Regeneration of Nonhuman Primate Hearts With Human Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

BACKGROUND: The clinical application of human induced pluripotent stem cell-derived cardiomyocytes (CMs) for cardiac repair commenced with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of human induced pluripotent stem cell-derived CMs into the cardiac muscle layer, which has reportedly induced electrical integration, is unclear because of concerns about poor engraftment of CMs and posttransplant arrhythmias. Thus, in this study, we prepared purified human induced pluripotent stem cell-derived cardiac spheroids (hiPSC-CSs) and investigated whether their direct injection could regenerate infarcted nonhuman primate hearts. METHODS: We performed 2 separate experiments to explore the appropriate number of human induced pluripotent stem cell-derived CMs. In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks before transplantation and were designated as recipients of hiPSC-CSs containing 2×107 CMs or the vehicle. The animals were euthanized 12 weeks after transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6×107 CMs). RESULTS: Recipients of hiPSC-CSs containing 2×107 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle (fractional shortening at 4 weeks after transplantation: 26.2±2.1%; 19.3±1.8%; P<0.05), with a low incidence of posttransplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits (fractional shortening at 12 weeks after transplantation: 22.5±1.0%; 16.6±1.1%; P<0.01, left ventricular ejection fraction at 12 weeks after transplantation: 49.0±1.4%; 36.3±2.9%; P<0.01). The incidence of posttransplant arrhythmia slightly increased in recipients of hiPSC-CSs containing 6×107 CMs. CONCLUSIONS: We demonstrated that direct injection of hiPSC-CSs restores the contractile functions of injured primate hearts with an acceptable risk of posttransplant arrhythmia. Although the mechanism for the functional benefits is not fully elucidated, these findings provide a strong rationale for conducting clinical trials using the equivalent CM products.

Efficacy of exon-skipping therapy for DMD cardiomyopathy with mutations in actin binding domain 1.

Exon-skipping therapy is a promising treatment strategy for Duchenne muscular dystrophy (DMD), which is caused by loss-of-function mutations in the DMD gene encoding dystrophin, leading to progressive cardiomyopathy. In-frame deletion of exons 3-9 (Δ3-9), manifesting a very mild clinical phenotype, is a potential targeted reading frame for exon-skipping by targeting actin-binding domain 1 (ABD1); however, the efficacy of this approach for DMD cardiomyopathy remains uncertain. In this study, we compared three isogenic human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing Δ3-9, frameshifting Δ3-7, or intact DMD. RNA sequencing revealed a resemblance in the expression patterns of mechano-transduction-related genes between Δ3-9 and wild-type samples. Furthermore, we observed similar electrophysiological properties between Δ3-9 and wild-type hiPSC-CMs; Δ3-7 hiPSC-CMs showed electrophysiological alterations with accelerated CaMKII activation. Consistently, Δ3-9 hiPSC-CMs expressed substantial internally truncated dystrophin protein, resulting in maintaining F-actin binding and desmin retention. Antisense oligonucleotides targeting exon 8 efficiently induced skipping exons 8-9 to restore functional dystrophin and electrophysiological parameters in Δ3-7 hiPSC-CMs, bringing the cell characteristics closer to those of Δ3-9 hiPSC-CMs. Collectively, exon-skipping targeting ABD1 to convert the reading frame to Δ3-9 may become a promising therapy for DMD cardiomyopathy.

Mature human induced pluripotent stem cell-derived cardiomyocytes promote angiogenesis through alpha-B crystallin.

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be used to treat heart diseases; however, the optimal maturity of hiPSC-CMs for effective regenerative medicine remains unclear. We aimed to investigate the benefits of long-term cultured mature hiPSC-CMs in injured rat hearts. METHODS: Cardiomyocytes were differentiated from hiPSCs via monolayer culturing, and the cells were harvested on day 28 or 56 (D28-CMs or D56-CMs, respectively) after differentiation. We transplanted D28-CMs or D56-CMs into the hearts of rat myocardial infarction models and examined cell retention and engraftment via in vivo bioluminescence imaging and histological analysis. We performed transcriptomic sequencing analysis to elucidate the genetic profiles before and after hiPSC-CM transplantation. RESULTS: Upregulated expression of mature sarcomere genes in vitro was observed in D56-CMs compared with D28-CMs. In vivo bioluminescence imaging studies revealed increased bioluminescence intensity of D56-CMs at 8 and 12 weeks post-transplantation. Histological and immunohistochemical analyses showed that D56-CMs promoted engraftment and maturation in the graft area at 12 weeks post-transplantation. Notably, D56-CMs consistently promoted microvessel formation in the graft area from 1 to 12 weeks post-transplantation. Transcriptomic sequencing analysis revealed that compared with the engrafted D28-CMs, the engrafted D56-CMs enriched genes related to blood vessel regulation at 12 weeks post-transplantation. As shown by transcriptomic and western blot analyses, the expression of a small heat shock protein, alpha-B crystallin (CRYAB), was significantly upregulated in D56-CMs compared with D28-CMs. Endothelial cell migration was inhibited by small interfering RNA-mediated knockdown of CRYAB when co-cultured with D56-CMs in vitro. Furthermore, CRYAB overexpression enhanced angiogenesis in the D28-CM grafts at 4 weeks post-transplantation. CONCLUSIONS: Long-term cultured mature hiPSC-CMs promoted engraftment, maturation and angiogenesis post-transplantation in infarcted rat hearts. CRYAB, which was highly expressed in D56-CMs, was identified as an angiogenic factor from mature hiPSC-CMs. This study revealed the benefits of long-term culture, which may enhance the therapeutic potential of hiPSC-CMs.

Cardiac Regeneration Using Pluripotent Stem Cells and Controlling Immune Responses.

Pluripotent stem cell (PSC)-derived cardiomyocytes are a promising source of cells in myocardial regeneration therapy for end-stage heart failure. Because most previous reports have focussed on xenotransplantation models using immunocompromised animals, studies on immune rejection in allogeneic transplantation models are needed for preclinical and clinical applications. Human leukocyte antigen (HLA) plays an important role in allogeneic transplantation, and cell bank projects are currently underway worldwide to stock induced pluripotent stem cells (iPSCs) generated from healthy individuals with homozygous HLA haplotypes. However, it is difficult to stock iPSCs that match the entire population in these cell banks; thus, several groups have produced hypoimmunogenic PSCs by knocking out HLA. These HLA-knockout PSCs were able to avoid rejection by T cells but still suffered rejection by natural killer (NK) cells caused by 'missing self-recognition'. Recent studies have attempted to generate hypoimmunogenic PSCs with gene editing to inhibit NK cell activation. Regenerative medicine using autologous iPSCs can be an ideal transplantation therapy, but, currently, there are major hurdles to its practical application. Hopefully, further research will resolve these issues. This review provides an overview of the current understanding and progress in this field.

Intracoronary transplantation of pluripotent stem cell-derived cardiomyocytes: Inefficient procedure for cardiac regeneration.

Advances in stem cell biology have facilitated cardiac regeneration, and many animal studies and several initial clinical trials have been conducted using human pluripotent stem cell-derived cardiomyocytes (PSC-CMs). Most preclinical and clinical studies have typically transplanted PSC-CMs via the following two distinct approaches: direct intramyocardial injection or epicardial delivery of engineered heart tissue. Both approaches present common disadvantages, including a mandatory thoracotomy and poor engraftment. Furthermore, a standard transplantation approach has yet to be established. In this study, we tested the feasibility of performing intracoronary administration of PSC-CMs based on a commonly used method of transplanting somatic stem cells. Six male cynomolgus monkeys underwent intracoronary administration of dispersed human PSC-CMs or PSC-CM aggregates, which are called cardiac spheroids, with multiple cell dosages. The recipient animals were sacrificed at 4 weeks post-transplantation for histological analysis. Intracoronary administration of dispersed human PSC-CMs in the cynomolgus monkeys did not lead to coronary embolism or graft survival. Although the transplanted cardiac spheroids became partially engrafted, they also induced scar formation due to cardiac ischemic injury. Cardiac engraftment and scar formation were reasonably consistent with the spheroid size or cell dosage. These findings indicate that intracoronary transplantation of PSC-CMs is an inefficient therapeutic approach.

Restoring Dystrophin Expression with Duchenne Muscular Dystrophy Exon 45 Skipping in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Induced pluripotent stem cell (iPSC)-based disease model is a useful tool that can represent the pathophysiology of patient organs that are inaccessible due to invasiveness. Here, we present a method to induce differentiation of Duchenne muscular dystrophy (DMD) patient-derived iPSCs into cardiomyocytes and restore dystrophin expression by exon skipping using antisense nucleic acids. This involves a 20-day multi-step culture process for differentiation to cardiomyocytes, followed by exon-skipping experiments. Additionally, RT-PCR, western blotting, and immunocytochemistry are used to confirm the restoration of dystrophin expression.

Chemical Genetics Reveals a Role of Squalene Synthase in TGFß Signaling and Cardiomyogenesis.

KY02111 is a widely used small molecule that boosts cardiomyogenesis of the mesoderm cells derived from pluripotent stem cells, yet its molecular mechanism of action remains elusive. The present study resolves the initially perplexing effects of KY02111 on Wnt signaling and subsequently identifies squalene synthase (SQS) as a molecular target of KY02111 and its optimized version, KY-I. By disrupting the interaction of SQS with cardiac ER-membrane protein TMEM43, KY02111 impairs TGFß signaling, but not Wnt signaling, and thereby recapitulates the clinical mutation of TMEM43 that causes arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited heart disease that involves a substitution of myocardium with fatty tissue. These findings reveal a heretofore undescribed role of SQS in TGFß signaling and cardiomyogenesis. KY02111 may find its use in ARVC modeling as well as serve as a chemical tool for studying TGFß/SMAD signaling.

Transplantation of Pluripotent Stem Cell-Derived Cardiomyocytes into a Myocardial Infarction Model of Cynomolgus Monkey.

Recent evidence has provided exciting proof of concepts for the use of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) for cardiac repair; however, large animal studies, which better reflect human disease, are required for clinical application. Here, we describe how to create myocardial infarction in cynomolgus monkey followed by transplantation of PSC-CMs. This method ensures the establishment of a myocardial infarction model and enables reliable PSC-CM transplantation.

ß-Arrestin-Biased AT1 Agonist TRV027 Causes a Neonatal-Specific Sustained Positive Inotropic Effect Without Increasing Heart Rate.

The treatment of pediatric heart failure is a long-standing unmet medical need. Angiotensin II supports mammalian perinatal circulation by activating cardiac L-type Ca2+ channels through angiotensin type 1 receptor (AT1R) and ß-arrestin. TRV027, a ß-arrestin-biased AT1R agonist, that has been reported to be safe but not effective for adult patients with heart failure, activates the AT1R/ß-arrestin pathway. We found that TRV027 evokes a long-acting positive inotropic effect specifically on immature cardiac myocytes through the AT1R/ß-arrestin/L-type Ca2+ channel pathway with minimum effect on heart rate, oxygen consumption, reactive oxygen species production, and aldosterone secretion. Thus, TRV027 could be utilized as a valuable drug specific for pediatric heart failure.

Establishment of a heart-on-a-chip microdevice based on human iPS cells for the evaluation of human heart tissue function.

Human iPS cell (iPSC)-derived cardiomyocytes (CMs) hold promise for drug discovery for heart diseases and cardiac toxicity tests. To utilize human iPSC-derived CMs, the establishment of three-dimensional (3D) heart tissues from iPSC-derived CMs and other heart cells, and a sensitive bioassay system to depict physiological heart function are anticipated. We have developed a heart-on-a-chip microdevice (HMD) as a novel system consisting of dynamic culture-based 3D cardiac microtissues derived from human iPSCs and microelectromechanical system (MEMS)-based microfluidic chips. The HMDs could visualize the kinetics of cardiac microtissue pulsations by monitoring particle displacement, which enabled us to quantify the physiological parameters, including fluidic output, pressure, and force. The HMDs demonstrated a strong correlation between particle displacement and the frequency of external electrical stimulation. The transition patterns were validated by a previously reported versatile video-based system to evaluate contractile function. The patterns are also consistent with oscillations of intracellular calcium ion concentration of CMs, which is a fundamental biological component of CM contraction. The HMDs showed a pharmacological response to isoproterenol, a ß-adrenoceptor agonist, that resulted in a strong correlation between beating rate and particle displacement. Thus, we have validated the basic performance of HMDs as a resource for human iPSC-based pharmacological investigations.

Pluripotent Stem Cells for Cardiac Regeneration　- Current Status, Challenges, and Future Perspectives.

Loss of myocardium permanently impairs cardiac function because the adult mammalian heart has limited regenerative capacity. Strategies to regenerate injured heart tissue include the transplantation of multiple types of stem cells. Among them, pluripotent stem cells (PSCs) are a promising option because of their unlimited self-renewal and unequivocal cardiomyogenic ability. To date, advances in stem cell biology allow generation of relatively homogeneous human PSC-derived cardiomyocytes (CMs). In this regard, preclinical studies of PSC-CM transplantation in rodents and larger animal models have provided convincing proof-of-concept results, triggering clinical studies in multiple countries. However, a few important uncertainties are yet to be addressed, warranting further investigation before clinical implementation of this novel therapy. An overview of the potential of stem cell therapy to provide new CMs for cardiac regeneration is presented.

Heart regeneration using pluripotent stem cells.

Pluripotent stem cells (PSCs), which include embryonic and induced pluripotent stem cells (ESCs and iPSCs, respectively), have great potential in regenerative medicine for heart diseases due to their virtually unlimited cardiogenic capacity. Many preclinical studies have described the functional benefits after transplantation of PSC-derived cardiomyocytes (PSC-CMs). However, transient ventricular arrhythmias were detected after injection into non-human primates and swine ischemic hearts; as engrafted PSC-CMs form an electrical coupling between host and graft, the immature characteristics of PSC-CMs may serve as an ectopic pacemaker. We are entering a critical time in the development of novel therapies using PSC-CMs, with the recent first clinical trial using human iPSC-CMs (hiPSC-CMs) being launched in Japan. In this review, we summarize the updated knowledge, perspectives, and limitations of PSC-CMs for heart regeneration.

Increased predominance of the matured ventricular subtype in embryonic stem cell-derived cardiomyocytes in vivo.

Accumulating evidence suggests that human pluripotent stem cell-derived cardiomyocytes can affect "heart regeneration", replacing injured cardiac scar tissue with concomitant electrical integration. However, electrically coupled graft cardiomyocytes were found to innately induce transient post-transplant ventricular tachycardia in recent large animal model transplantation studies. We hypothesised that these phenomena were derived from alterations in the grafted cardiomyocyte characteristics. In vitro experiments showed that human embryonic stem cell-derived cardiomyocytes (hESC-CMs) contain nodal-like cardiomyocytes that spontaneously contract faster than working-type cardiomyocytes. When transplanted into athymic rat hearts, proliferative capacity was lower for nodal-like than working-type cardiomyocytes with grafted cardiomyocytes eventually comprising only relatively matured ventricular cardiomyocytes. RNA-sequencing of engrafted hESC-CMs confirmed the increased expression of matured ventricular cardiomyocyte-related genes, and simultaneous decreased expression of nodal cardiomyocyte-related genes. Temporal engraftment of electrical excitable nodal-like cardiomyocytes may thus explain the transient incidence of post-transplant ventricular tachycardia, although further large animal model studies will be required to control post-transplant arrhythmia.

Circulating re-entrant waves promote maturation of hiPSC-derived cardiomyocytes in self-organized tissue ring.

Directed differentiation methods allow acquisition of high-purity cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs); however, their immaturity characteristic limits their application for drug screening and regenerative therapy. The rapid electrical pacing of cardiomyocytes has been used for efficiently promoting the maturation of cardiomyocytes, here we describe a simple device in modified culture plate on which hiPSC-derived cardiomyocytes can form three-dimensional self-organized tissue rings (SOTRs). Using calcium imaging, we show that within the ring, reentrant waves (ReWs) of action potential spontaneously originated and ran robustly at a frequency up to 4 Hz. After 2 weeks, SOTRs with ReWs show higher maturation including structural organization, increased cardiac-specific gene expression, enhanced Ca2+-handling properties, an increased oxygen-consumption rate, and enhanced contractile force. We subsequently use a mathematical model to interpret the origination, propagation, and long-term behavior of the ReWs within the SOTRs.

Amelioration of intracellular Ca2+ regulation by exon-45 skipping in Duchenne muscular dystrophy-induced pluripotent stem cell-derived cardiomyocytes.

Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by frameshift mutations in the DMD gene. DMD involves cardiac muscle, and the presence of ventricular arrhythmias or congestive failure is critical for prognosis. Several novel therapeutic approaches are being evaluated in ongoing clinical trials. Among them, exon-skipping therapy to correct frameshift mutations with antisense oligonucleotides is promising; however, their therapeutic efficacies on cardiac muscle in vivo remain unknown. In this study, we established induced-pluripotent stem cells (iPSCs) from T cells from a DMD patient carrying a DMD-exon 46-55 deletion, differentiated the iPSCs into cardiomyocytes, and treated them with phosphorodiamidate morpholino oligomers. The efficiency of exon-45 skipping increased in a dose-dependent manner and enabled restoration of the DMD gene product, dystrophin. Further, Ca2+-imaging analysis showed a decreased number of arrhythmic cells and improved transient Ca2+ signaling after exon skipping. Thus, exon-45 skipping may be effective for cardiac involvement in DMD patients harboring the DMD-exon 46-55 deletion.

Pluripotent Stem Cell-Derived Cardiomyocyte Transplantation for Heart Disease Treatment.

PURPOSE OF REVIEW: Cardiovascular disease is the leading cause of mortality worldwide. Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) have great potential to treat heart disease, owing to their capacity of engraftment and remuscularization in the host heart after transplantation. In the current review, we provide an overview of PSC-CMs for clinical transplantation. RECENT FINDINGS: Studies have shown that PSC-CMs can survive, engraft, and form gap junctions after transplantation, with functional benefit. Engrafted PSC-CMs matured gradually in host hearts. Only in a large animal model, transient ventricular arrhythmias were detected, mainly because of the ectopic pacing from the grafted PSC-CMs. Although intense immunosuppression is unavoidable in xenotransplantation, immunosuppression remains necessary for MHC-matched allogenic non-human primate PSC-CMs transplantation. This review offers insights on how PSC-CMs contribute to functional benefit after transplantation to injured non-human primate hearts. We believe that PSC-CM transplantation represents a potentially novel treatment for ischemic heart diseases, provided that several technological and biological limitations can be overcome.

Therapeutic Potential of Pluripotent Stem Cells for Cardiac Repair after Myocardial Infarction.

Myocardial infarction occurs as a result of acute arteriosclerotic plaque rupture in the coronary artery, triggering strong inflammatory responses. The necrotic cardiomyocytes are gradually replaced with noncontractile scar tissue that eventually manifests as heart failure. Pluripotent stem cells (PSCs) show great promise for widespread clinical applications, particularly for tissue regeneration, and are being actively studied around the world to help elucidate disease mechanisms and in the development of new drugs. Human induced PSCs also show potential for regeneration of the myocardial tissue in experiments with small animals and in in vitro studies. Although emerging evidence points to the effectiveness of these stem cell-derived cardiomyocytes in cardiac regeneration, several challenges remain before clinical application can become a reality. Here, we provide an overview of the present state of PSC-based heart regeneration and highlight the remaining hurdles, with a particular focus on graft survival, immunogenicity, posttransplant arrhythmia, maintained function, and tumor formation. Rapid progress in this field along with advances in biotechnology are expected to resolve these issues, which will require international collaboration and standardization.

Human Pluripotent Stem Cell-Derived Cardiac Tissue-like Constructs for Repairing the Infarcted Myocardium.

High-purity cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are promising for drug development and myocardial regeneration. However, most hiPSC-derived CMs morphologically and functionally resemble immature rather than adult CMs, which could hamper their application. Here, we obtained high-quality cardiac tissue-like constructs (CTLCs) by cultivating hiPSC-CMs on low-thickness aligned nanofibers made of biodegradable poly(D,L-lactic-co-glycolic acid) polymer. We show that multilayered and elongated CMs could be organized at high density along aligned nanofibers in a simple one-step seeding process, resulting in upregulated cardiac biomarkers and enhanced cardiac functions. When used for drug assessment, CTLCs were much more robust than the 2D conventional control. We also demonstrated the potential of CTLCs for modeling engraftments in vitro and treating myocardial infarction in vivo. Thus, we established a handy framework for cardiac tissue engineering, which holds high potential for pharmaceutical and clinical applications.

Impact of extracellular matrix on engraftment and maturation of pluripotent stem cell-derived cardiomyocytes in a rat myocardial infarct model.

Pluripotent stem cell-derived cardiomyocytes show great promise in regenerating the heart after myocardial infarction; however, several uncertainties exist that must be addressed before clinical trials. One practical issue is graft survival following transplantation. Although a pro-survival cocktail with Matrigel has been shown to enhance graft survival, the use of Matrigel may not be clinically feasible. The purpose of this study was to test whether a hyaluronan-based hydrogel, HyStem, could be a substitute for Matrigel. Human induced pluripotent stem cell-derived cardiomyocytes diluted with HyStem alone, HyStem plus pro-survival factors, or a pro-survival cocktail with Matrigel (PSC/MG), were transplanted into a rat model of acute myocardial infarction. Histological analysis at 4 weeks post transplantation revealed that, among the three groups, recipients of PSC/MG showed the largest graft size. Additionally, the grafted cardiomyocytes in the recipients of PSC/MG had a more matured phenotype compared to those in the other two groups. These findings suggest that further studies will be required to enhance not only graft size, but also the maturation of grafted cardiomyocytes.