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JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY VOL. 67, NO. 18, 2016 ª 2016 BY THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION ISSN 0735-1097/$36.00 PUBLISHED BY ELSEVIER http://dx.doi.org/10.1016/j.jacc.2016.01.083 THE PRESENT AND FUTURE STATE-OF-THE-ART REVIEW Translation of Human-Induced Pluripotent Stem Cells From Clinical Trial in a Dish to Precision Medicine Nazish Sayed, MD, PHD,a,b,c Chun Liu, PHD,a,b,c Joseph C. Wu, MD, PHDa,b,c ABSTRACT The prospect of changing the plasticity of terminally differentiated cells toward pluripotency has completely altered the outlook for biomedical research. Human-induced pluripotent stem cells (iPSCs) provide a new source of therapeutic cells free from the ethical issues or immune barriers of human embryonic stem cells. iPSCs also confer considerable advantages over conventional methods of studying human diseases. Since its advent, iPSC technology has expanded with 3 major applications: disease modeling, regenerative therapy, and drug discovery. Here we discuss, in a comprehensive manner, the recent advances in iPSC technology in relation to basic, clinical, and population health. (J Am Coll Cardiol 2016;67:2161–76) © 2016 by the American College of Cardiology Foundation. S Listen to this manuscript’s ince the initial discovery that bone marrow either adult stem cells or ESCs based on their origins cells possess regenerative capacity through and differentiation potentials. The unusual capacities clonal expansion, which laid the foundation of ESCs to proliferate without senescing (i.e., self- for the field (1), regenerative medicine has come a renewal) and to form all cell types of the embryo long way. As a type of stem cell, these bone marrow (i.e., pluripotency) made them unique and valuable cells have the capacity for both self-renewal and dif- resources for studying cell fate and tissue develop- ferentiation into different cell types. Earlier attempts ment. Initially, work on pluripotent stem cells to understand the pluripotency of the inner cell mass (PSCs) was conducted using human ESCs (5); howev- focused on the developmental capacity of nuclei by er, the requirement to destroy early-stage embryos cloning in frogs (2), cloning in adult mammalian cells in the process of ESC derivation made their use ethi- (3), derivation of mouse and human embryonic stem cally controversial. In addition, practical consider- cells (ESCs) (4,5), and generation of ESCs and somatic ations hindered their medical applications, because cell fusion (6). Similarly, MyoD, a mammalian tran- any cells or tissues generated from human ESCs by scription factor, was found capable of converting definition would be allotransplants into the recipient fibroblasts to myocytes, which led to the concept of patient, requiring possible life-long immunosuppres- master regulators, transcription factors that deter- sion therapy. mine lineage specification (7). Using these paradigms, The discovery by Takahashi et al. (8,9) that a small subsequently discovered stem cells were classified as set of reprogramming factors (e.g., Oct4, Sox2, Klf4, From the aStanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California; bInstitute for Stem Cell audio summary by Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California; and the cDepartment of Med- JACC Editor-in-Chief icine, Division of Cardiology, Stanford University School of Medicine, Stanford, California. This work was supported by the Na- Dr. Valentin Fuster. tional Institutes of Health (NIH; R01 HL126527, NIH R01 HL123968, NIH R01 HL128170, and NIH R01 HL130020) to Dr. Wu, and by an American Heart Association scientist development grant (13SDG17340025), NIH-National Heart, Lung, and Blood Institute Progenitor Cell Biology Consortium pilot grant (SR00003169/5U01HL099997), and a Stanford Cardiovascular Institute seed grant to Dr. Sayed. Dr. Wu is cofounder of Stem Cell Theranostics; and has received a research grant from Sanofi. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Manuscript received November 20, 2015; revised manuscript received January 26, 2016, accepted January 26, 2016. 2162 Sayed et al. JACC VOL. 67, NO. 18, 2016 MAY 10, 2016:2161–76 Human iPSCs for Precision Medicine ABBREVIATIONS and nuclear taking into account his or her unique genetic makeup AND ACRONYMS reprogramming of mouse (8) and adult hu- (30). The goal is to understand the complex mecha- man cells (9) to pluripotency became a land- nism of diseases so that made-to-order treatment mark development in regenerative medicine. plans could be designed for each patient based on Termed induced pluripotent stem cells (iPSCs), their condition. In this review paper, we aim to they promised a source of therapeutic cells highlight the promise of iPSCs for expanding basic free from the ethical issues or immune bar- science research and generating novel therapeutic riers of human ESCs while retaining similar agents for clinical and public health applications. CM = cardiomyocyte EC = endothelial cell ESC = embryonic stem cell iPSC = induced pluripotent stem cell iPSC-CM = induced pluripotent c-myc; OSKM) can induce properties, such as self-renewal and plurip- stem cell–derived cardiomyocyte otency. Since that initial discovery, a number PSC = pluripotent stem cell of advances have been introduced to enhance both the efficiency of iPSC produc- RPE = retinal pigment tion and the safety of the resultant lines. epithelial These improvements include the use of chemical agents to enhance efficiency (BIX-01294, valproic acid, RG108, AZA [5-aza-2 0 -deoxycytidine], dexamethasone, TSA [trichostatin A], and A-83-01) (10,11), use of alternative cell sources for reprogramming (embryonic, fetal, and adult fibroblasts; neural stem cells; adipose stem cells; keratinocytes; and iPSC TECHNOLOGY: EMERGING CONCEPTS The greatest hallmark of iPSC technology lies in its simplicity and reproducibility. Although the past decade has shown great improvement in making iPSCs safer and more efficacious, which is critical for pushing the technology toward clinical application, the mechanisms involved in efficient generation of iPSCs are just emerging. These evolving concepts raise important fundamental questions that will be discussed in the following sections. blood cells) (12–14), use of various factors that could HOW CAN A FEW TRANSCRIPTION FACTORS TURN replace the reprogramming factors (15,16), use of BACK THE CELLULAR CLOCK? Many studies have vectors that can be excised from the genome (17,18), tried to address this question, but the general use of “nonintegrating” vectors (19,20), supplemen- consensus is that the initial activity of the core plu- tation micro- ripotency genes (OSKM) has a snowball effect that ribonucleic acids (21), use of recombinant proteins/ results in simultaneous activation of the entire peptides to reprogram somatic cells (22,23), and most endogenous network of pluripotency genes and in- recently, a purely chemical approach (24). hibition of lineage-specific genes within the reprog- of reprogramming factors with With the concerted efforts of the academic com- rammed somatic cells. The initial phase of munity, the past decade has seen a tremendous push reprogramming is associated with cells undergoing toward the efficient generation of safer PSCs, with the metabolic changes and genome-wide alterations in hope that one day iPSCs could be used for regenera- histone marks and methylation, followed by a late tive medicine in the clinic (25). Although halted maturation phase that causes defined changes in temporarily, the clinical trial using iPSC-derived nuclear structure, the cytoskeleton, and signaling retinal pigment epithelial cells (iPSC-RPEs) for treat- pathways (31,32). Indeed, by looking closely at these ment of macular degeneration shows how far we have mechanisms, researchers can now obtain nearly per- come in developing clinical-grade stem cells (26). In fect iPSCs by clearing previous roadblocks to reprog- the short run, iPSC technology will likely offer more ramming (33). avenues to understand the pathophysiology of dis- ARE THESE iPSCs THE SAME AS ESCs? An important eases and discover new therapeutic molecules. Spe- question that comes up repeatedly is whether iPSCs cifically, for disease modeling, iPSCs can be generated have the same genetic and epigenetic landscape as from patients who carry certain genetic mutations ESCs, and whether differentiated cells (e.g., CMs) and then differentiated into disease-relevant cell from these 2 types of stem cells behave similarly. types, such as cardiomyocytes (iPSC-CMs) (27–29). Despite several previous studies suggesting differ- Similarly, iPSC-derived cells can then be subjected to ences in gene expression or deoxyribonucleic acid high-throughput screens to discover new therapeutic (DNA) methylation between iPSCs and ESCs (34–36), small molecules or conduct drug toxicity assays. the majority of iPSC and ESC clones are largely Lastly, iPSC technology might enable personalized indistinguishable (37–39). More recent reports sug- therapies (i.e., cell or tissue replacement without the gest that both cell types have identical molecular and need for immunosuppression, and use of drugs functional characteristics, with variations in their tailored according to each patient’s genes, environ- genetic backgrounds (i.e., different donors for iPSCs ment, and life-style). This is precision medicine, an and ESCs) accounting for most of their regulatory initiative with the intent to cure each patient by differences (40). Sayed et al. JACC VOL. 67, NO. 18, 2016 MAY 10, 2016:2161–76 COULD Human iPSCs for Precision Medicine TRANSDIFFERENTIATION BE THE NEXT variants that distinguish affected patients from is the direct unaffected subjects are located in noncoding regions reprogramming of one somatic cell type to another of the human genome with limited alignment to desired cell type as an alternative approach to iPSCs. genomes of animals used as model systems, we run Termed transdifferentiation, this concept relies on the risk that even after introducing the same human the premise that we could achieve rapid reprogram- genetic variant in the animal model, we might fail to ming of a desired cell type by entirely avoiding the recapitulate the human disease phenotype (49). Thus, iPSC stage (41). Several groups have shown successful there is a compelling need to model human diseases transdifferentiation of fibroblasts to various types of using human samples. Since the groundbreaking STEP? Another emerging concept somatic cells, such as neurons (42), hepatocytes (43), creation of the first human iPSCs (9), many groups CMs (44), and endothelial cells (45,46). However, a have applied this technology to model human dis- current major drawback to the use of direct reprog- eases, with amyotrophic lateral sclerosis and various ramming for clinical purposes is the tremendous dif- congenital diseases among the first to be modeled ficulty of obtaining sufficient numbers of target cells using patient-specific iPSCs (12,50,51). Since these that fully recapitulate the properties of the desired reports, many have attempted to study human dis- cells (47). In addition, transdifferentiation has thus eases using iPSCs from patients with neurodegener- far failed to generate a pure population of desired ative cells, which makes it difficult for them to be used for (53–56), muscular dystrophies (57,58), and hemato- diseases (50,52), cardiovascular disorders disease modeling and drug development (48). Simi- logic disorders (59,60), to name a few. Cardiovascular larly, in contrast to iPSCs, which once reprogrammed diseases that have been studied are summarized in can be easily maintained and differentiated to a Table 1. desired cell type, the transdifferentiation approach M o d e l i n g a b r o k e n h e a r t : u s e o f i P S C s . Cardio- would require restarting the entire reprogramming myopathy is a complex disease of the heart that has a process for each experiment. pathogenesis that includes myocardial infarction, APPLICATIONS OF iPSCs genetic mutations, endocrine disease, and drug toxicities. Historically, animal models have been used to understand the pathophysiology of cardiovascular There are many human diseases that have limited diseases and discover new therapeutics; however, treatment options because of either a lack of relevant because of significant differences in cardiovascular tissue samples or a lack of information regarding genetics and physiology between humans and ani- disease progression. Thus, researchers traditionally mals, there is a compelling need for more accurate have relied on in vitro assays or animal models to models to understand cardiac diseases. The ability to understand disease progression and develop thera- differentiate iPSCs to disease-relevant derivatives, peutic interventions. These model systems use either such as iPSC-CMs (27,77), now offers a valuable op- small animals (e.g., rats and mice) or large animals portunity to derive patient-specific cells for the pur- (e.g., dogs, pigs, and nonhuman primates). These pose of cardiac disease modeling and drug discovery research designs are premised on the ability of the (28,78). animal model to mimic human subjects pathophy- Human iPSC-CMs can serve as a disease model in a siologically and to eventually develop end-stage dis- dish by retaining the genetic variance present in the ease like that seen in humans. However, because of patient and eventually exhibiting the phenotypic differences in cardiovascular anatomy and physi- features of the disease in vitro. Indeed, several ology (e.g., humans and rodents diverged w75 million groups have used this iPSC-CM technology to model years ago), animal models often do not accurately cardiac channelopathies, such as long-QT syndromes reproduce human pathophysiology in meaningful (53–55,71,79,80), Timothy syndrome (73), LEOPARD ways. Because iPSCs can be derived from healthy and syndrome (51), catecholaminergic polymorphic ven- diseased patients, they can be a robust alternative to tricular tachycardia (81,82), arrhythmogenic right animals for modeling human diseases (Figure 1). ventricular dysplasia (74,83), familial hypertrophic Indeed, several models of iPSCs have been generated cardiomyopathy (62), and familial dilated cardiomy- and explored for studying various human diseases, as opathy (64–66). In addition to serving as an aid in the outlined in the next section. understanding of genetic cardiomyopathies, iPSC- DISEASE MODELING. The inherent properties of CMs have been used to model acquired or extrinsic iPSCs for self-renewal and differentiation into any cardiac diseases caused by endocrine, metabolic, and cell type make them an ideal candidate for mo- neuromuscular dysfunction. For example, iPSC-CMs deling human diseases. Because many of the genetic are being used in cell-based assays to model 2163 2164 Sayed et al. JACC VOL. 67, NO. 18, 2016 MAY 10, 2016:2161–76 Human iPSCs for Precision Medicine F I G U R E 1 Potential Applications of Patient-Specific iPSCs Disease-specific target cells differentiated from patient-specific induced pluripotent stem cells (iPSCs) have 3 major applications: disease modeling, regenerative therapies, and drug discovery/toxicity studies. iPSCs from patients with genetic mutations could be corrected via genome editing to yield healthy target cells for cell therapy. Both corrected and uncorrected target cells could be used for disease modeling or drug screening. RBC ¼ red blood cells. susceptibility to drug-induced cardiotoxicity (84,85), indicate that they resemble fetal rather than adult thereby allowing researchers to predict adverse drug CMs. Structurally, compared with adult human CMs, responses more accurately in patients with different these iPSC-CMs look much smaller in size and have genetic backgrounds. Similarly, iPSC-CMs have been less sarcomeric organization with decreased force used to understand the disease progression of dia- generation (91–93). This could diminish their utility betic cardiomyopathy (86), viral myocarditis (87), for modeling adult-related heart diseases. Table 2 cardiac hypertrophy (88), and genetic polymorphism- summarizes the differences between immature and induced cardiac ischemia (89). mature CMs. Disease modeling using iPSC-CMs has been feasible i P S C - C M s n e e d t o m a t u r e . In the developing heart, because of ever-improving differentiation protocols networks of diverse factors tightly regulate car- (27,29,90). Importantly, although the phenotypic diomyocyte maturation, including mechanical, elec- characteristics of the iPSC-CMs are similar to those of trical, and biochemical signals. Although relatively primary human CMs with respect to their molecular, simple expedients, such as keeping iPSC-CMs in cul- electrophysiological, ture for prolonged periods (97) or growing them on properties, these mechanical, functional and metabolic characteristics also specific substrates (119), can enhance some aspects of Sayed et al. JACC VOL. 67, NO. 18, 2016 MAY 10, 2016:2161–76 their structure and function, much research now focuses on uncovering factors and pathways that drive T A B L E 1 List of Cardiac Diseases Modeled With iPSCs their maturation (120). Agents and cues that have been explored to induce maturation include microRNAs (121,122), chromatin and histone proteins (123), Disease ments would be necessary to unravel the complexity RYR2 Arrhythmia and abnormal Ca2þ signaling No (61) HCM MYH7 Abnormal Ca2þ handling, disorganized sarcomeres, and electrophysiological irregularities No (62) DCM LMNA Nuclear senescence and cellular apoptosis No (63) TNNT2 Altered regulation of Ca2þ, decreased contractility, and abnormal distribution of sarcomeric a-actinin No (64,65) TTNtv Sarcomere abnormalities, impaired contractility and response to stress No (66) DES Abnormal desmin aggregations, aberrant Ca2þ handling, and beating rate No (67) PLN Abnormal Ca2þ handling, electrical instabilities, and increased cardiac hypertrophy markers Yes (68) could potentially allow us to bring iPSC-CMs to maturity in a dish, with closer resemblance to the adult human heart. However, it is unlikely that cells grown under in vitro conditions will fully resemble cells residing in intact living subjects because of differences in environmental milieu, whether cardiac or noncardiac. Hence, the expectation that iPSC-CMs will be identical to mature primary human CMs might be unrealistic. iPSCs and genome engineering: a perfect match. The MYBPC3 Contraction defects No (69) BTHS TAZ Sarcomere assembly and myocardial contraction abnormalities No (70) LQT1 KCNQ1 Prolonged duration of AP, aberrant electrophysiological Kþ current, and protective action of beta-blockade No (71) LQT2 KCNH2 Prolonged AP duration, reduced cardiac potassium current I (Kr), and increased arrhythmogenicity Yes (54,55,71) LQT3 SCN5A Prolonged AP duration and persistent Naþ current No (72) LQT8 CACNA1C Defects in Ca2þ signaling and arrhythmia No (73) ARVD PKP2 Exaggerated lipogenesis, desmosomal distortion No (74) Pompe disease GAA Glycogen accumulation and abnormal ultrastructure Yes (75) FRDA FXN Impaired iron homeostasis, mitochondrial damages and aberrant Ca2þ signaling No (76) LEOPARD syndrome PTPN11 Abnormal cell size and sarcomeric organization No (51) finger nucleases, transcription activator–like effector nuclease, and the clustered regularly interspaced short palindromic repeat (128), has further advanced the application of iPSC technology to precision medicine. These genetic engineering techniques allow for DNA to be inserted, replaced, or removed from a genome by use of nucleases that create specific double-strand breaks at preferred locations. Endogenous cellular mechanisms then repair these breaks by homologous recombination (129). With these tools, it is now possible to generate human cellular disease models in a precise and predictable manner. Indeed, genome editing has been used to introduce genetic alterations to create cardiac disease models or correct genetic mutations in iPSC-CMs to model cardiac diseases. Single genetic mutations responsible for cardiomyopathies, such as dilated Barth syndrome, long-QT (Ref. #) (61) of full cardiac maturation. Unraveling these pathways emergence of genome editing tools, such as zinc Gene Correction No made in generating more mature iPSC-CMs. Additional future studies using high-throughput experi- Features Arrhythmia and abnormal Ca2þ signaling and biochemical cues (126,127). By decoding these signaling pathways, substantial progress has been Locus CASQ2 CPVT DNA methylation (124), metabolic energetics (125), cardiomyopathy, 2165 Human iPSCs for Precision Medicine syn- drome, and Duchenne muscular dystrophy, have been corrected by use of these genome-editing tools (68,70,71,130,131), which suggests the feasibility of this approach for disease modeling. However, these AP ¼ action potential; ARVD ¼ arrhythmogenic right ventricular dysplasia; BTHS ¼ Barth syndrome; Ca2þ ¼ calcium; CPVT ¼ catecholaminergic polymorphic ventricular tachycardia; DCM ¼ dilated cardiomyopathy; FRDA ¼ Friedreich ataxia; HCM ¼ hypertrophic cardiomyopathy; iPSCs ¼ induced pluripotent stem cells; Kþ ¼ potassium; LEOPARD ¼ lentigenes, electrocardiographic conduction defects, ocular hypertelorism, pulmonary stenosis, abnormalities of the genitals, retarded growth, and deafness or hearing loss; LQT ¼ long-QT syndrome; Naþ ¼ sodium. engineered nucleases could introduce unintended genomic alterations; for example, in addition to cleaving the on-target site, they might have off-target REGENERATIVE MEDICINE. The use of PSCs, such as effects. Similarly, other challenges, such as delivery ESCs and iPSCs, for cell transplantation or organo- methods and efficiency, still need to be sorted out genesis has captured the imagination of the lay pub- (132). Taken together, this tag-team technology of lic, but it remains a difficult task to achieve from a patient-specific iPSCs and genome editing could lay scientific standpoint. The limitations of organ trans- the groundwork required to achieve the bold initia- plantation, which is plagued by lack of organ avail- tive of precision health (30). ability and problems with immunorejection, have led 2166 Sayed et al. JACC VOL. 67, NO. 18, 2016 MAY 10, 2016:2161–76 Human iPSCs for Precision Medicine T A B L E 2 Molecular, Electrophysiological, and Metabolic Profile of iPSC-CMs and Mature Cardiomyocytes Parameters Morphology Immature CMs Mature CMs (Ref. #) Shape Round or polygonal Rod and elongated Size 20–30 pF 150 pF (94) (95) Nuclei per cell Mononucleated w25% Multinucleated (96) Multicellular organization Disorganized Polarized (95) Sarcomere appearance Disorganized Organized (95) Sarcomere length Shorter (w1.6 mm) Longer (w2.2 mm) (97) (98) Sarcomeric proteins MHC b>a b >> a Titin N2BA N2B (99) Troponin I ssTnI cTnI (100) Sarcomere units Electrophysiology Z-discs and I-bands Formed Formed H-zones and A-bands Formed (prolonged differentiation) Formed (95) M-bands and T-tubules Absent Present Resting membrane potential w60 mV 90 mV (93) Upstroke velocity w50 V/s w250 V/s (101) Amplitude Small Large (95) Spontaneous automaticity Exhibited Absent (95) Present Absent (101) (102) (97) Action potential properties Ion currents Hyperpolarization-activated pacemaker (If) Sodium (INa) Low High Inward rectifier potassium (IK1) Low or absent High (101) Transient outward potassium current (Ito) Inactivated Activated (103) ATP-sensitive Kþ current (IK,ATP) Not reported Present (104) L- and T-type calcium (ICa,L and ICa,T), rapid and slow rectifier potassium currents (IKr and IKs), Naþ-Caþ exchange current (INCX) and acetylcholine-activated Kþ (IK,ACh) Similar to adult CMs (93) Conduction velocity Propagation of signal Slower (w0.1 m/s) Faster (0.3–1.0 m/s) (105) Gap junctions Distribution Circumferential Polarized to intercalated disks (106) Calcium handing Ca2þ transient Inefficient Efficient (107) Amplitudes of Ca2þ transient Small and decrease with pacing Increase with pacing (108) Excitation-contraction coupling Slow Fast (109) Contractile force wnN range/cell wmN range/cell (110) Low or absent Normal (111) Positive Negative (112) (113) Ca2þ-handling proteins CASQ2, RyR2, and PLN Force-frequency relationship Mitochondrial bioenergetics Mitochondrial number Low High Mitochondrial volume Low High (113) Mitochondrial structure Irregular distribution, perinuclear Regular distribution, aligned (114) Mitochondrial proteins DRP-1 and OPA1 Responses to b-adrenergic stimulation Low High (115) Metabolic substrate Glycolysis (glucose) Oxidative (fatty acid) (116) Response Lack of inotropic reaction Inotropic reaction (117) Cardiac alpha-adrenergic receptor ADRA1A Absent Present (118) ATP ¼ adenosine triphosphate; CM ¼ cardiomyocyte; iPSCs ¼ induced pluripotent stem cells; MHC ¼ major histocompatibility complex. Sayed et al. JACC VOL. 67, NO. 18, 2016 MAY 10, 2016:2161–76 Human iPSCs for Precision Medicine researchers to seek alternative approaches (9,15). animals would be required to reach statistical power Both ESCs and iPSCs could differentiate into any cell and allow more in-depth mechanical analyses of the type in the body and thus could potentially replace engrafted heart, and the ventricular arrhythmias diseased or dysfunctional cells. Advances in culturing noted in these animals after transplantation must be technology and differentiation protocols have pro- addressed before human trials can be conducted duced much safer and clinically relevant cells with (148,149). In an effort to overcome the issues of poor minimal tumorigenic risk (25). This enabled several engraftment or survival of transplanted cells, another groups to initiate clinical trials using PSCs, with most study showed that a cytokine-loaded 3-dimensional targeting eye diseases (26). Of the 13 clinical trials fibrin patch with iPSC-derived cardiovascular cells being conducted, 9 are for ESC-RPE or iPSC-RPE cells (iPSC-CMs, iPSC-derived endothelial cells [ECs], and for macular degeneration, which causes the progres- iPSC-derived smooth muscle cells) had a better sive deterioration of light-sensing photoreceptors in chance of improving cardiac function in a pig model the eye (133). Previous work had shown that iPSC-RPE of myocardial infarction (146). One current clinical and ESC-RPE cells were safe and functional in trial is actively recruiting patients to test the use of pre-clinical models of macular degeneration (134,135). human ESC-derived cardiac progenitor cells in pa- Similarly, iPSCs generated from patients with retinitis tients with heart failure (150). On the basis of the pre- pigmentosa, a condition that involves retinal degen- clinical data showing a significant improvement in eration, were able to differentiate to rod photore- cardiac function caused by the paracrine effects of the ceptor cells and have beneficial effects (136,137). transplanted cardiac progenitor Despite the excitement and momentum for the use of ongoing clinical trial is using fibrin patches that iPSC derivatives in clinical trials, a cautious approach contain Isl-1þ SSEA-1 þ cells. The first clinical case is advisable, because one of the clinical trials focused report from this trial has found improvement in the cells (151), this on treating macular degeneration with iPSC-RPE cells patient’s functional outcomes and evidence of new- was recently halted because of genetic mutations in onset contractility of the transplanted area (152); the derived autologous cells. In addition, pre-clinical however, the evaluation of efficacy remains specula- data related to iPSCs have shown great promise for tive, because these ESC-fibrin patches were implan- various other ailments, including hematopoietic dis- ted at the time of a coronary artery bypass graft orders, spinal cord and musculoskeletal injury, and procedure. Accordingly, more patients need to be liver damage (138–141). Because a comprehensive recruited, and randomized trials need to be per- detailing of all these is beyond the scope of this re- formed in the future. view, we discuss only a few applications to cardiac Tissue engineering: to the rescue of cell therapy. It has diseases in the following sections. become quite clear in the past few years that without P a t c h i n g t h e b r o k e n h e a r t . Cardiovascular dis- optimal bioengineering tools, the use of PSC-CMs for eases remain the leading cause of mortality and regenerative medicine might be limited. This is morbidity in humans. PSC-derived cardiac tissue evident given that previous studies using PSC-CMs or might help restore diseased areas of the heart. progenitor cells have seen only moderate success Indeed, early work showed that PSC-CMs not only because of the failure of cells either to survive formed myocardial grafts but also electrically coupled transplantation or to fully engraft into the host and partially remuscularized the infarcted areas and myocardium, which limits possible gains in cardiac improved myocardial performance in small animal function models (142–145). Similarly, PSC-CMs have shown engrafted cardiac cells with the host myocardium promise in some pre-clinical large animal models increases the risk of ventricular arrhythmias caused (146). In a recent study, Chong et al. (147) reported by electrical heterogeneity (147,148,154). A patch- remuscularization of the infarcted region when hu- based approach for cardiac repair might be more ad- man ESC-CMs were injected into nonhuman primate vantageous than intramyocardial injections, because models of myocardial infarction. These grafts showed the patch could simultaneously act as a substrate to synchrony with the host myocardium, with regular strengthen the injured myocardium and prevent calcium transients and electrical coupling after adverse remodeling, as well as provide a template for transplantation; however, ventricular arrhythmias cells to survive and even proliferate. With advances were noted in these animals after transplantation, in tissue bioengineering, diverse biomaterials have and the study failed to show an improvement in their been found with the potential to enhance cardiac cell cardiac function. Clearly, significant hurdles still therapy (155). These biomaterials (including collagen, need to be overcome to translate this pre-clinical fibrin, alginates, silk, decellularized heart matrix, and report to a clinical trial. A much larger cohort of synthetic polymers) now allow researchers to deliver (153). Similarly, misalignment of the 2167 2168 Sayed et al. JACC VOL. 67, NO. 18, 2016 MAY 10, 2016:2161–76 Human iPSCs for Precision Medicine cells, retain them in situ, and use them to replace scar rofecoxib (Vioxx, Merck & Co., Kenilworth, New Jer- tissue with a large number of stem cell derivatives. In sey), the gastrointestinal prokinetic drug cisapride addition to being a vehicle for cell delivery, cardiac (Propulsid; Johnson & Johnson, New Brunswick, New patches have been shown to stimulate endogenous Jersey), and the broad-spectrum antibacterial drug paracrine factors that assist in cardiac repair (156,157). grepafloxacin (Raxar, GlaxoSmithKline, Brentford, Although there is much enthusiasm for the use of United Kingdom), all of which were withdrawn engineered heart tissue constructed from PSC-CMs because of clinical cardiac or arrhythmogenic toxic- for cardiac repair, there are some limitations that ities (161). With the pharmaceutical industry invest- need to be addressed, including how to engineer tis- ing w$2 billion per new drug over a period of 10 to 15 sues that are simultaneously large enough to provide years (162), this situation creates a tremendous benefits but small enough to prevent death of the burden on the health-care system. The drug with- transplanted cells because of lack of oxygen and nu- drawals are attributable in part to drug safety studies trients. Even transplanted engineered heart muscle being evaluated in less than ideal nonhuman animal constructs showed poor survivability because of lack cells and models after years of laboratory and pre- of vascularization (158). Hence pre-vascularization of clinical testing (Figure 2). The pharmaceutical in- the in vitro heart tissue could potentially make these dustry typically conducts its cardiotoxicity studies of engineered patches more closely resemble the native new drugs using in vitro cell lines, such as Chinese human myocardium and allow survival of thicker hamster ovary (CHO) cells and human embryonic tissues. Indeed, the addition of human ECs to ESC- kidney 293 (HEK293) cells overexpressing the I Kr CMs enhanced formation of vessel-like structures protein hERG (human ether-à-go-go-related gene) and CM proliferation in vitro (110). Similarly, the (163,164). However, these in vitro cell lines do not addition of a vascular patch composed of human ESC- replicate human CMs well, and the overexpression of ECs and ESC-derived smooth muscle cells to a porcine a single cardiac ion channel in these cells does not model of myocardial infarction resulted in significant recapitulate the complex channel biology in CMs. improvements in (159). Taken Because of these limitations, there is an inherent risk together, the continuing progress made in tissue en- of cardiotoxic drugs slipping through initial screens gineering to promote cell engraftment and survival only to be withdrawn from the market after patients after transplantation is expected to take us yet have experienced harm and large investments have another step closer to the clinical use of PSC-CMs in been incurred or wasted. Moreover, the converse regenerative medicine. might also be true. It is possible that pre-clinical cardiac function DRUG DISCOVERY AND TOXICITY SCREENING. In the past, immortalized cell lines or experimental animal models have been used to screen therapeutic agents for human diseases with limited success because of differences from the actual human setting. For example, it has always been difficult to analyze and compare data between mice and humans for cardiac diseases given the considerable variation in their heart rates (e.g., mice exhibit a higher heart rate of 500 to 600 beats/min compared with human heart rates of 60 to 80 beats/min) and electrophysiological properties (e.g., mice have shorter action potentials than humans because of differences in cardiac ion channels) (160). Studies using a patient’s primary heart tissue are helpful but are limited because of lack of donor availability, which makes in vitro modeling using this approach difficult and hampers its use in the evaluation of drug efficacy and toxicity. testing of certain drugs using in vitro cell lines might show toxicity, only for the same drug to be later shown to be safe and efficacious for human usage (e.g., verapamil) (84,161). This could lead to valuable drugs not being marketed or developed at all because of inaccurate forecasting of their likely toxic effects by use of conventional approaches. With all these known roadblocks in conventional drug screening, there is a compelling need to use functional human CMs for drug screening. PSC-CMs have now been comprehensively characterized as a good model for cardiotoxicity studies. Using a standard protocol, electrophysiological responses of PSCCMs to a selection of known drugs that affect the hERG channel have shown comparable data to conventional hERG by use of in vitro assays (165,166). These studies indicate that PSC-CMs could be adopted as a standard model for cardiotoxicity studies. Importantly for pharmaceutical companies, this Conventional drug screening: a long, arduous model could be a suitable addition to their conven- r o a d . There has been a long list of high-profile drugs tional drug screening methods. that have been withdrawn from the market because of iPSC-based drug screening: precise and personal. Every their off-target and on-target toxicity to the heart, patient has a unique genetic background and reacts including the nonsteroidal anti-inflammatory drug differently to medications. Despite presenting with Sayed et al. JACC VOL. 67, NO. 18, 2016 MAY 10, 2016:2161–76 2169 Human iPSCs for Precision Medicine F I G U R E 2 Conventional Versus iPSC-Based Drug Discovery (A) The conventional drug discovery pathway is a very inefficient process with a high attrition rate. The majority of candidate drugs never reach the market because of safety and efficacy issues. This is attributable in part to the lack of appropriate drug development models that could accurately predict drug efficacy and in part to dependence on animal models of disease that do not replicate human pathophysiology. (B) By comparison, induced pluripotent stem cell (iPSC) technology allows highthroughput screening of therapeutic molecules by providing disease-specific drug development models that are generated from the patients themselves. This makes iPSC technology a much better predictive tool and enables well informed decisions to be made earlier in the drug development process. FDA ¼ U.S. Food and Drug Administration; iPSC-CM ¼ induced pluripotent stem cell–derived cardiomyocyte. similar symptoms, patients might have different un- In addition, PSC-CMs could be cultured in a dish for derlying causes of the disease, but conventionally, extended periods of time (167), and large-scale pro- they will still receive the same medication on the duction of PSC-CMs is feasible from healthy control basis of their symptoms. iPSC-based drug screening subjects or patients with various cardiac diseases technology might allow evaluation of a personalized (Table 1). This iPSC-based model could provide a therapy for every patient, an approach known as valuable tool for the pre-clinical screening of candi- precision medicine (30). dates for whom a treatment would have therapeutic With the advent of iPSCs (9) and the improvements value and for screening candidates who might have in differentiating iPSCs to functional CMs (27,29,90), off-target cardiac toxicity in any individual genotype, it is now feasible to generate functional human CMs taking us one step closer to the goal of bringing pre- for drug screening, thereby avoiding the issues asso- cision medicine to the clinic (Figure 2). ciated with earlier, less relevant screening systems Although PSC-CMs provide an excellent platform and minimizing the time and cost for drug develop- for drug screening and toxicology studies (165), they ment. These ESC-CMs and iPSC-CMs have been currently have several limitations. For example, PSC- thoroughly characterized to show similar electro- CMs are generally immature and resemble fetal CMs physiological, biochemical, and pharmacological both structurally and electrophysiologically (92,93). functions that certify them as bona fide CMs (101). This cellular immaturity issue might seriously limit 2170 Sayed et al. JACC VOL. 67, NO. 18, 2016 MAY 10, 2016:2161–76 Human iPSCs for Precision Medicine drug development with PSCs. To overcome this limi- correctly identify a subset of patients with specific tation, several approaches have been developed to diseases who will respond to the drugs under study. mature PSC-CMs, including differentiating them on Similarly, depending on the effectiveness of the drug 3-dimensional patches, which could enhance their in clinical trials, we might also gain the ability to contractile apparatus (120,168). Others have tried to diagnose sporadic diseases among a subset of pa- achieve microRNAs tients. Given that iPSC-based clinical trials are in their (miR-1/miR-499, Let7) to improve metabolic ener- infancy, much work is still needed to rapidly and getics (121,125) or by subjecting the cells to electrical economically develop personalized iPSCs. and mechanical stimulation (169). In addition, uni- PRECISION MEDICINE: THE RIGHT DRUG FOR THE axial stretching of engineered heart muscle made RIGHT PATIENT AT THE RIGHT DOSE. The practice of from PSC-CMs could enhance their viability and medicine is undergoing a fundamental change. maturation and has been used for drug screening Rather than a generalized approach, the diagnostic (170). Despite their resemblance to fetal CMs and their and therapeutic strategies are now patient oriented, maturity by overexpressing labeling as “immature” CMs, these PSC-CMs have with a focus on precision medicine (30). This involves been used successfully to study drug efficacy and integrating the patient’s “omics” information (e.g., toxicity (84,85). genomic, epigenomic, transcriptomic, and metab- iPSC CLINICAL TRIALS: TOWARD MACROMEDICINE. As olomic) with advances in medical technology to tailor described previously, iPSC technology has revolu- therapeutic options for each individual patient. With tionized the concept of precision medicine. In the the advent of iPSC technology, this endeavor of pre- past, because of the lack of available human samples, cision medicine is now feasible. drug toxicity screening was difficult and relied There is an overwhelming consensus that cardiol- heavily on immortalized cell-based assay or in vivo ogy will lead the development of precision medicine, animal models. Advances in iPSC technology could after decades of research have revealed many of the provide a steady supply of functional cells for molecular pathways that cause cardiomyopathies. pre-clinical screening of drugs. Moreover, iPSCs pro- Moreover, the use of iPSC-derived cardiovascular vide a unique platform that enables researchers to cells has now enabled researchers to understand the model diseases on a patient-by-patient basis. This underlying mechanisms of many cardiac diseases that micromedicine approach makes it possible to under- were difficult to model because of lack of patient stand disease progression at an individual patient samples. This new understanding has started to in- level and thus to screen for optimal pharmacological fluence diagnostic and therapeutic strategies for car- drugs individually. Beyond analyses performed for diac diseases by the use of drugs that target specific individual patients, high-throughput assays for drug molecular drivers. For example, the phenotype of toxicity that use human iPSC-CMs (171) can also be iPSC-CMs derived from patients with long-QT syn- conducted, supporting the role that iPSC technology drome was found to be affected by b-adrenergic re- can play in macromedicine, under which iPSC-based ceptor blockers (53). Similarly, a drug for malignant medicine could be applied to cohorts of patients hyperthermia, dantrolene, was found to restore the (172). In these clinical trials, iPSCs generated from abnormal Ca2þ sparks and arrhythmogenic phenotype patients could be differentiated into functional cells of iPSC-CMs derived from patients with catechol- and then used to analyze the efficacy of drugs (Central aminergic polymorphic ventricular tachycardia (81). Illustration). On this basis, patients could be classified Such genotype-guided therapy is precise and, if as drug responders or nonresponders, and only those applied on a large scale, can transform patient care. patients responding to the specific drugs would be To achieve a deeper understanding of complex moved to the next phase of the clinical trial. Thus, genetic cardiomyopathies, many more cardiac ge- iPSCs could provide valuable patient stratification nomes need to be analyzed. This will require ad- based on each patient’s responsiveness to the drug in vances in medical technology that can help us better clinical trials. Moreover, iPSCs could help us identify understand heart disease and that are capable of specific markers in the drug responders that corre- predicting an optimal treatment plan for each patient. spond to important patient subpopulations, thereby With the availability of next-generation sequencing boosting the success rate in clinical trials by and pre-selecting Taken pharmacogenomics can provide such valuable infor- patients who will benefit. bioinformatics, patient-specific iPSC-based together, iPSC technology has the potential to signifi- mation. A patient’s iPSC-CMs could help identify the cantly contribute to or even revolutionize macro- genetic basis of the disease, followed by disease medicine, for the first time allowing us to quickly and modeling, finally leading to the identification of JACC VOL. 67, NO. 18, 2016 MAY 10, 2016:2161–76 Sayed et al. Human iPSCs for Precision Medicine CENTRAL ILLUSTRATION iPSC Clinical Trial: From Micromedicine to Macromedicine Sayed, N. et al. J Am Coll Cardiol. 2016;67(18):2161–76. Induced pluripotent stem cell (iPSC) technology has contributed to both micromedicine and macromedicine. This includes everything from disease modeling and drug development based on cellular and molecular analyses of individual patients to iPSC clinical trials for stratification based on cellular and molecular analyses of a cohort of patients. Importantly, it could allow for the creation of a more precise clinical trial by identifying a subset of patients with a specific disease who optimally respond to the drugs under investigation, thereby boosting success rates by pre-selecting these drug responders. DNA ¼ deoxyribonucleic acid; iPSC-CM ¼ induced pluripotent stem cell–derived cardiomyocyte; RNA ¼ ribonucleic acid. 2171 2172 Sayed et al. JACC VOL. 67, NO. 18, 2016 MAY 10, 2016:2161–76 Human iPSCs for Precision Medicine pharmacogenetic effective drug biomarkers therapy. This that can facilitate information, once platform to conduct drug-screening assays and to discover novel therapeutics. However, a major collected, can be matched with drug discovery concern thus far is the immature status of the PSC- screens to predict the most effective drug treatment CMs. Various approaches, including cardiac tissue (combination) for each person. In conclusion, patient- bioengineering, have been utilized to bring these specific iPSC-based PSC-CMs to maturity. Finally, the availability of pharmacogenomics have the potential to identify genetically affected human tissues is very limited. patient-specific genetic loci that are responsible for Genome-edited PSC lines have now made it possible disease and help develop the optimal individualized to recapitulate the human disease in a dish by use of therapeutic strategy for each patient. various gene-editing tools, such as endonucleases iPSC disease modeling and CONCLUSIONS AND FUTURE PERSPECTIVES Since the initial basic discovery by Takahashi et al. (8,9) that a set of 4 transcription factors could reprogram mouse and human somatic cells to pluripotency, the field of iPSCs, free from the ethical issues associated with ESCs, has successfully expanded to different avenues of regenerative medicine. To date, iPSCs have 3 major applications, in disease modeling, drug discovery, and regenerative medicine (zinc finger nucleases or transcription activator–like effector nuclease) or palindromic repeats (clustered regularly-interspaced short palindromic repeat). Libraries of disease-specific cells can now be generated by creating allele-specific patient iPSC lines with which to conduct drug testing and toxicity studies. In summary, iPSC technology is not only revolutionizing science but will also fundamentally alter our healthcare system and the future of medicine by making it proactive, predictive, preventive, and personalized. (173). Human iPSC-CMs have been used extensively to ACKNOWLEDGMENTS The characterize and model human heart diseases (174). knowledge Joseph Gold, Jon Stack, and Blake Wu for There have been more than 700 publications in the critical reading of the manuscript and Amy Thomas for past few years that have expanded their use to addi- preparing the illustrations. Because of space con- tional human diseases (both monogenic and poly- straints, the authors could not include all of the rele- genic). These advances in cardiac disease modeling vant citations on the subject matter, for which they have been made possible because of tissue-specific apologize. authors gratefully ac- differentiation protocols that allowed researchers to derive healthy and disease-specific CMs (Table 1). In REPRINT REQUESTS AND CORRESPONDENCE: Dr. addition to disease modeling, several groups have Joseph C. Wu, Stanford Cardiovascular Institute, Stanford tried to inject PSC-CMs into small and large animal University School of Medicine, 265 Campus Drive G1120B, models of myocardial infarction, with varying levels Stanford, California 94305-5454. E-mail: joewu@stanford. of success. Concerted efforts are now under way to edu. 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Quantified proarrhythmic potential of selected human embryonic stem cell-derived cardiomyocytes. Stem Cell Res 2010;4:189–200. biology: glimpse of the past, present, and future. Circ Res 2014;114:21–7. KEY WORDS drug discovery, human-induced pluripotent stem cells, macromedicine, micromedicine, personalized medicine