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

Abstract

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.

1. INTRODUCTION

Heart disease is one of the leading causes of death worldwide.¹⁾ Cardiomyocytes are terminally differentiated cells; however, their poor reusability has made it extremely difficult to restore injured cardiac function using currently available therapies. Approximately four billion cardiomyocytes are present in the heart of an adult human, and around one billion are lost during a typical myocardial infarction.²⁾ Currently, heart transplantation is the only effective treatment for severe heart failure, but a donor shortage remains a serious problem. During the progress of myocardial infarction, neutrophil infiltration occurs within 24 h after coronary artery occlusion, followed by recruitment of monocytes with a peak at 72 h.³⁾ The formation of granulation tissue takes place simultaneously with the removal of dead tissue, and progressively resulting in the development of scar tissue.⁴⁾ Regenerative therapies using somatic stem cells, including bone marrow-derived cells,⁵⁾ mesenchymal stem cells,^6–8) and cardiomyocyte stem cells,⁹⁾ have been performed to treat infarcted hearts. When bone marrow-derived mononuclear cells are transplanted, an angiogenesis effect is observed based on an increase in the secretion of inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor-alpha.¹⁰⁾ However, these somatic stem cells have poor ability to differentiate into cardiomyocytes and cannot remain for long term after transplantation.¹¹⁾ These cell therapeutic effects are thought to be due to humoral factors secreted by the transplanted cells to exert a so-called paracrine effect.¹²⁾ Another recently emerging strategy for myocardial regeneration treatment is through the use of pluripotent stem cells (PSCs). PSCs are an ideal tool for regeneration of the heart, which requires a large number of cardiomyocytes. They have the advantages of nearly endless proliferative capacity and the ability to differentiate into almost all cell types, including cardiomyocytes. Here, we describe the recent progress in myocardial regeneration therapy using PSCs and address future tasks to allow its clinical application.

2. ESTABLISHMENT OF PSCs

In 1981, Evans and Kaufman succeeded in isolating PSCs directly from delayed mouse embryos¹³⁾; the cells were originally named EK cells after the scientists’ names, but are now more commonly known as embryonic stem cells (ESCs). These ESCs are karyotypically normal, able to induce teratocarcinoma when injected into syngeneic mice, and able to form embryoid bodies (EBs) in culture. Within 1 month of the first report, Martin¹⁴⁾ described the isolation of mouse ESCs from an inner cell mass derived from mouse blastocysts. Following the success of establishing mouse ESC lines, Thomson et al.¹⁵⁾ reported in 1998 that human ESCs could be established from blastocysts. Human ESCs have since generated substantial interest as a good candidate source for tissue regeneration. However, the use of human ESCs is laden with ethical concerns, which has restricted ESC research and clinical applications.

In 2006, Takahashi and Yamanaka¹⁶⁾ introduced four factors (Oct3/4, Sox2, c-Myc, and Klf4) into murine fibroblasts via retroviral infection, which successfully directly reprogrammed differentiated adult somatic cells to the embryonic-like stage, and were designated as induced pluripotent stem cells (iPSCs). The following year, that group succeeded in generating iPSCs from human fibroblasts using the same four factors as applied to mouse iPSCs.¹⁷⁾ The emergence of iPSCs can avoid the most significant concerns related to the destruction of embryos required in ESC research since it only involves the genetic reprogramming of somatic cells. In addition, iPSCs have particular advantages related to immunological aspects that allow them to be used in autologous transplantations.

3. DIFFERENTIATION of PSCs INTO CARDIOMYOCYTES

Mouse ESCs can be transdifferentiated into cardiomyocytes by forming three-dimensional cell aggregates in suspension culture conditions. These EBs then start beating spontaneously.¹⁸⁾ However, this kind of cardiac differentiation of human ESCs is rare, typically yielding <1% cardiomyocytes. In 2001, Kehat et al.¹⁹⁾ collected EBs after suspension culture of 10 d, then were replated on gelatin-coated plates and cultured with Dulbecco’s modified Eagle’s medium containing 20% fetal bovine serum to generate human ESC-derived cardiomyocytes. Through this modification, the cardiac differentiation efficiency was slightly enhanced, and almost 8% of all EBs showed a contractile property. In 2003, the Mummery research group first demonstrated the induction of cardiomyocyte differentiation in human ESCs that did not undergo as spontaneous cardiogenesis by co-culturing with END-2 cells, visceral endoderm-like cells derived from the mouse.²⁰⁾ They subsequently provided a protocol based on serum-free co-culture with END-2 cells or by exposing human ESC-conditioned medium of END-2 cells to generate approximately 25% cardiomyocytes.²¹⁾ To enhance the purity of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), various protocols have been exploited that mimic the embryonic heart development environment. These protocols induce cardiomyocytes mainly by stimulating or suppressing three signal transductions, nodal/activin, bone morphogenic protein (BMP), and Wnt signaling, which are discussed in turn below.

3.1. Nodal/Activin

Nodal is a member of the transforming growth factor-beta (TGF-β) family of growth factors and is an essential factor for the formation of the gastrulation and germ layer.²²⁾ In the absence of Nodal expression, no mesoderm is formed. Another protein in the TGF-β superfamily, activin, also has strong mesoderm-inducing action. In 2007, Laflamme and colleagues demonstrated a method of myocardial differentiation using activin A as a substitute for Nodal.²³⁾

3.2. BMP

Similar to Nodal and activin, BMP is a peptide belonging to the TGF-β superfamily but acts as a ventralization signal at the gastrulation invagination stage instead of having an effect on early mesoderm formation.²⁴⁾ In vivo, BMP was shown to suppress cardiogenesis on the early embryo²⁵⁾; thus, BMP signaling suppresses cardiomyocyte differentiation in the early stage and promotes differentiation in the later stage.

3.3. Wnt Signaling

There are two Wnt signaling pathways: Canonical and noncanonical. Each pathway plays an important role in cardiogenesis. The canonical Wnt signaling pathway is essential for normal gastrulation and mesoderm formation in the early development of embryo.²⁶⁾ Like BMP, Wnt/β-catenin signaling was reported to play a time-specific biphasic role in myocardial differentiation.²⁷⁾ However, in contrast to BMP signaling, early canonical Wnt signaling stimulates mesoderm induction, while inhibiting cardiogenesis at later time points.^27–30)

4. TRANSPLANTATION OF PSC-DERIVED CARDIOMYOCYTES

Preclinical studies conducted to date using PSC-CMs for cardiac repair are summarized in Table 1. Multiple studies involved the transplantation of hPSC-CMs as suspensions of dispersed cells into an infarcted myocardium. The Gepstein group³¹⁾ transplanted undifferentiated ESCs, ESC-CMs, or noncardiac ESC derivatives into an immunosuppressed rat heart that they previously induced a myocardial infarction. Interestingly, only recipients of hESC-CMs showed attenuation of the left ventricular remodeling process measured by echocardiography. Laflamme et al.²³⁾ transplanted hESC-CMs with cell-surviving factors including Matrigel into athymic rats, and confirmed a substantially large graft myocardium. They also showed that transplantation of hESC-CMs, unlike noncardiac derivatives, improved cardiac contractile function after myocardial infarction by cardiac magnetic resonance imaging. In addition, we previously reported that transplanted hESC-CMs into guinea pigs, with a heart rate that is relatively more matched to that of the human heart compared to other rodents, could electrically couple to the host hearts based on observation of the fluorescent calcium indicator, GCaMP.³²⁾ Collectively, those studies demonstrated that after transplantation into small animal models, hESC-CMs improved cardiac function and they also described decreased incidence of posttransplant arrhythmia. In 2014, the Murry group³³⁾ transplanted 1 × 10⁹ hESC-CMs into nonhuman primate hearts that resulted in long-term survival with all of the grafts becoming electrically integrated with the host heart. Unlike previous studies that used small animals, they observed posttransplant ventricular arrhythmia. Subsequently, our group³⁴⁾ performed allogeneic transplantation of nonhuman primate iPSC-CMs, which improved the contractile function of the infarcted heart but also significantly increased the incidence of ventricular arrhythmia. These evidences thus far suggest that PSC-CMs may cause posttransplant arrhythmia when transplanted into large animal models with a good match in both heart size and beating rate of the host and donor.

Table 1. Preclinical Studies of Pluripotent Stem Cell-Derived Cardiomyocyte Transplantation

Year (reference)	Host animal	Cell type	Cell number (cells)	Time of Tx (post-MI)	Cell delivery method	Observation period (weeks)	Cardiac function	Post-Tx arrhythmia
200731)	Rat	hESC-CMs	1.5 × 106	7–10 d	Intramyocardial direct injection	4	Improved	—
200723)	Rat	hESC-CMs	1 × 107	4 d	Intramyocardial direct injection	4	Improved	—
201232)	Guinea pig	hESC-CMs	1 × 108	10 d	Intramyocardial direct injection	4	Improved	—
201237)	Pig	hiPSC-CMs	2.5 × 107	28 d	Patch	8	Improved	—
201438)	Rat	hiPSC-CMs/ECs/MCs	2 × 106	7 d	Patch	8	Improved	—
201446)	Guinea pig	hESC-CMs	8 × 107	28 d	Intramyocardial direct injection	4	No change	—
201433)	Monkey	hESC-CMs	1 × 109	14 d	Intramyocardial direct injection	12	No change	+
201634)	Monkey	Monkey iPSC-CMs	4 × 108	14 d	Intramyocardial direct injection	12	Improved	+
201639)	Guinea pig	hESC-CMs	5 × 106	7 d	Patch	4	Improved	—

MI, myocardial infarction; Tx, transplantation; hESC, human embryonic stem cell; CMs, cardiomyocytes; hiPSC, human induced pluripotent stem cell; ECs, endothelial cells; MCs, vascular mural cells.

Despite the success in the engraftment and electrical coupling of PSC-CMs with the host myocardium, some doubts remain about the fact that a simple injection of dispersed cells alone will form an organized, highly aligned cardiac tissue structure. PSC-CM grafts are typically comprised of small, irregularly contoured islands of immature cardiomyocytes. Accordingly, substantial efforts have been made to form a cardiac tissue-like structure with tissue engineering strategies. Okano et al.³⁵⁾ developed a temperature-responsive culture dish, making it possible to collect cells in sheet form with retained structure and function in a noninvasive manner. Moreover, Sekine et al.³⁶⁾ reported that cell sheet transplantation yielded greater cell survival than cell injection. Kawamura et al.³⁷⁾ transplanted a hiPSC-CMs sheet to a porcine model of ischemic cardiomyopathy and observed graft survival and mechanical benefits. Masumoto et al.³⁸⁾ created cardiac tissue sheets consisting of hiPSC-CMs, endothelial cells, and vascular mural cells, which were transplanted into a rat model of myocardial infarction and showed improvement of cardiac contractile function. Weinberger et al.³⁹⁾ created human-engineered heart tissue (hEHT) derived from iPSC-CMs/endothelial cells and human endothelial cell patches (hEETs). After transplanting hEHT, hEETs, and cell-free patches onto the left ventricular wall of cryo-injured guinea pig hearts, only the hEHT showed remuscularization of the scar tissue and electrical coupling to the host heart, resulting an improvement of cardiac contractile function. However, the Murry group reported that tissue-engineered cardiac patches did not couple electrically with the host heart.⁴⁰⁾ Thus, electrical integration of a cardiac tissue-like structure transplanted into the heart needs further investigation.

Another possible strategy to deliver PSC-CMs is intracoronary injection of a cell suspension. Many studies have reported the injection of somatic stem cells such as bone marrow cells (BMCs) into the coronary arteries, and clinical trials are underway. In 2002, Strauer et al.⁴¹⁾ reported an improvement of cardiac function in 10 patients with myocardial infarction after intracoronary injection of autologous mononuclear BMCs. In the September 2006 issue from the New England Journal of Medicine, three papers were published related to the intracoronary administration of autologous BMCs in patients suffering from myocardial infarction. Two of them demonstrated improvement of cardiac function,^42,43) while the other one did not show any effect.⁴⁴⁾ In addition, intracoronary injection of mesenchymal stem cells performed after myocardial infarction was reported to improve both cardiac function and coronary blood flow.⁴⁵⁾ But the effects of PSC-CMs transplanted by coronary injection on engraftment of an infarcted myocardium remain unexplored.

Accumulated evidences suggest that transplantation of PSC-CMs improves cardiac contractile function in an acute or subacute myocardial infarction model; however, limited studies have been conducted with a chronic myocardial infarction model. We did not find any mechanical benefits of hESC-CMs transplantion in a guinea pig model 4 weeks after cryo-injury of the heart compared to vehicle-treated animals.⁴⁶⁾ On the other hand, Kawamura et al.³⁷⁾ observed mechanical improvement in a porcine chronic myocardial infarction model. Therefore, further studies are required involving a chronic myocardial infarction model that reflects more accurately the patient population who are at the end-stage of heart failure.

5. CURRENT LIMITATIONS AND FUTURE TASKS

In 2015, the Menasche group reported the first clinical trial of the use of human ESCs in myocardial regeneration.⁴⁷⁾ They implanted 4 × 10⁶ myocardial progenitor cells generated from ESCs into a fibrin patch and transplanted the epicardium with cardiac bypass surgery. Prior to surgery, as the patient received an implantable cardioverter defibrillator, no arrhythmia occurred after transplantation and tumor formation was not observed during 3 months following the operation. Thus, PSCs show good promise in myocardial regeneration therapy. Nevertheless, several problems remain to be addressed before their practical application in routine medical care.

5.1. Poor Survival of Transplanted Cardiomyocytes

Poor survival of grafted cells is one of the major factors hindering the therapeutic effects of cell transplantation.⁴⁸⁾ Therefore, the key to a successful regeneration treatment is to promote the efficient engraftment of transplanted cells. Here, we describe the three main factors that could prevent the survival of transplanted cardiomyocytes as potential targets for improvement: Inflammation; anoikis; and ischemia.

5.1.1. Inflammation

Myocardial infarction causes an intense inflammatory response.⁴⁹⁾ In the acute phase, once the myocardial cells are destroyed, they undergo phagocytosis and proteolysis to remove debris from the injured tissue. The infarcted myocardium activates various signaling pathways as Toll-like receptor signaling, resulting in the generation of reactive oxygen species that induce cytokine and chemokine upregulation. In the chronic phase, fibrosis in the infarcted region progresses, forming an extremely difficult environment for the survival of transplanted cells. Suzuki et al.⁴⁸⁾ reported that suppression of IL-1β, an important proinflammatory cytokine, doubled the number of transplanted cells after 72 h. Similarly, Murtuza et al.⁵⁰⁾ demonstrated that inhibition of IL-1 increased the survival rate of the transplanted cells after 3 weeks by more than 6-fold, accompanied by a decrease in cardiac hypertrophy and improvement in the left ventricular ejection fraction in the infarcted murine myocardium. Thus, inflammation control is one of the keys to a successful graft survival.

5.1.2. Anoikis

One of the main reasons of the death of graft cells is apoptosis by activation of the Jun N-terminal kinase pathway⁵¹⁾ triggered by the loss of matrix attachments, which is known as anoikis.⁵²⁾ Several cell scaffold materials such as Matrigel,²³⁾ hyaluronan acid-based extracellular matrix,^53–55) and thermosensitive and biodegradable hydrogels⁵⁶⁾ have been tested for their ability to control anoikis. Laflamme et al.²³⁾ reported that a prosurvival cocktail including Matrigel with hESC-CMs could significantly suppress anoikis and enhance graft survival. Moreover, Uesugi and colleagues discovered new low molecular-weight compounds with an anoikis-inhibitory effect.^57,58)

5.1.3. Ischemia

Zhang et al.⁵⁹⁾ revealed that ischemia was one of the main contributors to cell death after transplantation. They transplanted cardiomyocytes into vascularized 2-week-old cardiac granulation tissue or into a normal myocardium. They observed respectively 53 and 86% of reduction of cell death, compared to the acute necrotic myocardium, by measuring the terminal deoxynucleotide transferase deoxyuridine triphosphate (dUTP)-nick-end labeling staining (a marker of apoptosis). Consistent with that finding, activation and/or upregulation of heme oxygenase-1 was shown to reduce the infarct size and protect grafted cardiomyocytes from oxidative stress.^60,61) Additionally, heat-shock treatment improved cell tolerance to oxidative stress.^62,63)

5.2. Immunogenicity

In most of the preclinical studies, human PSC-CMs were transplanted into genetically or drug-induced immunodeficient animal models^33,34); however, few studies have evaluated the immune response in a more clinically relevant model. Presumably, a PSC-CMs graft can survive without immune rejection via the same protocol used in whole-heart transplantation, but this approach would increase the risks of infection and malignancy.⁶⁴⁾ Accordingly, substantial research has been carried out to reduce the use of immunosuppressants and avoid these risks. One possible strategy is now emerging through the banking of diverse human leukocyte antigen (HLA)-typed PSCs,⁶⁵⁾ which would allow for the matching of HLA types in allogeneic transplantation. Our group showed that major histocompatibility complex-matched allogeneic transplantation of nonhuman primate iPSC-CMs resulted in a long-term graft survival with relatively mild immunosuppression.³⁴⁾ In addition, recent progress in genome-editing technology has provided HLA knock-out PSC lines, which could also presumably prevent an HLA-induced immune response. Thus it is theoretically possible to create autologous cell-based therapies with iPSC technology, although the estimated costs required to create, test, and store these human iPSCs are currently prohibitive for the widespread application of this technique.

5.3. Tumor Formation

Tumor formation is also a major concern of the application of PSCs in myocardial regeneration therapy, because cardiac regeneration requires a large number of cardiomyocytes differentiated from PSCs. If a cell population contains a certain proportion of undifferentiated cells, the risk of teratoma formation will increase along with the number of transplanted cells. Tumorigenesis after transplantation in an undifferentiated state has been reported for both ESCs⁶⁶⁾ and iPSCs.⁶⁷⁾ Although teratoma formation has not been reported in PSC-derived cardiomyocytes, it is necessary to establish a differentiation protocol capable of producing high-purity cardiomyocytes and to confirm their long-term safety after cell transplantation.

5.4. Arrhythmia

Since arrhythmic events are the leading cause of death in patients with heart failure,⁶⁸⁾ a careful attention should be paid to the possibility of posttransplant arrhythmia. There are several advantages^32,69) and disadvantages⁷⁰⁾ regarding posttransplant arrhythmia after transplantation of PSC-CMs in small animal models. As mentioned in the previous section, recent studies using large animal models have shown that transplantation of PSC-CMs innately induces ventricular arrhythmia. Although the underlying mechanisms remain unclear, it could be caused by reentry, automaticity, or triggered activity of PSC-CMs.⁷¹⁾ Thus, more studies are required to clarify these mechanisms to achieve control over posttransplant arrhythmia.

5.5. Immature Phenotype of PSC-Derived Cardiomyocytes

Human iPSC-derived cardiomyocytes have depolarized maximum diastolic potentials,⁷²⁾ lack t-tubules,⁷³⁾ exhibit mitochondrial dyfunction,⁷⁴⁾ and have weak expression of the cardiac maturation marker cTnI even when cultured for 9 months in vitro.⁷⁵⁾ In vivo, although transplanted cardiomyocytes have been reported to mature to a certain extent,⁷⁶⁾ the grafted cardiomyocytes have a weaker expression of cardiac muscle-specific proteins than the host myocardial tissue and show a clearly distinct tissue structure. Furthermore, we reported that the expression of gap junction proteins such as connexin 43 was relatively weak in the graft myocardium, and there was nonuniformity of electrical conduction in the grafted myocardium as the heart rate increased.^32,34) Several reports suggested that this electrical heterogeneity was the main cause of posttransplant ventricular arrhythmia.^34,76)

6. CONCLUSION

Research on cardiac repair using PSCs has progressed rapidly in recent years owing to the hard work of multiple worldwide investigators, and the first clinical trial of ESCs is now ongoing.⁷⁷⁾ However, substantial challenges remain to be resolved before clinical application. For example, new technology for the manufacturing of iPSCs and the differentiation/purification of cardiomyocytes is required at a lower cost. Moreover, this research is currently being conducted at multiple independent institutions involving various protocols; therefore, more collaboration to establish a standardized protocol will be required for use in clinical trials.

Conflict of Interest

S.O. is an employee of Myoridge, Inc. Y.S. has no conflict of interest.

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