of cancer: gaps between in vitro and in vivo in induced pluripotent stem cells modeling

Current recognition of cancer disease

Cancer stem cell

Gaps between in vitro and in vivo

Induced cancer stem cells with tissue specificity

Induced cancer stem cells and metastasis

Novel concepts of cancer in conclusion

Index

Advances in Stem Cell Biology

Series Editor

Alexander Birbrair

Copyright

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Notices

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

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

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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

Noor Hayaty Abu Kasim

Faculty of Dentistry, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia

Said M. Afify

Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama city, Okayama, Japan

Division of Biochemistry, Chemistry Department, Faculty of Science, Menoufia University, Shebin EL-koum, Menoufia, Egypt

Pranay Agarwal,     Department of Orthopaedic Surgery, School of Medicine, Stanford University, Stanford, CA, United States

Nidhi Bhutani,     Department of Orthopaedic Surgery, School of Medicine, Stanford University, Stanford, CA, United States

Michela Bruschi,     Department of Orthopaedic Surgery, School of Medicine, Stanford University, Stanford, CA, United States

I-Ping Chen,     Department of Oral Health and Diagnostic Sciences, School of Dental Medicine, University of Connecticut Health, Farmington, CT, United States

Lyndah Chow,     Center for Immune and Regenerative Medicine, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Ft. Collins, CO, United States

Robert A. Colbert,     Pediatric Translational Research Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, United States

Shankhajit De,     Department of Kidney Development, The Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan

Steven Dow,     Center for Immune and Regenerative Medicine, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Ft. Collins, CO, United States

Saritha S. D'Souza,     National Primate Research Center, University of Wisconsin Graduate School, Madison, WI, United States

Andreas Faissner,     Ruhr University Bochum, Department of Cell Morphology and Molecular Neurobiology, Bochum, Germany

Jeremy Fischer,     Pediatric Translational Research Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, United States

Nazmul Haque

Department of Oral Biology and Biomedical Sciences, Faculty of Dentistry, MAHSA University, Selangor, Malaysia

TotiCell Limited, Dhaka, Bangladesh

Ghmkin Hassan

Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama city, Okayama, Japan

Department of Microbiology and Biochemistry, Faculty of Pharmacy, Damascus University, Damascus, Syria

James H. Hui

Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

NUS Tissue Engineering Programme, Life Sciences Institute, National University of Singapore, Singapore

University Orthopaedics, Hand and Reconstructive Microsurgery Cluster, National University Health System, Singapore

Fuyuki F. Inagaki,     Department of Surgery, National Center for Global Health and Medicine, Tokyo, Japan

Natsuko F. Inagaki,     Department of Lipid Signaling, National Center for Global Health and Medicine, Tokyo, Japan

Akhilesh Kumar,     National Primate Research Center, University of Wisconsin Graduate School, Madison, WI, United States

Mavis Loberas

Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

NUS Tissue Engineering Programme, Life Sciences Institute, National University of Singapore, Singapore

Yongquan Luo,     Pediatric Translational Research Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, United States

Alison Manchester,     Center for Immune and Regenerative Medicine, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Ft. Collins, CO, United States

Nicholas J. Maragakis,     Johns Hopkins University School of Medicine, Baltimore, MD, United States

Elly Munadziroh,     Department of Dental Material, Faculty of Dental Medicine, Universitas Airlangga, Surabaya Indonesia

Fatemeh Navid,     Pediatric Translational Research Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, United States

Ryuichi Nishinakamura,     Department of Kidney Development, The Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan

Ernst J. Reichenberger,     Center for Regenerative Medicine and Skeletal Development, Department of Reconstructive Sciences, University of Connecticut Health (UConn Health), Farmington, CT, United States

Lars Roll,     Ruhr University Bochum, Department of Cell Morphology and Molecular Neurobiology, Bochum, Germany

Shyam Kishor Sah,     Center for Regenerative Medicine and Skeletal Development, Department of Reconstructive Sciences, University of Connecticut Health (UConn Health), Farmington, CT, United States

Akimasa Seno,     Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama city, Okayama, Japan

Masaharu Seno,     Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, Okayama city, Okayama, Japan

Igor Slukvin

National Primate Research Center, University of Wisconsin Graduate School, Madison, WI, United States

Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States

Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, WI, United States

Pratiwi Soesilawati,     Department of Oral Biology, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia

Akshaya Srinivasan

Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

NUS Tissue Engineering Programme, Life Sciences Institute, National University of Singapore, Singapore

Arens Taga,     Johns Hopkins University School of Medicine, Baltimore, MD, United States

Yi-Chin Toh

School of Mechanical, Medical and Process Engineering, Science and Engineering Faculty, Queensland University of Technology, Brisbane, QLD, Australia

Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia

About the editor

Alexander Birbrair

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

Preface

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

This volume iPSC-derived Progenitors 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 form different types of progenitors in vitro, enabling us to study the cellular and molecular mechanisms involved in the biology of these progenitors. 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 a variety of progenitor cells, tissues and organs using state-of-the-art techniques. These advantages facilitated identification of key targets and definition of the molecular basis of several disorders. Thus, this book is an attempt to describe the most recent developments in the area of iPSCs 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 progenitors from iPSCs. Twelve chapters written by experts in the field summarize the present knowledge about iPSC-derived progenitors.

Lars Roll and Andreas Faissner from Ruhr University Bochum discuss iPSC-derived neural stem cells. James H. Hui and colleagues from National University of Singapore describe iPSC-derived craniofacial mesenchymal progenitor cells. Arens Taga and Nicholas J. Maragakis from Johns Hopkins University School of Medicine compile our understanding of iPSC-derived astrocyte progenitors. Natsuko F Inagaki and Fuyuki F Inagaki from National Center for Global Health and Medicine update us with what we know about iPSC-derived mesothelial progenitors. Ernst J. Reichenbergera and colleagues from University of Connecticut Health summarize current knowledge on generating keratinocyte progenitors from iPSCs. Robert A. Colbert and colleagues from National Institutes of Health address the importance of iPSC-derived bone progenitors. Nidhi Bhutani and colleagues from Stanford University talk about iPSC-derived chondrocyte progenitors. Elly Munadziroh and colleagues from Universiti Kebaangsan Malaysia focus on iPSC-derived dental pulp stem cells. Shankhajit De and Ryuichi Nishinakamura from Kumamoto University give an overview of the use of iPSC-derived nephron progenitors to generate kidney organoids. Steven Dow and colleagues from Colorado State University introduce the use of iPSC-derived mesenchymal stem cells in the management of inflammatory bowel disease. Bruce E. Torbett and Igor Slukvin from University of Wisconsin Medical School speak about iPSCs as a potential source of hematopoietic stem cells. Finally, Masaharu Seno and colleagues from Okayama University present iPSCs as a source for cancer stem cells to use in oncology research.

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: Induced pluripotent stem cell–derived neural stem cells

Lars Roll, and Andreas Faissner     Ruhr University Bochum, Department of Cell Morphology and Molecular Neurobiology, Bochum, Germany

Abstract

Induced pluripotent stem cells (iPSCs), derived from reprogrammed somatic cells, have revolutionized stem cell research. This chapter is dedicated to neural stem cells (NSCs) and their potential for basic research and clinical applications. After short introducing sections about neurogenesis in vivo and the extracellular matrix as important compartment for extrinsic signaling, we present current in vitro models for NSCs. Cerebral organoids are cell aggregates that mimic aspects of early brain development. In the meantime, a number of disease models based on iPSCs and organoid cultures have been established and are presented here as well as examples of developmental studies. We close the chapter with ethical issues around NSC research and a future perspective.

Keywords

Cerebral organoid; Disease model; Extracellular matrix; Matrisome; Neural crest; Neural induction; Neural stem cell; Neural tube; Stem cell niche; Transdifferentiation

Introduction

Development of the nervous system in vivo

Extracellular matrix

Extracellular matrix and neural stem cells

Extracellular matrix and its in vitro counterparts

In vitro models for neural stem cells and their progeny

Two-dimensional cultivation

Three-dimensional organoid cultivation

Direct conversion

Examples for the use of in vitro models

Early development modeling

Disease modeling

Ethical aspects

Future trends

References

Introduction

The nervous system allows us to receive information from the environment, to analyze it, and to respond appropriately. After damage, for example, due to neurodegenerative diseases or after trauma, regeneration is often severely limited and means irreversible deficits for patients. Therefore, it is of keen interest to understand the cell types of the nervous system with regard to their formation during development, their function in the adult organism, and their reaction to stress and damage. Current concepts to study this system with neural stem cells (NSCs), derived from (induced) pluripotent stem cells, are presented in this chapter. As many steps in cultivation protocols follow principles observed in vivo, the first part of the chapter gives a short overview of neurogenesis and gliogenesis. The extracellular matrix (ECM) is highlighted in the second part, as it represents an important source of extrinsic signals that regulate the cell fate in the developing, but also in the adult nervous system. Afterward, we describe two-dimensional (2D) and three-dimensional (3D) cultivation protocols and comment on the advantages and disadvantages of each approach. We give examples for applications in the context of developmental studies and disease modeling. We close the chapter with a discourse about ethical aspects of the work with NSCs and give an outlook on future perspectives.

Development of the nervous system in vivo

Development of the nervous system in the early embryo starts with the induction of the neuroectoderm by a structure called Hensen's node (Shparberg et al., 2019). First, the neural plate is formed (Fig. 1.1A). In response to sonic hedgehog (SHH) signals, released by the underlying notochord, the neural plate begins to bend and gives rise to the neural groove (Nikolopoulou et al., 2017). The structure invaginates and merges at the borders to generate the neural tube. Cells from the edges of the neural groove, termed neural fold, form the neural crest. After neural tube formation, this structure is located between the neural tube and the closed surface ectoderm.

Looking at the cellular level, the neural tube is formed by long, radially shaped NSCs, first referred to as neuroepithelial cells and later termed radial glia (Fig. 1.1B). They will later give rise to the cell types of the central nervous system. Neural crest cells migrate out and differentiate into various cell types, including those of the peripheral nervous system and melanocytes, the pigmented cells of the skin (Mayor and Theveneau, 2013).

How do NSCs form the nervous system with its cell types? NSCs expand the stem cell pool in the neural tube and later give rise to progenitor cells (Fig. 1.1C). Neurons are formed from progenitors during the embryonic phase of neurogenesis. This is followed by gliogenesis, which is completed after birth and describes the differentiation of oligodendrocytes and astrocytes (Kriegstein and Alvarez-Buylla, 2009). A specialized cell type are the ependymal cells, ciliated cells that line the ventricle walls and contribute to the stem cell niche. They are also derived from radial glia (Redmond et al., 2019). In the peripheral nervous system, the myelinating cells are called Schwann cells instead of oligodendrocytes. Additional cell types such as microglia and endothelia are present in the mature nervous system, but it is important to note that they are not derived from the NSC pool. Instead, they are formed outside the nervous system and migrate in (Ginhoux and Prinz, 2015).

Figure 1.1 Neurulation and regional identity of cells in the developing nervous system. (A) Steps of neural tube formation. Signals from Hensen's node induce formation of the neural plate (yellow) from surface ectoderm (gray). Subsequently, this region folds in response to SHH signals from the notochord and invaginates to form the neural groove. The structure bends and merges at the borders, called neural folds (green), and eventually gives rise to the neural tube. Cells from the neural folds form the neural crest, a structure located dorsally between the neural tube and the surface ectoderm. (B) Cellular organization of the neural tube and neural crest cells. Long, radially shaped cells form the neural tube and generate the cell types of the central nervous system, whereas cells of the neural crest migrate out and differentiate into different cell types, including those of the peripheral nervous system. (C) Neural stem cells (first named neuroepithelial cells, later radial glia) first expand the stem cell pool and later give rise to progenitor cells. The embryonic phase of neurogenesis is followed by the phase of gliogenesis, which is completed after birth. (D) Gradients of different soluble factors generate a positional code that defines the regional identity of the cells. The scheme shows the early central nervous system at the three-vesicle stage. The three vesicles will form the brain, whereas the thin, caudal part will differentiate into the spinal cord. BMP, bone morphogenetic protein; DKK, dickkopf; p, progenitor; SHH, sonic hedgehog; WNT, wingless-type MMTV integration site family.

How are NSCs instructed to acquire an appropriate cell fate with regard to their position in the developing organism? Gradients of different soluble factors, called morphogens, in combination generate a positional code that defines the regional identity of the cells (Tao and Zhang, 2016; Fig. 1.1D). Examples for such factors are bone morphogenetic protein (BMP), dickkopf (DKK), SHH, and wingless-type MMTV integration site family (WNT). Fig. 1.1D shows the early central nervous system at the three-vesicle stage. Cells in the three vesicles (pros-, mes-, and rhombencephalon) will give rise to the brain, whereas the thin, caudal part will differentiate into the spinal cord (Ishikawa et al., 2012).

The next section is dedicated to the ECM, which provides the basis for extracellular signaling.

Extracellular matrix

As mentioned above, external signals have a strong impact on the cell fate and on the behavior of the cells. In the developing nervous system, numerous processes have to be orchestrated to form this complex structure and to ensure its proper function. Aspects such as differentiation of stem cells into specific cell types with numerous subpopulations, migration of cells to their destination, nerve fiber growth, and also the formation of functional synapses (Gottschling et al., 2019) are only few examples. In this context, we want to emphasize on the ECM, as it provides the basis for external signals. Binding of a ligand to its receptor triggers intracellular signaling cascades, thereby regulating gene expression or cytoskeletal rearrangement (Hastings et al., 2019).

The ECM is formed of molecules that are secreted by cells into the extracellular space. Interactions of these molecules form a complex 3D network that provides signals to the cells. Depending on the tissue, the function of the ECM and therefore its composition varies considerably. For example, ECM in cartilage is rich in collagen fibers and optimized to absorb mechanical forces. On the contrary, the ECM of the nervous system is less eye-catching, although it has a number of important functions as we have already described. Here, the ECM is poor in fibrous collagens and instead based on a hyaluronan backbone (Burnside and Bradbury, 2014). These long carbohydrate chains are anchored in the cell membrane via hyaluronan synthase (Fig. 1.2), bind huge amounts of water, and are responsible for the viscous and gel-like consistency of the ECM (Perkins et al., 2017). The other constituents of the ECM interact directly or indirectly with hyaluronan and with each other. Glycoproteins like laminins, tenascin-C, or tenascin-R are characterized by a glycosylated core protein, which means that relatively small, branched carbohydrates are attached to the protein. Proteoglycans like aggrecan, neurocan, or receptor protein tyrosine phosphatase (RPTP) β/ζ additionally have at least one long, unbranched carbohydrate (glycosaminoglycan) chain bound to the core protein. Based on the carbohydrate composition, it can be distinguished between chondroitin sulfates, heparan sulfates, and other glycosaminoglycans (Bandtlow and Zimmermann, 2000). Interactions are not limited to ECM molecules. These are able to bind additional factors such as growth factors or other small molecules and thereby let the ECM function as a reservoir for such molecules. An important example is fibroblast growth factor, which is regulated by heparan sulfate proteoglycans (Sarrazin et al., 2011). The term matrisome refers to all genes that code for ECM molecules, based on bioinformatics. The core matrisome comprises about 300 molecules; the list of associated factors is even longer (Hynes and Naba, 2012).

Diversity and complexity of the ECM are further increased by several mechanisms, which enables fine-tuning of the signals that act on a cell. Many proteins are the result of alternative splicing, which means that different isoforms of a protein are generated from a single gene (Baralle and Giudice, 2017). Also the degree of glycosylation can differ, and the sulfation pattern of such carbohydrates is regulated independently (Iozzo, 1998). But synthesis of ECM molecules is not the only mechanism to generate diversity. Proteolytic cleavage of ECM molecules at specific sites is a prevalent way to regulate the function of signaling molecules. Matrix metalloproteinases, under tight control, cleave ECM molecules at specific positions (Rempe et al., 2016).

Figure 1.2 Extracellular matrix (ECM) in the nervous system. A simplified network with typical components is illustrated. Interacting molecules form a complex, three-dimensional network. It is based on long hyaluronan chains (carbohydrates) that are anchored in the membrane via their synthesizing enzyme, hyaluronan synthase. Glycoproteins like tenascin-C and tenascin-R or proteoglycans like aggrecan and neurocan can bind directly or indirectly (via link proteins) to the hyaluronan backbone. Beside classical matrix molecules, the ECM binds additional factors such as growth factors and thereby can function as a reservoir for these molecules. Eventually, binding of a molecule to its receptor triggers an intracellular cascade that regulates gene expression, cytoskeletal rearrangement, and other processes.

Extracellular matrix and neural stem cells

Several matrix molecules are associated with NSCs and are expected to be part of a niche-like environment that supports an undifferentiated state of the cells. Among those are laminins, tenascin-C, and proteoglycans of the chondroitin sulfate and heparan sulfate proteoglycan classes (Kazanis and ffrench-Constant, 2011; Faissner et al., 2017; Roll and Faissner, 2019). A specific chondroitin sulfate motif, the so-called DSD-1 epitope, is present on RPTPβ/ζ isoforms and defined by a distinct sulfation pattern (Clement et al., 1998). This structure is expressed by NSCs, and its manipulation affects proper stem cell proliferation (von Holst et al., 2006). These and other ECM molecules contribute to an environment that allows NSCs to maintain their stem cell properties (Faissner and Reinhard, 2015).

Extracellular matrix and its in vitro counterparts

A widely used tool for in vitro cell cultures is Matrigel, a gelatinous ECM produced by Engelbreth-Holm-Swarm sarcoma cells. Its composition is not clearly defined, but laminins and collagens have been identified as major components (Hughes et al., 2010).

Artificial matrices have been developed to generate a 3D environment for stem cells in vitro. These comprise hydrogels and polyacrylamide-based gels (Tibbitt and Anseth, 2009). Such defined gels can be supplemented with ECM-related peptides to mimic the ECM in vitro (Jarocki et al., 2019).

After this short introduction about the developing nervous system and underlying signaling molecules, current stem cell-based approaches are presented in the next section. Many of the protocols make use of the signaling molecules that had been identified in vivo before.

In vitro models for neural stem cells and their progeny

Numerous approaches have been developed in the past that are intended to model nervous system development in vitro. The possibility to reprogram somatic cells to a pluripotent state, first established by the group of Yamanaka for mouse (Takahashi and Yamanaka, 2006) and one year later for human cells (Takahashi et al., 2007), paved the way for a new era of stem cell research. Especially the work with human pluripotent cells without destroying an embryo is of great value.

Two-dimensional cultivation

First we want to describe an exemplary protocol that enables to generate neurons and other cell types, mainly based on 2D, adherent culture (Fig. 1.3A). Induced pluripotent stem cells (iPSCs) are cultivated adherently and allowed to form colonies. Subsequently, the cells are detached from the surface and allowed to aggregate. Within days, embryoid bodies form that contain cells of the three germ layers (i.e., endo-, meso-, and ectoderm). The ectodermal, more specifically the neuroectodermal cell fate is supported by neural induction medium. The cell aggregate, now called neural aggregate, consists of cells with neuroectodermal fate.

Neural aggregates are placed on laminin-coated cell culture plastic and adhere, cells migrate out and some of them form neural rosettes on the surface. These in vitro structures resemble the early neural tube. Neural rosette cells represent a population of early NSCs that are able to establish a relatively complex structure like the neural rosettes (Elkabetz et al., 2008). In the following steps, NSCs from the rosette structures are isolated and detached from the cell culture plastic. These neural stem/progenitor cells have lost the capability to form rosettes when allowed to adhere again. They are driven to differentiate and form the different cell types of the nervous system, depending on the composition of the differentiation medium. Specific subtypes of neurons can be generated, for example, dopaminergic neurons (Zhang and Zhang, 2010; Tao and Zhang, 2016). In many cases, protocols established with embryonic cells can be transferred to iPSCs.

Figure 1.3 Generation of neural cell types derived from iPSCs in vitro. (A) Protocol to generate neurons and other cell types mainly based on two-dimensional, adherent cultivation. Adherent pluripotent cells (iPSCs) are detached from the coated surface and allowed to aggregate. Within a few days, embryoid bodies form that contain cells of the three germ layers (ecto-, meso-, and endoderm; indicated by colors). The neuroectodermal cell fate is promoted by neural induction medium, the structure is now called neural aggregate. Neural aggregates are placed on coated cell culture plastic and some of the cells form neural rosettes, which resemble the neural tube. In the following steps, neural stem cells from the rosette structures are driven to differentiate and eventually form the different cell types of the nervous system. (B) Cerebral organoid protocol. Starting with adherent pluripotent cells, all following steps of the protocol exploit the self-organizing capacity of the cells in their three-dimensional environment. Embryoid bodies are generated from iPSCs and after a few days the medium is replaced by