Induced Pluripotent Stem Cell E-Book
Download this e-book to learn about using iPSCs as research models.
Induced pluripotent stem cells (iPSCs) have come a long way since their discovery in 2006 (Yamanaka et al., 2006). Considering their use in developmental studies, drug discovery, and cell therapies, iPSCs are powerful tools in many researchers' arsenals.
This e-book provides a thorough introduction to researchers who are new to iPSCs or are considering integrating them into future research.
In this e-book you will find:
- Strategies for successful iPSC and iPSC-derived culture
- A summary of efforts towards iPSC standardization
- Case studies for investigating and validating human disease solutions
- Future directions and scalability of using iPSCs in your research
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Pages 4 - 5, Introduction to iPSCs
Introduction to iPSCs
Human induced pluripotent stem cells (iPSCs) are now at the forefront of human disease and development research, facilitating highly representative modeling and the reliable development of new therapies. Rapidly advancing stem cell resources and technologies have enabled the investigation of disease in tissue- and genetics-specific contexts, in models from patient-derived sources, and in the context of development that was not previously possible. Adoption of iPSC culture in the laboratory requires education on appropriate cell sources, characteristics, maintenance, quality control, and study design. However, advances in reagents, resources, and methods have made iPSC culture simpler and more accessible, making this an opportune time to incorporate iPSCs into laboratory workflows.
Development of iPSCs as a research tool
Stem cells are undifferentiated cells that can give rise to multiple cell types in an organism and have the capacity for self-renewal.1 While totipotent stem cells can differentiate into any cell of an organism, pluripotent stem cells (PSCs) can produce all germ layers of an organism except for extraembryonic structures, e.g. the placenta. Once PSCs differentiate into one of the germ layers, they are considered āmultipotentā and can only become cells that make up that germ layer. Pluripotent and, subsequently, multipotent cells then differentiate into specialized cells under specific physiological or cell culture conditions. Potency, or the range of cell types a PSC can differentiate into, is therefore reduced with each step of differentiation.
Embryonic stem cells (ESCs), derived from the inner cell mass of a blastocyst, fall within the PSC category. The isolation and culture of ESCs from mice was first reported in the early 1980s.2,3 Over the next 20 years, methods for the differentiation of ESCs into multiple lineages were developed. In 1998, the isolation and culture of human ESCs inspired great interest in their potential use as a source of tissues for transplantation and cell-based therapies.4,5 Early methods for the differentiation of ESCs into hematopoietic, vascular, and cardiac cells included embryoid body formation, growth on stromal cells, and growth on extracellular matrix.4 However, the undefined nature of differentiation factors involved in these methods led to the generation of undesired cell types. The use of fetal calf serum in cultures, which contains undefined factors, was replaced by the use of defined growth factors for lineage-specific differentiation.4 The continued development of ESC culture and differentiation protocols included the use of animal-free culture conditions.
ESCs have been used for organoid development to study embryonic and fetal development.6 Advances in ESC culture have also led to the derivation of cloned embryonic stem cells using somatic cell nuclear transfer for potential use in cell therapy and research (humans) and reproductive cloning (non-human animals).6 Because of ethical concerns regarding embryonic sources of human ESCs, the potential for abnormal development, and immune reactivity to allogenic ESCs, ESC-based cell therapy clinical trials and the development of ESC-based models and therapies have proven difficult.7 As a result, alternative technologies, such as iPSCs, have been developed that provide human PSCs from somatic sources and allow for the derivation of stem cell cultures from patients with known diseases.
Human iPSCs are generated artificially from somatic cells and exhibit functions that are similar to ESCs.1 The development and differentiation of iPSCs derived from patients has provided many in vitro disease models that previously could not be achieved. The ability to prepare iPSCs directly from individual patients makes them amenable to and useful for medical applications and disease models, including models of diseases arising from developmental or germline genetic causes. Other diseases, such as neurological or cardiac disorders, were not previously amenable to in vitro modeling using traditional techniques because of a lack of access to diseased tissues from living patients and difficulty in culturing post-mortem tissues.8 For these and other diseases, iPSC-based modeling has become fundamental in research and drug development. Subsequently, several new drug candidates that were discovered using iPSC-based screening are now in development and clinical pipelines.9 Models derived from iPSCs can be highly representative of human disease pathobiology compared to primary cells or non-human models.9 In addition, iPSC models highly predictive of drug response may reduce the need for animal use. iPSC culture has also become increasingly accessible to research and drug development laboratories because of simplified access and the availability of validated and standardized iPSCs, reagents, and resources. As a result, iPSCs have become a key platform for research and drug testing.
Pages 6 - 7, In Practice: iPSC Cultures
Research and drug development problems that are addressed by iPSCs
The use of human iPSCs in research and drug development is improving our understanding of disease and its effective treatment. Previous limitations in how closely models have represented the pathobiology, genetics, and progression of disease are being minimized through the use of patient-derived human iPSC models. Further, the power of disease modeling to discriminate true mechanisms of disease and the clinical relevance of therapeutic targets have been strengthened by human iPSCs because of their complementarity to primary cell culture and animal models.
Relevance to human disease
Many primary cell culture and animal models differ from human disease states in ways that prevent clinical extrapolation of results. For example, primary tissue culture models of neurological diseases, such as Alzheimerās disease (AD), present issues with accurate representation of the disease process and pathology. Tissues that are obtained post-mortem do not allow the study of disease progression or therapeutic intervention.19 Proliferating human and rodent cells also do not accurately model neurodegenerative processes or represent neurons, which are post-mitotic.19 Moreover, the heterogeneity in AD etiology among patients and the spectrum of multiple small genetic or epigenetic changes contributing to disease cannot be feasibly recapitulated through genetic manipulation of non-diseased cells.20 There are also concerns regarding the disease relevance of animal models, as mammalian models are not ideally matched to human evolution or genetics. As an example, sporadic AD risk-associated gene products, including CD33, TREM2, MS4A6A, and CR1 microglial surface proteins, only share approximately 50% identity between humans and mice.19,21
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Complementarity to other models
The use of multiple test systems, including human and animal models, has increased the success of efforts to discover disease mechanisms and test therapies. While there is a need to study neurodegenerative disease in in vivo systems, the difficulty of accurate modeling in animals presents the need for appropriate human-derived models. iPSC-derived models can be used to identify and study a range of human neurodegenerative disease phenotypes prior to validation and further study in animal models.32,33 For example, cell lines and three-dimensional cultures derived from iPSCs have been used to discover mechanisms of protein aggregate pathologies as well as vascular (blood-brain barrier) and immunologic phenotypes in AD.32,34,35 Laperle et al. recently discovered potential therapeutic targets for Parkinsonās disease using patient-derived iPSCs and dopaminergic iPSC-derived cultures with validation in murine models.33 Ayabe et al. used both iPSC-derived neuronal cells and mouse cells to mechanistically implicate the modulation of mitochondrial function in beta-lactolin preventive therapy for AD.36 Patient-derived iPSCs are a particularly useful addition to programs investigating neurodevelopment, as stem cell differentiation programs can replicate pathways engaged in embryogenesis and organismal development.32 Mechanisms identified using these models can be further studied using genetic manipulation and treatment in vitro and in vivo.
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Reduction of animal use
High cost, time consumption, ethical considerations, and suboptimal translation to humans have led to efforts to replace or reduce animal use in toxicology and preclinical testing of drugs. Since 2007, the National Research Council and the National Academies of Science, Engineering and Medicine have encouraged a transition from animal testing to mechanisms of action models to assess the risks associated with experimental drugs. More recently, the US Food and Drug Administration (FDA) has outlined plans for advancing novel techniques using in vitro human-derived and in silico platforms for discovery and toxicology through its Alternative Methods Working Group.42 The stated goal of the FDA is to āreplace, reduce and/or refineā animal use in preclinical testing. Patient-derived iPSC models for hepatotoxicity, neurotoxicity, cardiotoxicity, and airway epithelial toxicity/carcinogenicity are examples of alternative methods given in recent FDA reports.42 These methodologies are recognized by the FDA as having the potential for obtaining better predictive and mechanistic insights from preclinical studies.
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Page 8, In Practice: iPSC Cultures
Strategies for successful iPSC and iPSC-derived culture
While there are clear benefits of implementing human iPSC culture in research and drug development, the generation of meaningful data requires attention to specific quality control and experimental design considerations. These include ensuring genetic heterogeneity and stability, sufficient sample size, thorough characterization, and appropriate maturation of cell lines. These aspects of iPSC-based research are discussed herein.
Minimizing clonal variation and genetic instability
Phenotypic and genetic variation between cell lines and controls is a critical issue in any cell-based model and applies to iPSC culture similarly. While the traditional use of family- and gender-matched healthy control subjects can be susceptible to heterogeneity, the use of isogenic iPSC cell lines, which are generated using gene editing of well-characterized iPSCs from healthy controls or from the patient line with genetic correction, can minimize the problems associated with cell line variation.7 However, genetic instability acquired during culture remains a risk with iPSCs. This risk also exists with established cultures derived from primary cells, but is particularly important with PSCs because of their self-renewing capacity. With cell-based therapy, genetic instability can present the risk of introducing pathogenic changes. To mitigate this risk, therapies with engineered suicide genes exist that can eliminate cells with chromosomal abnormalities.7,44 An example of a suicide gene is an inducible thymidine kinase that can be activated using ganciclovir and is linked to cyclin D1, which is activated upon cell cycle progression to ablate proliferative cells.40 Therefore, suicide genes in cell-based therapies can effectively eliminate the evolution of tumor-initiating cells after transplantation, facilitating the safety of these treatment modalities.
Increasing sample size
As a wide variety of genetic backgrounds and causal genetic variations underlie certain diseases, large cohorts are required to elucidate disease etiology and clinical translation, especially when the underlying genetics are unknown. Interpretation of results obtained from studies that incorporate only a few cell lines derived from iPSCs can be confounded by genetic, epigenetic, and clonal variation between lines.7 Increasing the number of iPSC lines can minimize confounding ānoiseā in order to be sensitive to disease-relevant signals.7 High cost, low donor availability, and insufficient resources can be obstacles to in-house development of sufficiently large and well-characterized panels of human iPSCs to study a particular disease. However, large numbers of well-characterized iPSC lines derived from patients with a variety of diseases and healthy controls are growing in availability. These resources increase confidence in results obtained from panels of iPSC lines and can be accessed through biobanks and repositories with searchable databases of available iPSC lines, such as the ones indicated in the Further reading and resources section.
Maturity
iPSC-derived cells can exhibit relatively immature phenotypes compared to adult tissues, even after differentiation.7 While immature phenotypes are amenable to the study of developmental or early-onset diseases, the representative study of late-onset diseases can be difficult using immature cells. Likewise, long-term disease-related alterations in terminally differentiated cells may be difficult to recapitulate longitudinally when the model is undergoing maturation over a time scale that is not representative of the disease process.
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