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April 28, 2015
Classically described as a cell’s ability to differentiate into all 3 germ layers—mesoderm, ectoderm, and endoderm—pluripotency has long been the elusive core of germinal development that, if properly controlled, could lead to organ and tissue regeneration in humans. In 2006, Takahashi and Yamanaka and colleagues1 advanced biomedical science with the discovery that pluripotency could be induced in adult cells using just 4 embryonic transcription factors. Their method for creating so-called induced pluripotent stem cells (iPSCs) was straightforward for many laboratories with the requisite molecular tools yet was incredibly bold in scope, earning Yamanaka a Nobel Prize in 2012. Now nearly a decade and more than 5400 related publications since the initial 2006 article, the iPSC field is still only beginning to realize its full potential.
Prior to 2006, the study of pluripotency, especially in human systems, was problematic because it relied largely on human embryonic stem cells. First isolated from the inner cell mass of human blastocytes by Thomson and colleagues2 in 1998, human embryonic stem cells fostered both enormous hope and controversy. Destruction of human embryos was unpalatable to many in the public. For scientists, the US government’s restrictions substantially limited the number of individual lines that could be derived with National Institutes of Health (NIH) funding. This latter point is best illustrated by the limited number of NIH registry-approved human embryonic stem cell lines (300), which represent less than 0.00001% of the world’s 7 billion people and thus do not capture the great majority of human genetic diversity. Induced pluripotent stem cells are easier to derive than human embryonic stem cells, are largely free of ethical problems, and have enabled a major expansion in the genetic diversity of pluripotent cell lines.
Kitchener D. Wilson, MD, PhD1,2; Joseph C. Wu, MD, PhD1,3,4
PHENOTYPING THE GENOME
While cell therapies are on the more distant horizon, a new direction that is achievable with current technologies has emerged: modeling of genetic diseases using iPSCs, or “personalized medicine in a dish.” For the first time, development of human disease can be observed at the single-cell level, presenting an opportunity to rapidly phenotype the genome. Such iPSC disease models, which are isogenic with the patient from whom they are derived, eliminate the need for primary human tissues that are difficult to acquire and maintain in culture.4 For example, iPSC-derived cardiomyocytes from patients with a gene mutation causing dilated cardiomyopathy have a phenotype that recapitulates the disease.5 Importantly, iPSC models of genetic diseases allow for direct observation of genetic variation on phenotype and minimize the influences of environment and lifestyle.
With recent innovations in genome editing such as the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system that allow for rational, targeted alterations of DNA in live cells, there is great synergy with iPSCs to phenotype the resulting edited genome. This could involve either introducing a DNA variant into healthy iPSCs and then monitoring for a disease phenotype upon differentiation or removing a DNA mutation and monitoring for a healthy phenotype (as has been demonstrated for α1-antitrypsin deficiency6). Edited iPSCs can also address the difficulties in discerning true disease-causing genetic mutations from benign variation, complementing standard in silico predictions and epidemiologic studies. In particular, genome editing of iPSCs can reveal the degree of pathogenicity, if any, that is caused by variants of unknown significance, which are an enormous problem for genome sequence interpretation.
PHARMACEUTICAL SCREENING AND DEVELOPMENT
Because cells differentiated from disease- and patient-specific iPSCs may exhibit disease processes at the single-cell level, they are an attractive option for screening therapeutic compounds (Figure). High throughput screens of pharmaceuticals against cells differentiated from disease iPSCs have allowed for rapid assessment of efficacy and toxicity.7 Additionally, given the genetic heterogeneity underlying many common diseases for which treatments are ineffective due to unpredictable patient response, iPSC disease models also present an opportunity to tailor therapies to the disease-causing DNA mutation. The identified compounds, or combinations of compounds, would then be broadly applicable to all patients carrying the same mutation. For these reasons, iPSCs have elicited interest from the pharmaceutical industry with the hope that research and development can be greatly streamlined. For example, disease-specific iPSC lines will help expedite identification of drug candidates and accelerate the screening of toxic and off-target effects. To this end, multiple pharmaceutical and governmental initiatives are currently constructing large iPSC banks for use in drug screening and disease modeling.8
Many well-known technical challenges remain with iPSCs. Reprogramming adult cells to the embryonic stage is often time consuming, resource intensive, and plagued by inconsistency. As a result, iPSCs are not yet attainable from large populations of individuals without significant resource investment. Changing the primary cell type (eg, fibroblasts, leukocytes, keratinocytes, etc), the specific cocktail of reprogramming factors (eg, small molecules, noncoding RNAs, or other genetic factors), or transfection method (eg, viral or nonviral) has not yet led to sufficient reduction in time and labor to justify wide-scale routine implementation, particularly in clinical settings.
Other challenges include the potential for acquired somatic mutations and chromosomal rearrangements during the induction of pluripotency, though there is mixed evidence regarding the significance of this problem. There have also been reports of genetic and epigenetic variation between iPSC lines that were derived using identical methods, as well as incomplete epigenetic reprogramming. Nonviral, nonintegrating reprogramming methods have addressed some of these issues, but the process continues to be somewhat stochastic and requires refinement.
In addition, even if iPSC derivation was radically efficient, subsequent regenerative therapies and disease modeling will continue to be limited to only those cell types for which differentiation-from-pluripotency protocols exist. Many of these protocols yield mixed populations of immature, partially differentiated cells and, as with reprogramming, can be laborious and resource intensive. Thus, iPSC disease modeling studies require substantial controls and replicates and carry the caveat that observed cellular phenotypes in vitro may not always be relevant to adult disease.
THE FUTURE: PRECISION MEDICINE AND IPSCS
In this new era of precision medicine, in which an individualized approach to disease is the goal, it is hoped that new data-driven disease taxonomies coupled with targeted therapies will transform medicine into an efficient and modern enterprise. Big data are frequently cited as the major thrust of this endeavor, which will integrate data from the genome, epigenome, exposome, and individual patients to produce more accurate diagnoses, targeted treatments, and improved health outcomes. Induced pluripotent stem cell models, with their power for elucidating the genome-epigenome-phenotype relationship, must also be included in these algorithms. Patient- and disease-specific iPSCs are clearly the ideal tool for efficiently characterizing the individual phenotype, while also disentangling the confounding influences of environment and lifestyle. In the coming decades, data gained from iPSCs could constitute one of the most promising primary building blocks of precision medicine.