iPSCs in Tissue Engineering

The series Advances in Stem Cell Biology is a timely and expansive collection of comprehensive information and new discoveries in the field of stem cell biology. iPSCs in Tissue Engineering, Volume 11 addresses how induced pluripotent stem cells (iPSCs) are being used to advance tissue engineering. Somatic cells can be reprogrammed into iPSCs by the expression of specific transcription factors. These cells have been transforming biomedical research over the last 15 years. This book will address the advances in research of how iPSCs are being used for the generation of different tissues and organs such as the lungs, trachea, salivary glands, skeletal muscle, liver, intestine, kidney, even the brain, and much more. This volume is written for researchers and scientists interested in stem cell therapy, cell biology, regenerative medicine, and tissue engineering and is contributed by world-renowned authors in the field.

• Provides overview of the fast-moving field of stem cell biology and function, regenerative medicine, and therapeutics
• Covers the engineering of the following organs: lungs, trachea, salivary glands, skeletal muscle, liver, intestine, kidney, even the brain, and more
• Is contributed from stem cell leaders around the world

• Language: English
• Publisher: Academic Press
• Release date: Aug 12, 2021
• ISBN: 9780128241905

Book preview

iPSCs in Tissue Engineering, Volume 11

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

Acknowledgment

Chapter 1. Pluripotent stem cell–derived brain-region-specific organoids

Introduction

Methodologies to generate and culture brain-region-specific organoids

Applications of brain-region-specific organoids

Future directions

Chapter 2. The construction of 3D cognitive networks from iPSCs through precise spatiotemporal specification

Introduction

Origins and the molecular identity of cortical interneurons (CINs)

The functional classification of CINs

The spatiotemporal cues for CIN specification reported in rodent models

Human iPSCs as a model to monitor cortical interneuron development

The unified approach in generating hPSC-differentiated MGE in vitro

Monitoring temporal changes with hiPSCs’ single-cell RNA-seq

PSC-induced organoids as a model for CIN migration

Limitations in adopting hPSC-derived organoid for modeling CINs

Chapter 3. Induced pluripotent stem cells for vascular tissue engineering

Induced pluripotent stem cells—a cell source with great potential

Cell sources applicable for reprogramming

Reprogramming somatic cells into iPSCs

Tissue engineering

Vascular tissue engineering

Scaffolds

Cell source for vascular tissue engineering

IPSCs in vascular tissue engineering

Differentiating iPSCs into vascular phenotypes

Future outlook

Chapter 4. Induced pluripotent stem-cell-derived corneal grafts and organoids

Introduction

Corneal structure and function

Corneal development and maintenance

Limbal stem cells (LSCs)

Limbal stem cell deficiency (LSCD)

Therapeutic strategies for the treatment of LSCD

Conjunctival limbal autografts (CLAU)

Cultured limbal epithelial transplantations (CLET)

Simple limbal epithelial cell transplantations (SLET)

Alternative strategies for the treatment of bilateral limbal stem cell deficiency

Induced pluripotent stem cells and their importance in ocular research and regenerative medicine

Directed differentiation of iPSCs into eye field clusters and corneal specification

iPSC-derived three-dimensional corneal organoids and their characteristics

iPSC-derived corneal epithelial grafts for regenerative applications

iPSC-derived corneal tissues for disease modeling and in vitro drug testing

Major challenges in using iPSCs derived corneal cells and tissues for regeneration

Future perspectives and conclusion

Chapter 5. Induced pluripotent stem cell–derived salivary glands

Introduction

Main text

Future directions

Conclusion

Chapter 6. Induced pluripotent stem cells for trachea engineering

Introduction

Trachea structure and mechanical properties

Scaffolds for trachea engineering

Cell sources for trachea engineering

Summary

Chapter 7. Looking back, moving forward: the renaissance, applications, and vascularization strategies of human-induced pluripotent stem cell–derived lung organoids

Introduction

The human lung

Human lung organoids

Back to the future: strategies for vascularization

Conclusions and perspectives on current challenges

Chapter 8. Skeletal muscle engineering using human induced pluripotent stem cells for in vitro disease modeling

Introduction

Human iPSCs as a promising tool for skeletal muscle modeling

Patient-derived iPSCs to simulate cellular pathology in skeletal muscle in vitro

Derivation of myogenic progenitors from human iPSCs

Sphere-based expansion of myogenic progenitors from human iPSCs

Two-dimensional (2D) micropatterned culture for modeling human skeletal myotubes

Three-dimensional artificial muscle to facilitate the formation of elongated myofibrils

Challenges to improve in vitro muscle modeling using patient-specific iPSCs

Conclusion

Chapter 9. iPSC bioprinting for musculoskeletal tissue

Introduction

How to differentiate iPSCs into musculoskeletal tissue cells

iPSC myogenic differentiation

iPSC chondrogenic differentiation

iPSC osteogenic differentiation

Bioprinting techniques

Bioprinting skeletal muscle

Cartilage and bone bioprinting

Bioprinting for cartilage tissue

Bioprinting for bone tissue

Conclusions

Chapter 10. IPSC-derived 3D human fatty liver models

Liver tissue

Human liver cell cultures

Liver disease models using hiPSC-Hep

Current status and future perspectives

Chapter 11. IPSC-derived intestinal organoids and current 3D intestinal scaffolds

Introduction to the intestinal tissue morphology

Organoid limitations

Ideal intestinal scaffold requirements

Current state-of-the-art in in vitro intestinal tissue models

Future perspectives

Chapter 12. Models of kidney glomerulus derived from human-induced pluripotent stem cells

Introduction

The glomerulus in the context of the nephron: basic anatomy and physiology

Modeling the glomerulus: insights from the embryological development

Modeling kidney development in vitro: generation of kidney organoids

Modeling glomerular development and function in vitro: glomerulus-on-a-chip and bioprinting

Future perspectives: innovations in bioengineering of kidney tissues from iPSCs

Chapter 13. Ureteric bud structures generated from human iPSCs

Introduction

Early studies generating UB-lineage cells

Establishment of differentiation methods for UB tissues

Generation of a disease model using iUB organoids

Hurdles to overcome

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.

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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. A 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. I owe her all success in my career and personal life.

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

Contributors

Rohan Bhattacharya

Department of Biomedical Engineering, Duke University, Durham, NC, United States

Center for Biomolecular and Tissue Engineering, Duke University, Durham, NC, United States

Dandan Cao

School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China

Shenzhen Key Laboratory of Fertility Regulation, Reproductive Medicine Center, The University of Hong Kong-Shenzhen Hospital, Shenzhen, Guangdong, China

Wai-Yee Chan,     School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China

Julia Deinsberger

Disease Modeling and Organoid Technology (DMOT) Research Group, Department of Dermatology, Medical University of Vienna, Vienna, Austria

Skin and Endothelium Research Division (SERD), Department of Dermatology, Medical University of Vienna, Vienna, Austria

Andreas M. Grabrucker

Bernal Institute, University of Limerick, Limerick, Ireland

Health Research Institute (HRI), University of Limerick, Limerick, Ireland

Cellular Neurobiology and Neuro-Nanotechnology Lab, Department of Biological Sciences, University of Limerick, Limerick, Ireland

Suyu Hao,     School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China

Miriel S.H. Ho,     The CReATe Fertility Centre, Toronto, ON, Canada

Mirabelle S.H. Ho,     The CReATe Fertility Centre, Toronto, ON, Canada

K.A. Kilian

School of Chemistry, Australian Centre for Nanomedicine, University of New South Wales, Sydney, NSW, Australia

School of Materials Science and Engineering, University of New South Wales, Sydney, NSW, Australia

Clifford L. Librach

The CReATe Fertility Centre, Toronto, ON, Canada

Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada

Department of Physiology, University of Toronto, Toronto, ON, Canada

Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada

Department of Gynecology, Women's College Hospital, University of Toronto, Toronto, ON, Canada

Savitri Maddileti,     Center for Ocular Regeneration, Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, LV Prasad Eye Institute, Hyderabad, Telangana, India

Shin-Ichi Mae,     Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan

Siobhan Malany,     Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, FL, United States

Sigita Malijauskaite

Department of Chemical Sciences, University of Limerick, Limerick, Ireland

Bernal Institute, University of Limerick, Limerick, Ireland

Indumathi Mariappan,     Center for Ocular Regeneration, Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, LV Prasad Eye Institute, Hyderabad, Telangana, India

Kieran McGourty

Department of Chemical Sciences, University of Limerick, Limerick, Ireland

Bernal Institute, University of Limerick, Limerick, Ireland

Health Research Institute (HRI), University of Limerick, Limerick, Ireland

Guo-li Ming

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States

Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States

Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States

Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States

Kenji Mishima,     Division of Pathology, Department of Oral Diagnostic Sciences, School of Dentistry, Showa University, Shinagawa-ku, Tokyo, Japan

Kai-Kei Miu,     School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China

John J.E. Mulvihill

Bernal Institute, University of Limerick, Limerick, Ireland

Health Research Institute (HRI), University of Limerick, Limerick, Ireland

School of Engineering, University of Limerick, Limerick, Ireland

Samira Musah

Department of Biomedical Engineering, Duke University, Durham, NC, United States

Center for Biomolecular and Tissue Engineering, Duke University, Durham, NC, United States

Department of Medicine, Division of Nephrology, Duke University School of Medicine, Durham, NC, United States

Phuong T.T. Nguyen,     Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States

Arinze Emmanuel Okafor,     Department of Biomedical Engineering, Duke University, Durham, NC, United States

Kenji Osafune,     Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan

Maddalena Parafati,     Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, FL, United States

Vinay Kumar Pulimamidi

Center for Ocular Regeneration, Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, LV Prasad Eye Institute, Hyderabad, Telangana, India

School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India

Samantha Robertson,     Department of Comparative Biosciences, University of Wisconsin, Madison, WI, United States

S. Romanazzo,     School of Chemistry, Australian Centre for Nanomedicine, University of New South Wales, Sydney, NSW, Australia

I. Roohani

School of Chemistry, Australian Centre for Nanomedicine, University of New South Wales, Sydney, NSW, Australia

Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia

School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia

Makoto Ryosaka,     Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan

Yasuo Saijo,     Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, Niigata-shi, Niigata, Japan

Hongjun Song

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States

Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States

Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States

The Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States

Masatoshi Suzuki

Department of Comparative Biosciences, University of Wisconsin, Madison, WI, United States

The Stem Cell and Regenerative Medicine Center, University of Wisconsin, Madison, WI, United States

Junichi Tanaka,     Division of Pathology, Department of Oral Diagnostic Sciences, School of Dentistry, Showa University, Shinagawa-ku, Tokyo, Japan

Sin-Ruow Tey,     Department of Comparative Biosciences, University of Wisconsin, Madison, WI, United States

Zhangting Wang,     School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China

Benedikt Weber

Disease Modeling and Organoid Technology (DMOT) Research Group, Department of Dermatology, Medical University of Vienna, Vienna, Austria

Skin and Endothelium Research Division (SERD), Department of Dermatology, Medical University of Vienna, Vienna, Austria

Qiliang Zhou,     Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, Niigata-shi, Niigata, 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 the Federal University of Minas Gerais in Brazil, where he started his own lab. His laboratory is interested in understanding how the cellular components of different tissues function and control disease progression. His group explores the roles of specific cell populations in the tissue microenvironment by using state-of-the-art techniques. His research is funded by the Serrapilheira Institute, CNPq, CAPES, and FAPEMIG. In 2018, Alexander was elected affiliate member of the Brazilian Academy of Sciences (ABC), and, in 2019, he was elected member of the Global Young Academy (GYA). He is the Founding Editor and Editor-in-Chief of Current Tissue Microenvironment Reports and Associate Editor of Molecular Biotechnology. Alexander also serves in the editorial board of several other international journals: Stem Cell Reviews and Reports, Stem Cell Research, Stem Cells and Development, and Histology and Histopathology.

Preface

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 cell (iPSC) biology from different perspectives. Therefore, the book was subdivided into several volumes.

This volume iPSCs in Tissue Engineering 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 for tissue engineering. Further insights into the biology of these cells will have important implications for the possible use of iPSCs as a source for newly engineered tissues. The authors focus on the modern state-of-the-art methodologies and the leading-edge concepts in the field of stem cell biology. In recent years, remarkable progress has been made in the obtention of iPSCs and their differentiation into several cell types, tissues, and organs using state-of-the-art techniques. These advantages facilitated identification of key targets and definition of the molecular basis of several disorders. Thus, this book is an attempt to describe the most recent developments in the area of iPSC biology, which is one of the rising hot topics in the field of molecular and cellular biology today. Here, we present a select collection of detailed chapters on what we know so far about the use of iPSCs for the engineering of different tissues. Thirteen chapters written by experts in the field summarize the present knowledge about iPSC potential in organ engineering.

Guo-li Ming and colleagues from the University of Pennsylvania discuss iPSC-derived brain region–specific organoids. Wai-Yee Chan and colleagues from the Chinese University of Hong Kong describe the construction of 3D cognitive networks through precise spatiotemporal specification. Julia Deinsberger and Benedikt Weber from the Medical University of Vienna compile our understanding of iPSCs for vascular tissue engineering. Indumathi Mariappan and colleagues from Hyderabad Eye Research Foundation update us with what we know about iPSC-derived corneal grafts and organoids. Junichi Tanaka and Kenji Mishima from Showa University summarize current knowledge on iPSC-derived salivary glands. Qiliang Zhou and Yasuo Saijo from Niigata University address the importance of iPSCs in Trachea Engineering. Clifford L. Librach and colleagues from the University of Toronto talk about the renaissance, applications, and vascularization strategies of human iPSC-derived lung organoids. Masatoshi Suzuki and colleagues from the University of Wisconsin focus on engineering skeletal muscle using human iPSCs. Iman Roohani and colleagues from the University of New South Wales give an overview of the use of iPSCs in bioprinting for musculoskeletal tissue engineering. Maddalena Parafati and Siobhan Malany from the University of Florida present iPSC-derived 3D human fatty liver models. Kieran McGourty and colleagues from the University of Limerick introduce what we know so far about iPSC-derived intestinal organoids and current 3D intestinal scaffolds. Samira Musah and colleagues from Duke University update us with information on models of kidney glomerulus derived from human iPSCs. Finally, Kenji Osafune and colleagues from Kyoto University summarize our current status on ureteric bud structures generated from human 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

Acknowledgment

The cover was kindly provided by Miriel Ho.

Chapter 1: Pluripotent stem cell–derived brain-region-specific organoids

Phuong T.T. Nguyen ¹ , Hongjun Song ² , ³ , ⁴ , ⁵ , and Guo-li Ming ² , ³ , ⁴ , ⁶       ¹Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States      ²Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States      ³Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States      ⁴Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States      ⁵The Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States      ⁶Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States

Abstract

Brain organoids derived from human pluripotent stem cells have emerged as invaluable tools to study human-specific features in normal brain development and diseases. Given that brain regions differ in cellular composition, molecular signature, and functional specialization, establishing brain-region-specific organoids is crucial for more precise in vitro modeling of human brains. Here, we review selected protocols for generating brain organoids with various regional identities from pluripotent stem cells, including dorsal forebrain, ventral forebrain, hippocampus, thalamus, hypothalamus, retina, midbrain, and cerebellum. These organoids possess many characteristics that resemble the fetal brain tissue of the corresponding brain regions. In addition, we highlight the utilities of using these protocols in elucidating neurodevelopmental processes, studying the impacts of genetic and environmental perturbations, and facilitating strategic development of therapies.

Keywords

Assembloids; Brain organoids; Brain tumors; Cerebellum; Dorsal forebrain; Hippocampus; Hypothalamus; Prenatal drug exposure; Regenerative medicine; Retina; Thalamus; Ventral forebrain; Zika virus

Introduction

Methodologies to generate and culture brain-region-specific organoids

Organoids with the prosencephalon (forebrain) origin

Dorsal forebrain organoids—cortex

Ventral forebrain organoids—MGE, LGE, and CGE

Medial forebrain organoids—choroid plexus, cortical hem, and hippocampus

Caudal forebrain organoids—thalamus

Rostral forebrain organoids—hypothalamus and retina

Organoids with mesencephalon (midbrain) origin

Midbrain organoids

Organoids with rhombencephalon (hindbrain) origin

Cerebellar organoids

Applications of brain-region-specific organoids

Normal human brain development

Investigating human-specific developmental events

Assembloids to study neuronal migration, axonal projections, and network connectivity

Neurodevelopmental disorders

Congenital brain malformations

Neuropsychiatric disorders

Neurodegenerative disorders

Prenatal exposure to adverse agents and hypoxic injury

Neurotropic viral infections

Drugs of abuse

Environmental contaminants

Pharmacological agents

Hypoxia

Brain tumors

Regenerative medicine

Future directions

References

Introduction

Brain organoids are cultured 3D structures, which are comprised of diverse neural cell types and derived from pluripotent stem cells (PSCs) (Di Lullo and Kriegstein, 2017). Human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) can be efficiently patterned to neuronal fate using a combination of small molecules or proteins. These cultured brain organoids recapitulate many essential aspects of human fetal brains, including the molecular signature, cellular composition, and cytoarchitecture (Di Lullo and Kriegstein, 2017). The tremendous advances in brain organoid technologies in recent years create a unique opportunity to explore human-specific features under healthy versus compromised conditions in a simplified and tractable experimental model system. For example, brain organoids can be genetically and pharmacologically manipulated for mechanistic studies while maintaining the complex genetic background of each individual. Since the developmental trajectories of various brain regions are highly intricate and different from each other, it is important to model this aspect of the human brain in vitro. Additionally, brain-region-specific organoids provide a useful platform to explore the mechanisms of neurological disorders, which may be dependent on the developmental origins of affected brain regions and the vulnerability of specific neuronal subtypes to different pathological insults. In this chapter, we review methodologies to generate brain-region-specific organoids from human PSCs and discuss how these in vitro 3D cultures are leveraged to deepen our understanding of human brain development and diseases as well as to explore novel therapeutic strategies.

Methodologies to generate and culture brain-region-specific organoids

Tremendous progress has been made over the past decade to develop and improve the methodologies to obtain brain organoids that more closely resemble the fetal human brain. Lancaster and colleagues first reported the generation of whole-brain organoids: starting from human ESC- or iPSC-derived neuroectodermal tissue embedded in Matrigel droplets, which were then transferred into a spinning bioreactor for enhanced oxygen, nutrient support, and keeping cultures in suspension (Lancaster et al., 2013). As no specific brain patterning cues were used in this original protocol, now also referred to as an unguided protocol, organoids generated by this approach contained heterogeneous population of cells, with identities of various brain regions, including the dorsal forebrain, ventral forebrain, midbrain, hindbrain, hippocampus, choroid plexus, retina, meninges, and other cell types of nonneural origin (Lancaster et al., 2013). The diverse cell types identified in the whole brain organoids were further confirmed by subsequent single-cell RNA sequencing studies, showing the presence of multiple subtypes of neural progenitor cells and neuronal cells, as well as cells expressing mesodermal makers (Camp et al., 2015; Quadrato et al., 2017). Interestingly, photosensitive cells in 8-month-old whole brain organoids were shown to respond to light stimulation, demonstrating certain functionality of this in vitro system (Quadrato et al., 2017). Since then, the field moved quickly to developing methodologies of generating more homogeneous brain-region-specific organoids with the application of tissue patterning factors, which are now termed guided protocols (Qian et al., 2019).

During embryogenesis, the neuroepithelium folds to form the neural tube, which gives rise to different regions of the brain and spinal cord (Ishikawa et al., 2012). The early developing neural tube is comprised of three primary vesicles including prosencephalon, mesencephalon, and rhombencephalon in order from rostral to caudal. Prosencephalon, also known as forebrain, is later subdivided into telencephalon and diencephalon (Ishikawa et al., 2012) (Fig. 1.1). In the developing telencephalon, the dorsal part gives rise to structures such as cerebral cortex, choroid plexus, cortical hem, hippocampus, and olfactory lobe, while the ventral part gives rise to lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE), caudal ganglionic eminence (CGE), and basal ganglia (Hébert and Fishell, 2008). Diencephalon is the embryonic origin of hypothalamus, thalamus, posterior pituitary, pineal gland, and optic cup (Martinez-Ferre and Martinez, 2012; Rizzoti, 2015; Adler and Canto-Soler, 2007). Mesencephalon, also known as midbrain, gives rise to structures such as substantia nigra and ventral tegmental area (Prakash and Wurst, 2006). Finally, rhombencephalon or hindbrain is subdivided into metencephalon and myelencephalon, which are the embryonic origins of cerebellum, medulla, and pons (Frank and Sela-Donenfeld, 2019). For more details, there are several excellent and comprehensive reviews of all brain regions and their corresponding embryonic origins (Ishikawa et al., 2012; Hébert and Fishell, 2008; Martinez-Ferre and Martinez, 2012; Rizzoti, 2015; Adler and Canto-Soler, 2007; Prakash and Wurst, 2006; Frank and Sela-Donenfeld, 2019). Different brain regions are patterned based on the gradients of factors such as fibroblast growth factors (FGFs), WNTs, and retinoic acid (RA) along anterior–posterior axes and WNTs, bone morphorgenetic proteins (BMPs), and sonic hedgehog (SHH) along dorsal–ventral axes (Tao and Zhang, 2016). Specific combinations of small molecules or proteins modulating these pathways have been used as potent cues to guide precise induction of PSCs toward brain-region-specific identity for 2D cultures (Tao and Zhang, 2016; Moya et al., 2014; Cutts et al., 2016). Direct differentiation of PSCs into brain-region-specific organoids is mostly based on this prior knowledge along with fine-tuning of the concentration and application timing during the early patterning phases (Fig. 1.1). In the following sections, we discuss methodologies of generating brain-region-specific organoids and highlight prominent features of each selected protocol. In general, all protocols start from patterning to a more homogeneous population of progenitor cells with identities of the corresponding brain regions, followed by suspension culture conditions and media for promoting growth and neuronal differentiation and maturation. A summary of culture protocols and their key features from selected publications is provided in Figs. 1.2–1.4.

Figure 1.1 Summary of the major patterning factors required for optimized formation of brain-region-specific organoids.Secondary brain vesicles, including telencephalon, diencephalon, mesencephalon, metencephalon, and myelencephalon, give rise to brain regions, such as dorsal forebrain, medial forebrain, ventral forebrain, retina, thalamus, hypothalamus, midbrain, cerebellum, and medulla. Patterning factors that are common among selected protocols for each brain region are listed here. More comprehensive list of patterning factors and how they are applied is shown in Figs. 1.2–1.4.

Organoids with the prosencephalon (forebrain) origin

Dorsal forebrain organoids—cortex

Cerebral cortex is the largest area of the brain important for higher cognitive functions. Human cerebral cortex to brain ratio is the largest among mammalian species due to the massive expansion of the cortical surface, and this brain structure is the basis for remarkable intellectual abilities in humans (Rakic, 2009). The developing human cortex arises from the dorsal telencephalon, which is comprised of inner neural progenitor zones and outer postmitotic neuronal layers (Dehay et al., 2015). Within the neural progenitor population, the cells are organized into three progenitor zones: ventricular radial glia (vRGs) in the ventricular zone (VZ), intermediate progenitors in the inner portion of the subventricular zone (SVZ), and outer radial glia (oRGs) in the outer portion of SVZ (oSVZ). Neurons generated from vRGs and oRGs migrate along radial fibers of these progenitors to form the cortical plate (CP), which is the outermost region of the cortex (Dehay et al., 2015). CP is structured as stratified cortical layers, and neural progenitors progressively commit to generating layer-specific neurons (layers II–VI) in an inside-out pattern, with later born neurons populating the outer layers with the exception of layer I neurons expressing REELIN, which are generated first (Shen et al., 2006). Unlike other cortical neurons, REELIN+ neurons can also be derived from cortical hem, ventral forebrain, and septum of the developing brain and control the radial migration of later born cortical neurons (Bielle et al., 2005).

Figure 1.2 Selected protocols for generating unguided brain organoids and region-specific dorsal and ventral forebrain organoids.Generally, the procedure starts with preinduction phase in which PSC cell reaggregates or whole cell colonies are cultured to form 3D embryoid bodies (EBs). Then, induction of brain-region-specific identities is often accomplished using two sequential steps. Finally, media containing neurotrophic factors and methods to enhance the diffusion of O2 and nutrients promote neuronal differentiation and survival in long-term culture. Above the line, media supplements are listed first, followed by patterning factors or neurotrophic factors, which are included in the brackets. KSR refers to knockout serum replacement. Below the line describes additional culture conditions other than the components of culture media. The timeline of each protocol is not drawn to scale, but rather aligned with the main phases of organoid cultures described above. Key features of each protocol are classified as cellular, molecular, or structural.

Figure 1.3 Selected protocols for generating medial, caudal and rostral forebrain organoids.Protocols for forebrain organoids are grouped by brain regions, including medial (M.), caudal (C.), and rostral (R.) forebrains. Firstly, PSC cell re-aggregates or whole cell colonies are cultured to form 3D embryoid bodies (EBs). Then, induction of brain region-specific identities is often accomplished using two sequential steps. Finally, media containing neurotrophic factors, and methods to enhance the diffusion of O2 and nutrients promote neuronal differentiation and survival in long term culture. Above the line, media supplements are listed first, followed by patterning factors or neurotrophic factors which are included in the brackets. GFF refers to growth factor free media. Below the line describes additional culture conditions other than the components of culture media. The timeline of each protocol is not drawn to scale. Key features of each protocol are classified as cellular, molecular, or structural.

Figure 1.4 Selected protocols for generating midbrain and hindbrain organoids.Similar to other brain-region-specific organoids, these organoids start from the formation of EBs by either reaggregation of cell suspension or whole stem cell colonies. These EBs are then subjected to multiple rounds of patterning factors before being transferred to media containing factors promoting neuronal differentiation. Above the line, media supplements are listed first, followed by patterning factors or neurotrophic factors, which are included in the brackets. Below the line describes additional culture conditions other than the components of culture media. The timeline of each protocol is not drawn to scale, but rather aligned with the main phases of organoid cultures described above. Key features of each protocol are classified as cellular, molecular, or structural.

In 2005, Sasai's laboratory was the first to identify factors required for efficiently inducing progenitors with a telencephalic identity from mouse ESCs without feeder cells or serum (Watanabe et al., 2005). Mouse ESCs were first dissociated, then slowly reaggregated, followed with a serum-free suspension culture method (SFEB or serum-free culture of embryoid body-like aggregates). In addition to the optimization of starting cell numbers in each aggregate, they showed that blocking Wnt and TGF-β signaling pathways by Dkk1 and LeftyA in the early phase maximized neural induction up to 90% efficiency (Watanabe et al., 2005). In the later phase, Wnt and Shh signaling were shown to be important for differentiation into dorsal forebrain fate (Pax6+/Foxg1+) and ventral forebrain fate (Nkx2.1+/Islet1+/Foxg1+), respectively (Watanabe et al., 2005). This method was subsequently applied to human ESCs to show that human telencephalic progenitors can be generated in 3D cultures (Watanabe et al., 2007) (Fig. 1.2).

Building upon the SFEB culture method, Eiraku and colleagues further improved the differentiation efficiency of mouse and human ESCs into telencephalic progenitors with a quick ESC reaggregation step (SFEBq) (Eiraku et al., 2008). This study empirically demonstrated the feasibility of generating brain-region-specific 3D aggregate cultures controlled by extrinsic patterning factors. They showed that dorsal cortical neuroepithelium patterned by WNT and TGF-β inhibition had the self-organizing capacity to form neural rosettes with apical–basal polarity and gave rise to neurons with a temporal order mimicking early corticogenesis in vivo (Eiraku et al., 2008) (Fig. 1.2). Marker expression and microarray global gene expression analyses of organoids generated from human ESCs or iPSCs confirmed their dorsal forebrain identity and at day 50 in culture they were most correlated with the first-trimester human cerebral cortex (Eiraku et al., 2008; Mariani et al., 2012). However, late-born upper cortical neurons were not present and the stratification of cortical layers was not observed using this approach. Moreover, PAX6+ neural progenitors decreased over time and became completely depleted around 100   days in culture, presumably due to a lack of oxygen and nutrient supplies as organoids grew larger in size (Eiraku et al., 2008). Indeed, growing these cortical organoids under 40% oxygen conditions appeared to be helpful for maintaining the progenitor zones up to 90   days and allowed for further development (Kadoshima et al., 2013). For example, the late-born upper cortical neurons and CP exhibited a tendency of inside-out structural patterning, which resembled the cytoarchitecture of human cortex in the early second trimester (Kadoshima et al., 2013). It is thought that evolutionary expansion of human cortex was enabled by the emergence of oRGs, a progenitor type that appears at a later stage of corticogenesis and is highly enriched in the developing human cortex, but not in the mouse cortex (Geschwind and Rakic, 2013; Lui et al., 2011). Indeed, some TBR2-/PAX6+/SOX2+ progenitors with oRG morphology were found in the outer portion of the SVZ using this optimized high oxygen culture condition (Kadoshima et al., 2013).

In an effort to establish a simpler and more reproducible method to culture cortical organoids, Pasca et al. directly patterned the intact human iPSC colonies to a cortical fate using dual SMAD inhibitors to block BMP and TGF-β signaling (Fig. 1.2), omitting the reaggregation step of dissociated PSCs used in SFEB or SFEBq methods (Chambers et al., 2009; Paşca et al., 2015). The aggregates were then cultured under suspension conditions in media containing FGF2 and EGF (epidermal growth factor) for 19   days without the addition of Matrigel, then switched to media containing neurotrophic factors to promote neuronal maturation (Paşca et al., 2015). Cortical organoids generated by this method exhibited well-separated progenitor zones and CP. At day 76, these organoids contained a large number of neurons expressing the mature neuronal marker NEUN, which normally appears in the fetal brain at 20   weeks of gestation. Whole transcriptional analysis showed a strong correlation between 76-day cortical organoids and the midgestation fetal human brain (Paşca et al., 2015). In addition to the generation of more mature neurons, a prominent feature of this protocol was robust astrogenesis. GFAP+ astrocytes can be observed as early as 50   days at a low percentage, which gradually increased to 20% of total cells by 180   days, with some functionality comparible to postnatal astrocytes (Paşca et al., 2015). In addition, a study by Lancaster and colleagues noted that generation of cortical organoids was significantly more consistent when organoids had a higher surface area to volume ratio, likely due to increased exposure to instructive factors (Lancaster et al., 2017). Thus, biodegradable microfilaments were used for structural support while stem cell aggregates underwent self-organization during neural induction, resulting in elongated organoids (Lancaster et al., 2017).

Although human-specific features of cortical development such as the appearance of oRGs could be observed in cortical organoids in early studies, the cell density was usually sparse with no distinct oSVZ layer (Lancaster et al., 2013; Kadoshima et al., 2013). In contrast, oRGs become the dominant neural progenitor pool for neurogenesis during late phases of human cortical development (Kriegstein et al., 2006; Sun and Hevner, 2014). To address this limitation, Qian and colleagues added an additional forebrain patterning step after initial neural induction of human iPSC colonies by dual SMAD inhibition, which was done by embedding the spherical structures into Matrigel for scaffolding in the presence of a WNT agonist and TGF-β inhibitor (Qian et al., 2016) (Fig. 1.2). This additional patterning step appeared to be essential for the expansion of the progenitor pool with forebrain identity close to 100% (Qian et al., 2016). The organoids were then cultured in miniaturized spinning bioreactors to enhance oxygen and nutrient penetration to promote survival and maturation of organoids. The key advantage of this protocol was the generation of a large number of oRGs in the late developing stages that formed a distinct and thick oSVZ layer. The oRGs expressed known markers, such as HOPX, FAM107A, and PTPRZ1, and displayed typical morphology with their radial processes contacting only the basal surface (Qian et al., 2016). Moreover, the CP in cortical organoids generated by this approach exhibited rudimentary layer separation with an inside-out pattern and showed more neuronal diversity, with neurons expressing TBR1+/CTIP2+ deep layer, SATB2+ upper layer, BRN2+/CUX1+ layer II/III, and REELIN+ layer I markers, along with several subtypes of GABAergic interneurons. Of note, the absence of NKX2.1+ ventral progenitors indicated that dorsal progenitors were capable of generating GABAergic neurons, a characteristic of the human but not mouse brain (Qian et al., 2016). Transcriptional analysis of these cortical organoids showed neuronal maturation over time and a strong correlation with human fetal brains, especially the prefrontal cortex. For example, 100-day-old organoids resembled the transcriptional signature of 24-week-old prefrontal cortex and 35-week-old frontal cortex (Qian et al., 2016).

Cortical organoids grow relatively large in size, up to several milliliters in diameter. An inherent problem of large 3D organoid cultures is a lack of vascularization that limits the diffusion of nutrients and oxygen, leading to hypoxia-induced cell death (Qian et al., 2019). Since the progenitor zones, as in the developing brain, are organized in the interior of cortical organoids, progenitor cells are more vulnerable to hypoxia and become depleted over time, hindering the in vitro investigation of later stages of human cortical development. Several strategies are therefore being developed to overcome this diffusion limit. The first approach is to expose the interior of organoids by slicing or cutting so that the central hypoxia-prone region has access to media and/or oxygen. For example, brain organoids were routinely cut in half by spring scissors to minimize hypoxia in the core without using spinning bioreactors (Watanabe et al., 2017). This method preserved the integrity of organoids up to 150   days in culture, but cortical layer separation was not apparent (Watanabe et al., 2017). Alternatively, brain organoids were sliced into 300-μm sections and grown under the air–liquid interface culture condition (Giandomenico et al., 2019). This ALI-CO (air–liquid interface cerebral organoid) methodology greatly improved neuronal survival under the adherent culture conditions that facilitated the formation of multiple axon tracts, including corticospinal tracts, but lost the cellular cytoarchitecture of progenitor and neuronal layers (Giandomenico et al., 2019). Qian and colleagues employed a similar slicing method to routinely slice the cortical organoids into thick 500-μm sections under suspension culture conditions to maintain the cellular cytoarchitecture of the entire progenitor zone and neuronal layers to sustain continuous neurogenesis (Qian et al., 2020). Single-cell RNA sequencing of these sliced neocortical organoids (SNOs) revealed clusters of distinct cell type identity including all cortical-layer-specific neurons, intermediate progenitors, and radial glial cells. Moreover, SNOs grown over 120   days exhibited well-separated upper and deep cortical layers, a phenomenon that occurs in the third trimester of human fetal brain development (Qian et al., 2020; Bayer and Altman, 2004; Nowakowski et al., 2017; Zhong et al., 2018; Ozair et al., 2018). The second approach for overcoming the diffusion limit is transplantation of organoids into the brain of host animals to achieve vascularization. For example, human brain organoids were grafted into adult immunodeficient mouse brains, and it was shown that host blood vessels grew into the organoids and supported long-term survival (Mansour et al., 2018). While human neurons from organoids formed connections with the mouse neurons and exhibited robust activity, the structural organization of progenitor zones largely disappeared soon after transplantation (Mansour et al., 2018; Daviaud et al., 2018). The third approach is to induce in vitro vascularization by introducing blood vessel forming cells into brain organoids (Pham et al., 2018; Cakir et al., 2019). However, because of a lack of circulation, the impact could be limited.

Despite considerable differences among various protocols for generating cortical organoids, the reproducibility of cortical organoid models was reflected by single-cell RNA sequencing of organoids using four different protocols (Velasco et al., 2019). With further improvement of culture conditions to grow these organoids, we will be able to better understand human-specific features of cortical development and mechanistic underpinnings at different stages, especially at later gestational stages, which are almost impossible to achieve in primary fetal tissues due to their limited availability for research.

Ventral forebrain organoids—MGE, LGE, and CGE

Cerebral cortex contains glutamatergic neurons and GABAergic interneurons. MGE, a region of the ventral forebrain, is the source of most cortical interneurons. These ventrally derived interneurons are born during midgestation in human brain development, migrate to the cortex, and undergo maturation up to early postnatal stages (Nicholas et al., 2013). Meanwhile, the LGE of the ventral forebrain generates interneurons that later migrate to the striatum, nucleus accumbens, and olfactory bulb (Wichterle et al., 2001). Shh signaling has been found to be essential for patterning the telencephalic progenitors into the ventral forebrain fate (Watanabe et al., 2005). Under SFEBq culture conditions, a low concentration of Shh agonist induced LGE identity, while a higher concentration favorably induced MGE identity in organoids derived from mouse ESCs (Danjo et al., 2011). For human ESCs or iPSCs, three groups used a combination of WNT inhibition and SHH activation to induce ventral forebrain differentiation after human PSCs were first patterned to the telencephalic fate (Birey et al., 2017; Bagley et al., 2017; Xiang et al., 2017) (Fig. 1.2). Organoids generated by this approach contained progenitor cells of both MGE and LGE/CGE region identity, expressing high levels of MGE marker NKX2.1 and LGE/CGE marker GSX2, but low levels of dorsal forebrain markers. Although pan GABAergic neuron markers such as GABA and GAD67 were detected early on, distinct GABAergic neuron subtype markers such as SOMATOSTATIN (SST), Neuropeptide Y (NPY), CALRETININ (CR), and CALBINDIN (CB) were detected later in culture, indicative of temporal progression and neuronal fate specification (Birey et al., 2017; Bagley et al., 2017; Xiang et al., 2017). Interestingly, the timing to detect subtypes of interneurons was quite different among these protocols, which could be due to the concentration and timing of the WNT and SHH manipulation used by different groups. Single-cell RNA sequencing of ventral forebrain organoids confirmed multiple cell types including ventral progenitors, GABAergic neurons, astrocytes, and oligodendrocyte precursor cells (Birey et al., 2017; Xiang et al., 2017).

Medial forebrain organoids—choroid plexus, cortical hem, and hippocampus

Hippocampus is the brain region that plays a critical role in consolidation of information from short-term memory to long-term memory, spatial navigation, and emotions (Anand and Dhikav, 2012). During human brain development, the dorsomedial telencephalon gives rise to medial pallium tissue, which in turn generates hippocampus. Choroid plexus is derived from the most dorsomedial portion of the telencephalon and produces cerebrospinal fluid for the central nervous system (Emerich et al., 2005). Cortical hem, which is lateral to choroid plexus, secretes WNTs and BMPs that are essential for hippocampal and choroid plexus patterning (Grove et al., 1998). Consistent with the in vivo patterning cues required for dorsalization, by carefully titrating the concentration of BMP4 and WNT agonist and optimizing the treatment time, organoids with early medial forebrain identity were generated from human ESCs based on SFEBq methods (Sakaguchi et al., 2015) (Fig. 1.3). These medial forebrain organoids contained a continuous neuroepithelium with choroid plexus, cortical hem, and hippocampal primordium-like tissue. However, bona fide hippocampal organoids containing distinct region-specific neurons were not generated even after culturing for 100   days, due to the difficulty in maintaining the structural integrity of organoids over the long term (Sakaguchi et al., 2015). Instead, dissociation of these medial forebrain organoids and further culturing under 2D adherent conditions allowed the detection of PROX1+ dentate granule-like neurons and KA1+ CA3 pyramidal-like neurons, which exhibited synaptic activity and network synchronization (Sakaguchi et al., 2015). Currently, there is no protocol reported for the generation of hippocampal organoids with the arealized dentate and CA neurons.

Caudal forebrain organoids—thalamus

Thalamus is the center for information relay between the cortex and the peripheral tissues that is critical for sensory-motor processing. Thalamic tissue arises from diencephalon, which is caudally adjacent to the telencephalon (Nakagawa, 2019). Xiang et al. first reported the method to differentiate human ESC aggregates into thalamic organoids by simultaneous induction of neuroectodermal fate and caudalization by dual-SMAD inhibition and insulin (Xiang et al., 2019) (Fig. 1.3). Thalamic region identity was then specified by MEK/ERK inhibitor and BMP7, which were used to prevent excess caudalization and promote thalamic differentiation, respectively (Xiang et al., 2019; Shiraishi et al., 2017). These thalamic organoids expressed typical markers of developing thalamus such as PAX6, OTX2, DBX1, and GBX2 as well as thalamus-specific marker TCF7L2. Single-cell RNA sequencing confirmed thalamic lineage of neural progenitors to neurons. Moreover, global gene expression analysis showed that thalamic organoids were more similar to the dorsal thalamus than other regions of the human fetal brain (Xiang et al., 2019).

Rostral