Methods in iPSC Technology

The series Advances in Stem Cell Biology is a timely and expansive collection of comprehensive information and new discoveries in the field of stem cell biology. Methods in iPSC Technology, Volume 9 addresses the different methods used for induced pluripotent stem cell (iPSC) formation, maintenance, expansion, and differentiation. The ability to reprogram different cell types to iPSCs offers an opportunity to generate pluripotent patient-specific cell lines that can help in the understanding of multiple human disorders. This volume addresses a variety of current methods used in the generation and manipulation of iPSCs, such as magnetic nanoparticles, piggyBac vectors, lentiviral vectors, bioscaffolds, somatic cell nuclear transfer, CRISPR/Cas9, bacteria, and much more. This 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 stem cell biology and function, regenerative medicine, and therapeutics
• Covers the different methods used for iPSC formation, maintenance, expansion, and differentiation
• Is contributed by world-renowned experts in the field

• Language: English
• Publisher: Academic Press
• Release date: Jun 2, 2021
• ISBN: 9780323856447

Book preview

Methods in iPSC Technology, Volume 9

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. Current reprogramming methods to generate high-quality iPSCs

Introduction

Reprogramming methods (Table 1.1)

Reprogramming factors

Commentary

Chapter 2. Using magnetic nanoparticles in iPSCs

Introduction

Nanotechnology and nanoparticles

Biomedical applications of magnetic nanoparticles

Transgene delivery to hiPSCs using magnetic nanoparticles

Transfection of hiPSC-CMs using magnetic nanoparticles

Conclusions and perspectives

Chapter 3. PiggyBac vectors in pluripotent stem cell research and applications

From a moth transposon to the piggyBac vector

Improved piggyBac systems for transgene expression in PSCs

Use of piggyBac vectors in PSCs

Concluding remarks and future perspectives: piggyBac in the genome editing era

Chapter 4. Lentiviral vectors as the delivery vehicles for transduction into iPSCs: shortcomings and benefits

Chapter 5. Decellularized liver extracellular matrix for iPSC-based liver engineering

Introduction

Development of decellularized liver matrix

iPSCs as a cell source for liver engineering

Liver engineering with decellularized matrix and iPSCs

Future directions

Chapter 6. Combining bioscaffolds and iPSCs in the treatment of neural trauma and Alzheimer’s disease

Introduction

Background of induced pluripotent stem cells

The investigation of bioscaffolds and iPSC in spinal cord and peripheral nerve injuries

Induced pluripotent stem cell and bioscaffolds for the therapy for Alzheimer’s disease

Chapter 7. Emerging strategies for scalable human induced pluripotent stem cell expansion and differentiation

Introduction

General considerations for hiPSC bioreactor culture

Novel bioreactors for hiPSC expansion and differentiation

Conclusions and future trends

Chapter 8. One plus one could be greater than two: combining the powers of somatic cell nuclear transfer with Yamanaka’s factors in generating clinical grade human pluripotent stem cells

A brief history of somatic cell nuclear transfer

The Magic egg

Epigenetics: memories make a cell

Transcription factors: you are what you express!

Embryonic stem cells

Induced pluripotent stem cells

Are iPSCs suitable for human applications?

Regeneration and cancer: both sides of a coin

One plus one could be greater than two: iPSC nucleus for nuclear transfer

iPSCNT and future prognostications

Chapter 9. Bacteria to form induced pluripotent stem cells

Introduction

Origin of eukaryotic cells

Bacterial influence on cellular homeostasis and plasticity

Cell reprogramming by lactic acid bacteria (LAB) in vitro

Prospects

Conclusion

Chapter 10. CRISPR/Cas9 technologies to manipulate human induced pluripotent stem cells

Introduction

Methodologies for CRISPR/Cas9-mediated genome editing

Applications of the CRISPR/Cas9 system in human pluripotent stem cells

Conclusion and future directions

Chapter 11. Scalable expansion of human pluripotent stem cells for biomanufacturing cellular therapeutics

Introduction

Cultivation environment and hPSC physiology

Challenges and conclusions

Chapter 12. Xeno-free cultivation of human induced pluripotent stem cells for clinical applications

Introduction

Historical overview of stem cells

Human pluripotent stem cells

Culture of human pluripotent stem cells

Clinical manufacturing

Clinical trials

Cell banks

Future prospects

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.

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

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ISBN: 978-0-323-85766-6

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

Contributors

Aylin Acun

Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States

Shriners Hospitals for Children, Boston, MA, United States

Mohammad Badrul Anam

Department of Developmental Neurobiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

HIGO Program, Kumamoto University, Kumamoto, Japan

Stem Cell-Based Tissue Regeneration Research and Education Unit, Kumamoto University, Kumamoto, Japan

Ryan Brice,     Department of Biological Sciences, Wichita State University, Wichita, KS, United States

Joaquim M.S. Cabral

Department of Bioengineering and iBB – Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal

Associate Laboratory i4HB – Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal

Young Cha,     Department of Psychiatry and Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, MA, United States

Ornit Chiba-Falek

Division of Translational Brain Sciences, Department of Neurology, Duke University Medical Center, Durham, NC, United States

Department of Neurobiology, Duke University Medical Center, Durham, NC, United States

Giuseppe Maria de Peppo,     The New York Stem Cell Foundation Research Institute, New York, NY, United States

Maria Giovanna Garone,     Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy

Shah Adil Ishtiyaq Ahmad

Department of Developmental Neurobiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

Stem Cell-Based Tissue Regeneration Research and Education Unit, Kumamoto University, Kumamoto, Japan

Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Tangail, Bangladesh

Arif Istiaq

Department of Developmental Neurobiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

HIGO Program, Kumamoto University, Kumamoto, Japan

Stem Cell-Based Tissue Regeneration Research and Education Unit, Kumamoto University, Kumamoto, Japan

Department of Stem Cell Biology, Faculty of Arts and Science, Kyushu University, Fukuoka, Japan

Naofumi Ito

Department of Developmental Neurobiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

Stem Cell-Based Tissue Regeneration Research and Education Unit, Kumamoto University, Kumamoto, Japan

Elena F. Jacobson,     Chemical and Biological Engineering, Tufts University, Medford, MA, United States

Hyunsoo Jang,     Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

Boris Kantor

Viral Vector Core, Duke University, Durham, NC, United States

Department of Neurobiology, Duke University Medical Center, Durham, NC, United States

Joshua Kehler,     Department of Biological Sciences, Wichita State University, Wichita, KS, United States

Chun-Hyung Kim

Department of Psychiatry and Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, MA, United States

Paean Biotechnology, Daejeon, Korea

Nam-Shik Kim,     Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

Kwang-Soo Kim,     Department of Psychiatry and Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, MA, United States

Mikiko Kudo

Department of Developmental Neurobiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

Department of Stem Cell Biology, Faculty of Arts and Science, Kyushu University, Fukuoka, Japan

Pierre Leblanc,     Department of Psychiatry and Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, MA, United States

Rhoda Mondeh-Lowor,     The New York Stem Cell Foundation Research Institute, New York, NY, United States

Jihoon Moon,     Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

Shintaro Nakayama

Department of Developmental Neurobiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

Stem Cell-Based Tissue Regeneration Research and Education Unit, Kumamoto University, Kumamoto, Japan

Department of Stem Cell Biology, Faculty of Arts and Science, Kyushu University, Fukuoka, Japan

Diogo E.S. Nogueira

Department of Bioengineering and iBB – Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal

Associate Laboratory i4HB – Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal

Kunimasa Ohta

Department of Developmental Neurobiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

HIGO Program, Kumamoto University, Kumamoto, Japan

Stem Cell-Based Tissue Regeneration Research and Education Unit, Kumamoto University, Kumamoto, Japan

Department of Stem Cell Biology, Faculty of Arts and Science, Kyushu University, Fukuoka, Japan

Carlos A.V. Rodrigues

Department of Bioengineering and iBB – Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal

Associate Laboratory i4HB – Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal

Alessandro Rosa

Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy

Center for Life Nano Science, Istituto Italiano di Tecnologia, Rome, Italy

Fawaz Saleh,     The New York Stem Cell Foundation Research Institute, New York, NY, United States

Madhusudana Girija Sanal,     Institute of Liver and Biliary Sciences, New Delhi, India

Jeffrey S. Schweitzer,     Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States

Demetrios M. Stoukides,     Chemical and Biological Engineering, Tufts University, Medford, MA, United States

Emmanuel S. Tzanakakis

Chemical and Biological Engineering, Tufts University, Medford, MA, United States

Clinical and Translational Science Institute, Tufts Medical Center, Boston, MA, United States

Basak E. Uygun

Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States

Shriners Hospitals for Children, Boston, MA, United States

Megan A. Yamoah,     Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, United States

Li Yao,     Department of Biological Sciences, Wichita State University, Wichita, KS, United States

Ki-Jun Yoon,     Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

Xiao-Dong Zhang

Department of Internal Medicine, School of Medicine, University of California, Davis, CA, United States

Department of Veterans Affairs, Northern California Health Care System, Mather, CA, United States

About the editor

Alexander Birbrair

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

Preface

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

This volume Methods in iPSC Technology offers contributions by known scientists and clinicians in the multidisciplinary areas of biological and medical research. The chapters bring up-to-date comprehensive overviews of current advances in the field. This book describes recent advances in the use of iPSCs to model several diseases 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-the-art methodologies and the leading-edge concepts in the field of stem cell biology. In recent years, remarkable progress has been made in the obtention of iPSCs and their differentiation into several cell types, tissues, and organs using state-of-the-art techniques. These advantages facilitated identification of key targets and definition of the molecular basis of several disorders. Thus, the present book is an attempt to describe the most recent developments in methods in iPSCs technology which is one of the rising hot topics in the field of molecular and cellular biology today. Here, we present a selected collection of detailed chapters on diverse techniques to generate, manipulate, expand, and differentiate iPSCs. Twelve chapters written by experts in the field summarize the present knowledge about methods in iPSCs technology.

Kwang-Soo Kim and colleagues from Harvard Medical School introduce current reprogramming methods to generate high-quality iPSCs. Megan A. Yamoah and Xiao-Dong Zhang from University of California discuss using magnetic nanoparticles in iPSCs. Maria Giovanna Garone and Alessandro Rosa from Sapienza University of Rome bring our attention to piggyBac vectors in pluripotent stem cell research. Boris Kantor and Ornit Chiba-Falek from Duke University describe lentiviral vectors as delivery vehicles for transduction into iPSCs. Aylin Acun and Basak E. Uygun from Harvard Medical School update us with what we know about decellularized liver extracellular matrix for iPSC-based liver engineering. Li Yao and colleagues from Wichita State University compile our understanding of the progress in combining bioscaffolds and iPSCs in the treatment of neural trauma and Alzheimer's disease. Joaquim M.S. Cabral and colleagues from University of Lisboa summarize current knowledge on emerging strategies for scalable human iPSC expansion and differentiation. Madhusudana Girija Sanal from Institute of Liver and Biliary Sciences talks about somatic cell nuclear transfer in iPSCs. Kunimasa Ohta and colleagues from Kyushu University focus on bacteria to form iPSCs. Ki-Jun Yoon and colleagues from Korea Advanced Institute of Science and Technology address the importance of CRISPR/Cas9 technologies to manipulate human iPSCs. Emmanuel S. Tzanakakis and colleagues from Tufts University update us tools for scalable expansion of human iPSCs for biomanufacturing cellular therapeutics. Finally, Giuseppe Maria de Peppo and colleagues from The New York Stem Cell Foundation Research Institute outline the necessity of xeno-free cultivation of human iPSCs for clinical applications.

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: Current reprogramming methods to generate high-quality iPSCs

Young Cha¹, Pierre Leblanc¹, Chun-Hyung Kim¹,², Jeffrey S. Schweitzer³, and Kwang-Soo Kim¹     ¹Department of Psychiatry and Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, MA, United States     ²Paean Biotechnology, Daejeon, Korea     ³Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States

Abstract

Induced pluripotent stem cell (iPSC) technology that could revert the fate of terminally differentiated somatic cells into embryonic stem cell–like status by using ectopic expression of four transcription factors (e.g., Oct4, Sox2, Klf4, and c-Myc) opened up a new era of stem cell research to study and treat intractable human diseases, including neurodegenerative disorders such as Parkinson’s and Huntington’s diseases. Nevertheless, our current understanding of the reprogramming process is incomplete and requires further investigation into the underlying mechanisms and development of optimal reprogramming techniques. In this chapter, we provide an overview of the current techniques and critical issues for further optimization of high-quality iPSC generation.

Keywords

Cell replacement therapy; Cell-penetrating peptide; Core pluripotency transcription factor; Disease modeling; Episomal vector; Genome-integrating viral reprogramming method; Induced pluripotent stem cells; Metabolic reprogramming factor; microRNA; Nonintegrating viral reprogramming method; Nonviral reprogramming method; Oocyte-specific factor; Recombinant protein; Small molecule; Synthetic mRNA

Introduction

Reprogramming methods

Genome-integrating viral reprogramming methods

Retrovirus

Lentivirus

Nonintegrating viral reprogramming methods

Adenovirus

Sendai virus

Nonviral reprogramming methods

Reprogramming using cell extracts or recombinant proteins

Minicircle vectors

Synthetic mRNAs

Episomal vectors

Reprogramming factors

Core pluripotency transcription factors

Oct4

Sox2

Klf4

Myc

Nanog

Lin28

Oocyte-specific factors

Chromatin remodeling factors

Pluripotency-associated factors

Additional transcription factors

Metabolic reprogramming regulators

MicroRNAs

Commentary

Acknowledgments

References

Introduction

In 2006, Shinya Yamanaka and his colleagues first reported on technology allowing terminally differentiated mouse somatic cells to be reprogrammed into embryonic stem cell (ESC)-like state henceforth referred to as induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006). Subsequently, Yamanaka’s and two other groups successfully reprogrammed human somatic cells into iPSCs using the same or similar sets of reprogramming factors (Park et al., 2008; Takahashi et al., 2007; Yu et al., 2007), offering the unprecedented possibility of generating disease- or patient-specific pluripotent stem cells without the ethical issues that plagued the destruction of human embryos required to obtain human ESCs. Thus, the introduction of iPSC technology not only revolutionized stem cell research for mechanistic studies of development and disease, but it also opened up a new paradigm for personalized cell therapy by providing the possibility of unlimited autologous cell sources (Hockemeyer and Jaenisch, 2016; Karagiannis and Eto, 2016; Li and Izpisua Belmonte, 2016; Mertens et al., 2016; Scudellari, 2016; Takahashi and Yamanaka, 2016; Tapia and Scholer, 2016). However, despite this enormous potential, iPSC technology remains in its early stages and significant obstacles must be overcome in order to realize its full potential. In this chapter, current techniques and some of the issues related to iPSC generation and establishment will be discussed.

Reprogramming methods (Table 1.1)

Genome-integrating viral reprogramming methods

Retrovirus

In the early days of iPSC research following the report by Yamanaka’s group, retroviruses were widely used as a delivery tool to introduce the classical reprogramming cocktail into target cells (Takahashi et al., 2007; Takahashi and Yamanaka, 2006). This retroviral gene delivery technology was based on simple gamma retroviruses of murine origin, largely the Moloney murine leukemia virus (MMLV) that allows relatively long-term expression of transgenes (Walther and Stein, 2000). However, MMLV-based retroviral vectors transduce only actively dividing cells, limiting their use in delivering reprogramming factors into either nondividing cells or slow-dividing cells (Miller et al., 1990). In addition, retroviral delivery of transgenes leads to their permanent integration into host genomes. Their expression is impacted by the genomic locations of the integrations, host cell type, promoter used, and viral cis-acting sequences, all potentially compromising reprogramming efficiency and iPSC quality, with potential unwanted outcomes such as tumor formation (Hu, 2014).

Lentivirus

James Thomson and colleagues introduced the use of lentiviral vectors to deliver an alternative reprogramming cocktail composed of Oct4, Sox2, Nanog, and Lin28 into somatic cells to establish human iPSCs (Yu et al., 2007). Unlike retroviral vectors, lentiviral vectors can deliver reprogramming cocktails into both actively proliferating cells and quiescent cells (Fassati, 2006). However, like retroviruses, they are also subject to insertional mutagenesis caused by vector integration into the host cells’ genome. To address or minimize these issues, CRE recombinase-based excisable lentiviral vectors or tetracycline-inducible lentiviral vectors have been developed (Soldner et al., 2009; Somers et al., 2010). In spite of the high reprogramming efficiency of this method, the stable integration of lentiviral vectors remains a significant health and safety concern as it can allow for potential reactivation of transgenes in tissues derived from reprogrammed cells, thus limiting future application of iPSCs created in this way for translational studies such as disease modeling or cell replacement therapies.

Nonintegrating viral reprogramming methods

Adenovirus

Adenoviruses are nonintegrating vectors and thus eliminate the problems associated with disruption of or retention in the host genome, but their reprogramming efficiency is less than 0.0012% in mouse and human cells (Stadtfeld et al., 2008; Zhou and Freed, 2009). At present, it is unclear why the reprogramming efficiency by adenoviruses is significantly lower than those by retro- and lentiviruses. Thus, considerable work to optimize expression and increase reprogramming efficiency would be required to make these vectors a practical tool.

Table 1.1

ASC, human adipose stromal cell; CB, human cord blood cells; DPC, human dental pulp cell; FL, mouse fetal liver cells; HDF, human dermal fibroblast; HEP, mouse hepatocytes; HFF, human fetal fibroblast; K, Klf4; L, Lin28; M∗, L-Myc; M, c-Myc; MEF, mouse embryonic fibroblast; MSC, human mesenchymal stem cell; N, Nanog; ND, not determined; O, Oct4; PBMC, human peripheral blood mononuclear cell; S, Sox2; shp53, p53 shRNA; T∗, SV40 large T; T, TERT; TTF, mouse tail-tip fibroblast; VPA, valproic acid.

Sendai virus

Sendai virus is an RNA virus that can be diluted out of cells within 10 passages postinfection and that does not enter the nucleus. In addition, since large amounts of proteins are produced by Sendai virus vectors, they can reprogram fibroblasts and blood cells at higher efficiencies (up to 1%) (Ban et al., 2011; Fusaki et al., 2009; Seki et al., 2010). However, a disadvantage of this method is that established iPSCs must be maintained for up to 10 passages at a higher temperature (39°C) to achieve complete clearance of the viruses from the cells. In addition, since the long-term effects of this virus in humans are not completely known, the safety of iPSCs generated by this method in humans remains to be examined.

Nonviral reprogramming methods

Reprogramming using cell extracts or recombinant proteins

Ideally, protein-based reprogramming could generate footprint-free and safe iPSCs. Almost two  decades ago, Philippe Collas and colleague reported the successful conversion of human fibroblasts into different cell types: Exposure of 293T cells to T-cell extracts resulted in cells displaying T-cell functions while exposing them to neuronal precursor extracts resulted in cells displaying neuronal functions (Hakelien et al., 2002). They also reported that treatment of fibroblasts with extracts of embryonic carcinoma cells can elicit functional reprogramming and differentiation plasticity (Taranger et al., 2005). A few years later, two groups additionally showed that treatment of human or mouse fibroblasts with mouse ESC extracts induced the generation of iPSCs (Bru et al., 2008; Cho et al., 2010). Although these studies provided the proof of evidence for protein (extracts)-based reprogramming, it is challenging to deliver hydrophilic, bioactive macromolecules such as proteins directly into the intracellular space, which requires crossing the hydrophobic plasma membrane. To counter this challenge, investigators introduced pretreatment of the cells with membrane-permeabilizing reagents favoring transient pore formation in the plasma membrane to facilitate delivery of macromolecules into target cells. However, this approach is highly cytotoxic, drastically reducing the efficiency of somatic cell reprogramming. Indeed, despite extensive optimization efforts, we found that introduction of mouse ESC extracts into permeabilized rat cells yielded only partially reprogrammed cells while viral transduction of reprogramming factor genes readily generated rat iPSCs (Chang et al., 2010). In 1988, Frankel and colleagues pioneered the addition of cell-penetrating peptides (CPPs) to macromolecules in order to facilitate their delivery into cells. They reported that the human immunodeficiency virus transactivator of transcription (HIV-TAT) protein can efficiently penetrate the cell membrane by a short basic segment residing at amino acid 48–60, activating HIV-specific genes (Frankel et al., 1988; Frankel and Pabo, 1988). Thereafter, other artificial CPPs were introduced consisting of positively charged short peptides, typically of 5–30 amino acids in length, which facilitated the transport of a wide variety of bioactive cargos across the cell membrane with minimal cytotoxicity via endocytosis-dependent or endocytosis-independent pathways (Fig. 1.1) (El-Sayed et al., 2009; Ziegler et al., 2005). Based on this concept, we and others reported the successful generation of iPSCs from human and mouse somatic cells, respectively (Kim et al., 2009; Zhou et al., 2009). In the study by Zhou et al. iPSCs were generated from mouse embryonic fibroblasts using purified 11 arginine (11R)-tagged recombinant reprogramming proteins (i.e., Oct4-11R, Sox2-11R, Klf4-11R, and c-Myc-11R) together with the histone deacetylase inhibitor, valproic acid (VPA) (Zhou et al., 2009). In contrast, we reported the successful generation of human iPSCs with direct delivery of 9R-fused four reprogramming proteins in the absence of any chemical treatment (Kim et al., 2009). These protein-based iPSCs displayed typical characteristics similar to those of ESCs such as in vitro and in vivo pluripotent differentiation potentials to form all three germ layer lineages. In addition, protein-based reprogramming method showed the enhanced genomic integrity of resulting iPSCs compared to viral-induced strategies (Park et al., 2014). Thereafter, many investigators additionally used the same approach and have attempted to improve it (Table 1.2). For instance, Han et al. showed that transduction of highly purified, five reprogramming proteins, including Oct4, Sox2, Klf4, c-Myc, and Nanog, shortened the timeline for emerging hiPSC colonies (Han et al., 2017). Other common CPPs, including TAT, were adopted for protein-based reprogramming strategy by several researchers (Caulier et al., 2017; Tang et al., 2011; Thier et al., 2010, 2012). Importantly, Zhang et al. reported that TAT-fused reprogramming proteins have a higher reprogramming efficiency compared to 11 arginine–fused proteins (Zhang et al., 2012a). In addition, Nemes et al. showed that GST- and nuclear localization signal peptide-conjugated reprogramming proteins facilitate membrane penetration and nuclear localization, leading to the enhanced iPSC generation from mouse fibroblasts compared to previous studies (Nemes et al., 2014). Despite these efforts, at present, the efficiencies of protein-based reprogramming are still much lower than those of viral reprogramming methods. Notably, Cooke and colleagues reported that the poor reprogramming efficiency of protein-based approach is related to the lack of activation of toll-like receptor (TLR) signaling, which can be induced by viral infection, and suggested that the efficiency of protein-based reprogramming can be improved by addition of TLR agonists (Lee et al., 2012). This interesting idea warrants further investigation by independent laboratories. Finally, it is worthwhile to note that it is technically challenging to obtain large amounts of highly concentrated CPP-fused reprogramming proteins, mainly due to their short half-life (e.g., c-Myc and Klf4). Successfully addressing these obstacles will make protein-based reprogramming approaches invaluable for the iPSC technology to develop to its full potential.

Figure 1.1  Proposed mechanisms of translocation of cell-penetrating peptide (CPP)-fused reprogramming proteins. The positively charged CPPs enable the fused recombinant proteins to be delivered into cells via endocytic pathways and/or direct penetration through plasma membrane. The CPP-fused reprogramming proteins move to nucleus and induce the expression of target genes, leading to the reprogramming process.

Table 1.2

GST, glutathione-S-transferase; HAF, human amniotic fluid cells; HDF, human dermal fibroblast; HEP, mouse hepatocytes; HFF, human fetal fibroblast; HSC, human hematopoietic stem cell; K, Klf4; L, Lin28; M, c-Myc; MEF, mouse embryonic fibroblast; N, Nanog; NLS, nuclear localization signal; O, Oct4; Poly I:C, polyinosinic-polycytidylic acid; R, arginine; RV, retrovirus; S, Sox2; TAT, transactivator of transcription; VPA, valproic acid.

Minicircle vectors

Minicircle vectors are composed of a eukaryotic expression cassette containing only a eukaryotic promoter and cDNAs of the reprogramming factors to be expressed. Oct4, Sox2, Nanog, and Lin28-expressing minicircle vectors have enabled the reprogramming of human adipose stromal cells (ASCs) into iPSCs with an efficiency of 0.005% in 28 days (Jia et al., 2010). However, no published report of successful reprogramming of somatic cells other than ASCs and neonatal fibroblasts has yet appeared, suggesting that the method needs further refinement.

Synthetic mRNAs

The generation of footprint-free iPSCs using synthetic mRNAs for the reprogramming factors was introduced by Warren et al. to reprogram fibroblasts (Warren et al., 2010). This strategy is superior to other nonviral protocols both in terms of reprogramming efficiency and kinetics. However, this method was labor intensive, requiring daily transfection of mRNAs for 16 days due to the short half-life of mRNAs. To address this issue, an improved method using a nonmodified mRNA cocktail with four transfections was developed by Poleganov et al. (2015), expanding its usefulness.

Episomal vectors

Transient expression of reprogramming factors is another footprint-free reprogramming approach. However, using standard plasmid vectors, the expression of these factors is of a duration too short to reprogram cells efficiently (Okita et al., 2008). This shortcoming was addressed by the OriP/EBNA-based episomal vector system offering stable expression of reprogramming factors for a longer period of time. A single transfection of three OriP/EBNA plasmids expressing Oct4, Sox2, Nanog, Klf4, c-Myc, Lin28, and SV40 large T antigen resulted in the reprogramming of human fibroblasts into iPSCs with ∼0.01% efficiency (Cheng et al., 2012; Okita et al., 2011; Schlaeger et al., 2015; Yu et al., 2009). These episomal vector systems allowed the generation of iPSCs from diverse human somatic cells, including newborn and adult fibroblasts, CD34+ cord blood, peripheral blood mononuclear cells, and dental pulp cells (Cheng et al., 2012; Okita et al., 2011; Schlaeger et al., 2015; Yu et al., 2009). Importantly, transfected episomal vectors were spontaneously lost within 12 passages. While it has the limitation that only limited cell types have thus far been successfully used as a source, this method, together with the mRNA method, represents the most promising candidates for generating clinical-grade iPSCs.

Reprogramming factors

Core pluripotency transcription factors

In its groundbreaking 2006 publication, Yamanaka’s group identified four pluripotency-associated genes, namely Oct4, Sox2, Klf4, and c-Myc (so-called Yamanaka factors), that are highly expressed in ESCs. These four genes had enough power to reprogram mouse and human fibroblasts to the ESC-like state (Takahashi et al., 2007; Takahashi and Yamanaka, 2006). Soon after, Thomson’s group also reported the conversion of human fetal and postnatal fibroblasts into human iPSCs using a different combination of reprogramming factors, including Oct4, Sox2, Nanog, and Lin28 (Yu et al., 2007). In addition, George Daley’s group also reprogrammed human fibroblasts into human iPSCs by adding hTERT and SV40 large T antigen to Yamanaka factors (Park et al., 2008).

Oct4

Oct4 (Octamer-binding transcription factor 4) appears to be the most important and irreplaceable factor involved in the reprogramming process. As a member of the POU (Pit-Oct-Unc) domain transcription factor family, Oct4 can activate the expression of its target genes through binding the octameric consensus sequence motif AGTCAAAT (Pan et al., 2002). Oct4 is commonly expressed in all ESCs and also plays a central role in determining the fate of pluripotent stem cells (PSCs); increased or decreased expression of Oct4 results in the differentiation of PSCs into endoderm, mesoderm, or trophectoderm (TE), respectively (Boiani and Scholer, 2005; Do and Scholer, 2009; Lee et al., 2008; Nichols et al., 1998; Niwa et al., 2000). Recent genome-wide analyses to identify the genomic binding sites of pluripotency transcription factors, including Oct4, revealed that Oct4 acts at many genomic locations, including promoters and enhancers, together with a variable but overlapping set of transcription factors (Chen et al., 2008; Kim et al., 2008; Sridharan et al., 2009). Unsurprisingly given its critical role in maintaining pluripotency, Oct4 activity is tightly regulated to ensure the continuity of the germline and proper differentiation of various tissues and organs. The expression pattern of Oct4 is very similar across species; Oct4 expression is present in mature oocytes, reaches a much higher level at the eight-cell stage, and then becomes restricted to the inner cell mass (ICM), reduced in the TE and primitive endoderm (PE), and finally is restricted only to primordial germ cells (Palmieri et al., 1994; Pesce et al., 1998).

Sox2

Sox2, a member of the SRY-related high-mobility-group box (Sox) family, is one of the original reprogramming factors used by Yamanaka and his colleagues (Abdelalim et al., 2014). During the early stages of development, Sox2 is expressed in both ICM and TE, whereas at the late blastocyst stage its expression becomes restricted to pluripotent epiblasts and multipotent cells of the extraembryonic ectoderm, but