Review Stem Cell-Based Therapy for Ischemic Heart ... - SAGE Journals

Cell Transplantation, Vol. 22, pp. 663–675, 2013 Printed in the USA. All rights reserved. Copyright  2013 Cognizant Comm. Corp.

0963-6897/13 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368912X655109 E-ISSN 1555-3892 www.cognizantcommunication.com

Review Stem Cell-Based Therapy for Ischemic Heart Disease Lien-Cheng Hsiao,*† Carolyn Carr,* Kuan-Cheng Chang,† Shinn-Zong Lin,‡§¶ and Kieran Clarke* *Cardiac Metabolism Research Group, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK †Division of Cardiology, Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan, ROC ‡Center for Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan, ROC §Department of Neurosurgery, China Medical University Beigang Hospital, Yunlin, Taiwan, ROC ¶Graduate Institute of Immunology, China Medical University, Taichung, Taiwan, ROC

Despite great advances in therapy over the past decades, ischemic heart disease (IHD) remains the leading cause of death worldwide because the decrease in mortality after acute myocardial infarction (AMI) leads to a longer life span in patients with chronic postinfarct heart failure (HF). There are no existing medical treatments that can cure chronic HF and the only currently available therapeutic option for end-stage HF is heart transplantation. However, transplantation is limited by the shortage of donor organs and patients require lifelong immunosuppression. In the past 10 years, stem cell-based cardiac therapy has been proposed as a promising approach for the treatment of IHD. There is a variety of potential stem cell types for cardiac repair and regeneration, including bone marrow cells (BMCs), resident cardiac stem cells (CSCs) and induced pluripotent stem cells (iPSCs). Stem cell-based therapy may comprise cell transplantation or cardiac tissue engineering (CTE), which might be an attractive alternative to solve the problems of low retention and poor survival of transplanted cells. This review focuses on the characteristics of stem cells from various sources and discusses the strategies of stem cell-based therapy for the treatment of IHD. Key words: Ischemic heart disease (IHD); Heart failure (HF); Stem cell; Cell transplantation; Cardiac tissue engineering (CTE)

INTRODUCTION Despite remarkable advances in pharmacological, interventional, and surgical therapies over the last few decades, cardiovascular disease (CVD) remains the leading cause of morbidity and mortality in many parts of the world (88). The main contributor to CVD is ischemic heart disease (IHD), which usually denotes a continuum of illnesses ranging from coronary atherosclerosis, myocardial infarction (MI), postinfarct heart failure (HF), and ultimately to end-stage HF (108). In terms of pathophysiology, early IHD (coronary atherosclerosis) is characterized by endothelial dysfunction and plaque formation, which is usually clinically asymptomatic. If the disease progresses without adequate treatment, however, it may eventually lead to acute myocardial infarction (AMI). AMI, also known as a heart attack, is most commonly caused by coronary occlusion resulting from a thrombus following the rupture

of an unstable atherosclerotic plaque (74). If the blocked coronary artery is not opened rapidly, cardiomyocytes (heart muscle cells) begin to die within the ischemic region of the myocardium, and eventually, fibrous noncontractile scar tissue is formed, causing progressive contractile dysfunction (43). Epidemiological statistics show that within 5 years following AMI, up to one-third of the surviving patients develop HF, defined as a state in which cardiac output is not adequate to support the body’s metabolic requirements (115,117). The inadequacy of cardiac function leads to the activation of compensatory mechanisms to maintain cardiac output. Over time, as the heart decompensates, patients inevitably develop end-stage HF, which in turn results in multiple organ failure from poor perfusion and ultimately death (43). Clinically, postinfarct HF accounts for the majority of hospitalization and deaths related to heart diseases, with patients with postinfarct HF having an estimated median

Online prepub date: October 4, 2012 Address correspondence to Carolyn Carr, D.Phil., Department of Physiology, Anatomy and Genetics, Sherrington Building, University of Oxford, Parks Road, Oxford OX1 3PT, UK. Tel: +44-01865 282247; Fax: +44-01865 282272; E-mail: [email protected] or Lien-Cheng Hsiao, M.D., Division of Cardiology, Department of Medicine, China Medical University Hospital, 2 Yude Road, Taichung, 40447, Taiwan, ROC. Tel: 886-4-22052121; Fax: 886-4-22053425; E-mail: [email protected]

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survival of ~4 years (38), as therapeutic options available for HF patients are limited. Medical treatment provided to date can decelerate, but not prevent, the process of deterioration to end-stage HF (43). Left ventricular assist devices (LVADs) can bridge the gap to receipt of a donor heart for transplant, via unloading the decompensated heart, but heart transplantation is the only therapeutic procedure available in the terminal stage of HF. However, the shortage of donor organs limits its application. Furthermore, patients who undergo transplantation are at high risk due to the complex surgery, and thereafter, they are required to take lifelong immunosuppressive medication. Thus, there is an urgent need for the development of a novel approach to treatment of IHD, including postinfarct and terminal HF. In the last decade, stem cell-based cardiac therapy has emerged as a promising strategy that aims to replace noncontractile fibrous scar tissue after MI and modulate remodeling. Thus, it is hoped that cell-based therapy could prevent subsequent HF following MI and repair and regenerate damaged myocardium in patients with postinfarct HF and even end-stage HF. Many preclinical studies have demonstrated that implanted stem cells can augment blood flow via neovascularization, modify scar tissue formation, and eventually improve cardiac function (27,74). So far, a number of clinical trials have been conducted using skeletal myoblasts, bone marrow mononuclear cells (BMMNCs), or mesenchymal stem cells (MSCs); however, results from these studies were mixed. Recently, resident cardiac stem cells (CSCs) and induced pluripotent stem cells (iPSCs) have drawn much attention due to their autologous origin and cardiomyogenic potential. Here, we review multiple stem cell sources that have been considered as candidates for the treatment of IHD. Additionally, stem cell-based strategies, consisting of cell transplantation and cardiac tissue engineering, will be addressed to compare their features and limitations. TYPES OF STEM CELL FOR CARDIAC THERAPY By definition, stem cells are undifferentiated cells that are clonogenic, self-renewing, and pluripotent or multipotent, capable of giving rise to all or multiple cell types in the body (27). Typically, stem cells can be divided into three broad categories: embryonic stem cells, derived from the inner cell mass of blastocysts; induced pluripotent stem cells, derived from adult differentiated cells through reprogramming by the introduction of pluripotent transcription factors; and adult stem cells, such as skeletal myoblasts, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, and cardiac stem cells present in adult tissues. Since 2000, a number of different cell types have been investigated to treat patients with AMI or chronic ischemic cardiomyopathy in clinical trials, including

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skeletal myoblasts, bone marrow mononuclear cells, circulating progenitor cells, and mesenchymal stem cells (27,36,74,94). However, these cell sources have presented mixed results, and no cell type has been proved to be the best candidate for cardiac repair and regeneration in clinical trials conducted to date (36,94). On the other hand, it is important to note that autologous CSCs, including c-kit+ cells and cardiosphere-derived cells, are already being investigated in phase I clinical trials. Encouragingly, the results of two clinical trials were recently released in Lancet, reporting the safety and efficacy of the use of resident CSCs in patients with ischemic heart disease (9,58). With better understanding of endogenous CSCs and advances in technology for generating iPSCs, it is hoped that CSCs and iPSCs could play crucial roles in the future of cardiovascular regenerative medicine. Each of these stem cells has its own advantages and disadvantages, which are briefly summarized in Table 1 below. Certainly, time will decide which stem cell type is the optimal choice in the end. Embryonic Stem Cells Embryonic stem cells (ESCs), the prototypical stem cell, can develop into all cell types in the body, including pancreatic b-cells, neural cells, and cardiomyocytes (11,103). The isolation of mouse embryonic stem cells was first reported in 1981 (31). Mouse ESCs (mESCs) were originally used to investigate embryonic development and establish genetically modified mice (129). Over time, researchers expanded their interest to the field of the regenerative therapy involving a stem cell-based approach (129). In 1998, the first human ESCs (hESCs) were isolated by Thomson et al. (110) and have subsequently been attracting significant interest as a potential cell source for regenerative medicine due to their pluripotent capability and proliferation potential. A number of studies showed that mESC- and hESCderived cardiomyocytes can survive and improve heart function when injected into infarcted myocardium in murine models (46,48,71,123). However, there are several undesirable limitations with the practical application of hESCs, such as ethical problems, teratoma formation, and immunological rejection, which have hampered the initiation of clinical trials in patients with cardiovascular disease (10,92). It is clear that a better understanding of molecular and genetic pathways for ESC differentiation and cardiac development could prevent contamination with undifferentiated ESCs, preventing teratoma formation when transplanted into the body (54,94). Alternatively, to overcome the ethical issues and immune rejection, induced pluripotent stem cells might present a more attractive alternate, as they are of autologous origin (103).

OVERVIEW IN CARDIAC STEM CELL THERAPY

Table 1.

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Advantages and Disadvantages of Various Stem Cell Types for Cardiac Repair

Cell Type

ESC

SM

BMC

CSC

Proliferative capacity Cardiomyogenic differentiation Arrhythmia Ethical problem Immune response Tumor formation Results in clinical trial

Yes Yes ND Yes Yes Yes No

Yes No Yes No No No Mixed

Yes Controversial No No No No* Mixed

Yes Yes No No No No Positive§

iPSC Yes Yes ND No No ND No

ESC, embryonic stem cell; SM, skeletal myoblast; BMC, bone marrow-derived cell; CSC, resident cardiac stem cell; iPSC, induced pluripotent stem cell; ND, not determined. *It has been reported that bone formation and substantial intramyocardial calcification in infarcted hearts occurred after delivery of bone marrow cells in animal models (12,125). §So far, there are two phase I clinical trials reporting that resident cardiac stem cells (c-kit+ cells or cardiosphere-derived cells) exerted beneficial effects when transplanted postinfarction in ischemic heart disease patients (9,58).

Induced Pluripotent Stem Cells Recently, induced pluripotent stem cells (iPSCs) have been generated using a novel technology, which involves the introduction of transcription factors related to pluripotency into adult terminally differentiated cells, such as dermal fibroblasts, causing them to revert to an embryonic stem cell-like stage (116). Takahashi et al. established that overexpression of four transcription factors [sex determining region Y box 2 (Sox2), octamer binding transcription factor 4 (Oct4), myelocytomatosis viral oncogene homolog (c-Myc), and Krüppel-like factor 4 (Klf4)] could convert mouse skin fibroblasts into pluripotent stem cells (104). Furthermore, differentiation of iPSCs into functional murine cardiomyocytes was demonstrated (66,84). In 2007, Yu et al. successfully reprogrammed human somatic cells to iPSCs using four genes including Sox2, Oct4, Nanog, and Lin28 (128), and these human iPSCs have been shown to have the potential to differentiate into functional cardiomyocytes (77,130). Importantly, despite subtle epigenetic differences associated with reprogramming, iPSCs strongly resemble ESCs in terms of morphology, differentiation capacity, gene expression profile, and teratoma formation (18). The use of iPSCs avoids the ethical dilemmas and immunological problems of ESCs, as they are derived from an autologous source; however, there remain concerns for clinical application because their generation employs viruses and oncogenes (54). Thus, for safety reasons, the development of approaches using nonvector pluripotent induction (45,81,127) without the need for oncogenes (76) might pave the way for clinical applications in the future (33,103,127). Skeletal Myoblasts Skeletal myoblasts or satellite cells, giving rise to skeletal muscle, were extensively studied in animal models

of myocardial infarction before entering the clinical arena, due to the advantages of their myogenic commitment, high expansion capacity in culture, good resistance to ischemia, and autologous origin (28,54,74). Reports from experimental studies have shown that implanted myoblasts resulted in ventricular wall thickening and increased contractility, thereby improving function in the infarcted myocardium (34,41,105,107). Based on the positive results, skeletal myoblasts were the first cell type to be examined in a human trial for cardiac repair (68). After the beneficial effects were shown in several pilot studies, further randomized controlled trials (RCT) using autologous skeletal myoblasts were conducted including MAGIC (Myoblast Autologous Grafting in Ischemic Cardiomyopathy), MARVEL-1 (To Assess Safety and Efficacy of Myoblast Implantation Into Myocardium Post Myocardial Infarction) and SEISMIC [Safety and Effects of Implanted (Autologous) Skeletal Myoblasts (MyoCell) Using an Injection Catheter]. The MAGIC study by Menasche et al. was terminated early because of a failure to reveal clinical efficacy and more events of arrhythmias in cell-treated patients undergoing coronary artery bypass grafting (CABG) for ischemic cardiomyopathy (67). In contrast, the MARVEL-1 trial by Lainscak et al. showed a mean increase of 90.9 m during a 6-min walk test 6 months after myoblast transplantation via catheter in patients with congestive heart failure, compared with a mean decrease of 3.7 m in the placebo group (49). Recently, the SEISMIC trial by Duckers et al. reported that injection of autologous skeletal myoblasts in patients with HF is safe and relieves symptoms based on a trend toward improved exercise tolerance in the cell-treated group despite no significant effect in left ventricular ejection fraction (LVEF) (29). However, despite improving cardiac function when transplanted into ischemic myocardium, these cells were unable to transdifferentiate into cardiomyocytes and integrate electromechanically with the host myocardium, thereby

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increasing the risk of sustained ventricular tachycardia, a life-threatening arrhythmia (54,69). To suppress ventricular arrhythmia in patients receiving skeletal myoblast therapy, prophylactic cardioverter-defibrillator implantation and/or amiodarone may need to be used (10). Collectively, in light of no cardiomyocyte regeneration, failure to integrate with host myocardium, potential lethal arrhythmia, and mixed results, further research is required prior to future clinical applicability. Bone Marrow-Derived Stem Cells The bone marrow is a heterogeneous tissue, consisting of different subpopulations, including hematopoietic stem cells (HSCs) and endothelial progenitor cells (around 2–4%), very rare mesenchymal stem cells (MSCs) (0.001–0.01% of the nucleated cells), and large proportions of committed progenitor cells and their specifically differentiated progeny (2,27,74,86). Technically, bone marrow-derived cells are easily accessible either by bone marrow aspiration or by isolation from peripheral blood after mobilization with cytokines such as stem cell factor (SCF) and/or granulocyte colony-stimulating factor (G-CSF) (33). These cells have attracted great attention as candidates for cell-based therapy because of their autologous origin, safety, ease of isolation, and reduced immunogenicity (MSCs) (4,122). The use of bone marrow mononuclear cells (BMMNCs), endothelial progenitor cells (EPCs), purified progenitor cells (CD34+ or CD133+), and MSCs in experimental and clinical studies has provided informative data related to human CVDs. (14,122). However, as they are multipotent, it should be noted that bone marrow-derived cells could differentiate into a variety of cell types when transplanted, thereby carrying a potential risk of bone, cartilage, and adipose tissue formation in the heart (101). It has been reported that unselected bone marrow cells resulted in substantial intramyocardial calcification and MSCs caused bone formation after transplantation into infarcted hearts in animal models (12,125). HSCs, identified by the expression of cell surface antigens such as CD34, CD133, c-kit (CD117), and stem cell antigen-1 (Sca-1), are lineage negative (Lin–) (120,121). These cells can be obtained from the bone marrow, umbilical cord, and peripheral blood, giving rise to all blood cell types (120). HSCs have been extensively studied and used to treat a variety of hematological disorders in the clinic, such as anemia, leukemia and lymphoma (33). A study by Orlic and colleagues showed that Lin–/c-kit+ bone marrow cells injected into infarcted myocardium of mice were able to generate new cardiomyocytes (82). However, other studies have been unable to demonstrate cardiomyocyte transdifferentiation of HSCs and cardiac function improvement in animal models of myocardial infarction (5,75,79). Furthermore, over the past years, several clinical trials of human HSCs have

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shown insignificant or no benefits in terms of ejection fraction (94,120). MSCs, identified by the surface marker expression of CD90, CD105, and CD73, are precursors of nonhematopoietic tissues, such as bone marrow, muscle, cartilage, adipose tissue, and heart, and have the capacity to give rise to fibroblasts, osteoblasts, chondroblasts, and adipocytes in vitro (15,26,119). Experimental results suggested that MSCs were able to transdifferentiate into cardiomyocyte-like cells under special culture conditions and in normal or injured myocardium in animals (7,15,57,111). In addition, it was shown that MSCs injected into infarcted myocardium could increase regional blood vessel density, prevent scar expansion, promote regional wall motion, and prevent ventricular remodeling (95,112). Interestingly, a recent report by Hatzistergos et al. demonstrated that allogeneic bone marrow MSCs stimulated the proliferation and differentiation of c-kit+ CSCs when injected into the swine model of MI (37). Clinical trials conducted in the past years have demonstrated safety and feasibility and displayed improvement in left ventricular function (17,118); however, these benefits are inconsistent (1). Currently, other clinical trials of MSCs in patients with IHD are still under way. For example, PROMETHEUS (Prospective Randomized Assessment of Mesenchymal Stem Cell Therapy in Patients Undergoing Surgery) is evaluating the safety and effectiveness of injecting MSCs into the heart in postinfarct patients who are undergoing CABG (http://www.clinicaltrials.gov/ct2/ show/NCT00587990). The POSEIDON-Pilot Study (The Percutaneous Stem Cell Injection Delivery Effects on Neomyogenesis Pilot Study) aims to study and compare the safety and therapeutic efficacy of allogeneic MSCs with autologous MSCs in patients with chronic ischemic cardiomyopathy (http://www.clinicaltrials.gov/ct2/show/ NCT01087996). So far, it remains controversial whether MSCs have the potential to truly transdifferentiate into cardiomyocytes (36,85,89). BMMNCs (CD34+ and CD133+) are the most common bone marrow cell type used in clinical trials for patients with AMI and ischemic cardiomyopathy over the last decade (101). In the early cohort and randomized pilot studies, BMMNCs were reported to result in a modest increase, of between 1% and 5%, in left ventricular ejection fraction (EF) at short-term follow up (3 to 6 months) (14,94). However, some clinical trials have not demonstrated the consistent results at long-term follow up (longer than 12 months) (20). For example, the REPAIR-AMI (Reinfusion of Enriched Progenitor Cells and Infarct Remodeling in Acute Myocardial Infarction), a RCT including 204 patients, showed that intracoronary delivery of BMMNCs following AMI increased EF by 2.5% (p = 0.01) compared with the control group at 4 months follow-up. The significant effect on EF was lost at 12 months follow-up;


however, decreased mortality was observed in the treatment group (93). Importantly, there are two recent clinical trials: BALANCE for AMI (Clinical Benefit and Long-Term Outcome After Intracoronary Autologous Bone Marrow Cell Transplantation in Patients With Acute Myocardial Infarction) and STAR-heart for ischemic cardiomyopathy (the acute and long-term effects of intracoronary stem cell transplantation in 191 patients with chronic heart failure) using BMMNCs, both showing a significant improvement in ejection fraction and survival rate in the treated group receiving BMMNC transplantation at 5 years follow-up, suggesting that BMMNC therapy results in significant and long-standing improvements in LV function and mortality in patients with AMI and chronic IHD (102,126). There are clinical trials using BMMNCs still in progress, such as TIME, REGENERATE-AMI, and REGENERATE-IHD. The TIME study aims to assess the safety and effect of timing of administration of BMMNCs in patients with AMI (http://www.clinicaltrials.gov/ct2/show/NCT00684021) (113). The REGENERATE-AMI study was designed to investigate the efficacy of early BMMNC transplantation within 6 h after successful primary angioplasty in AMI patients (http://www.clinicaltrials.gov/ct2/show/ NCT00765453). The REGENERATE-IHD is comparing the efficacy of BMMNC treatments using three different delivering methods, including indirect approach by mobilization using G-CSF, intracoronary infusion, and intramyocardial injection, in patients with ischemic HF (http:// www.clinicaltrials.gov/ct2/show/NCT00747708). It is hoped that more clinical studies will provide further insights into the therapeutic efficacy and help solve issues regarding bone marrow-derived cell transplantation in patients with IHD, including optimal cell type, cell dosing, and timing and route of delivery. Resident Cardiac Stem Cells The heart has traditionally been regarded as a terminally differentiated organ without the ability to regenerate itself. Recently, this dogma has been challenged by the discovery of a subpopulation of Lin– and c-kit+ cardiac stem cells (CSCs) resident in the rat heart, reported by Anversa and colleagues in 2003 (8). Furthermore, Anversa et al. developed methods for isolation and expansion of c-kit+ human CSCs (hCSCs) from small myocardial specimens. When injected into immunocompromised rats and mice, these cells differentiated into cardiomyocytes and improved the LV performance of infarcted hearts (6). Other groups have also identified likely CSC populations using different cell surface markers such as Sca-1 (80) and ATP-binding cassette transporter Abcg2 (62) or by the transcription factor insulin gene enhancer protein (Isl-1) (50). Furthermore, Messina et al. described a method to culture CSCs (grown as multicellular clusters, termed cardiospheres) to generate a mixed population

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expressing c-kit, Sca-1, and fetal liver kinase-1 (Flk-1) (70). In addition, another source of endogenous resident cardiac progenitor cells with regenerative potential for the adult heart is the epicardium, with several groups reporting the discovery of epicardium-derived myocardial and vascular progenitors in embryonic mouse and adult human heart (56,97,98,114,132). Importantly, these CSC populations have been found to have the potential to differentiate into cardiomyocytes and, in some cases, also into smooth muscle and endothelial cells (63). Marban et al. modified the protocol described by Messina’s group to substantially expand cardiospherederived cells (CDCs) in vitro and showed myocardial regeneration and functional improvement when these cells were injected into the infarcted mouse heart (99). In contrast to other populations of CSCs, cardiospheres and CDCs have been reported to contain a mixed population consisting of c-kit+ cardiac progenitor cells and cells expressing CD90 (mesenchymal-related) and CD31/CD34 (endothelial progenitor-related) markers (24,61,70,99). It is possible that the cardiac progenitor cells could readily engraft, differentiate, and function when transplanted into the injured myocardium in the presence of cardiac mesenchymal stem cells and endothelial progenitor cells via synergistic paracrine effects (16,61). Taken together, it is plausible that CSCs provide a desirable candidate cell for cardiac therapy in the clinical setting due to their endogenous origin and potential to develop into the three main cardiac lineages (23). To date, human trials of endogenous CSCs in patients with ischemic heart disease include SCIPIO (Cardiac Stem Cell Infusion in Patients with Ischemic Cardiomyopathy) and CADUCEUS (Cardiosphere-Derived Autologous Stem Cell to Reverse Ventricular Dysfunction). The SCIPIO trial by Bolli et al. was designed to examine the safety and efficacy of intracoronary delivery of autologous CSCs, which are expanded c-kit-expressing cells from right atrial appendages, in patients with ischemic cardiomyopathy. The initial results, published in the November 2011 issue of Lancet, are encouraging, confirming the safety and feasibility, and providing the evidence which shows that intracoronary infusion of autologous c-kit+ CSCs leads to a significant improvement in LV systolic function and a substantial reduction in scar size at 1 year of follow-up (9). Similarly, the CADUCEUS trial, led by Marban et al., aimed to investigate the effects of autologous CDC transplantation via the intracoronary route in patients with a recent MI and ischemic left ventricular dysfunction. The results, published in Lancet early in 2012, were that intracoronary infusion of autologous CDC contributed to significant increases in viable myocardium, regional contractility, and regional systolic wall thickening despite no significant change in LVEF, which might be explained by the

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fact that EF at baseline was only moderately impaired (39%), leaving little room for improvement by 6 months (58). Because of the positive findings, further research with longer follow-up and larger, phase II studies are required to confirm the true and persistent clinical benefits of c-kit+ CSCs and CDCs. STEM CELL-BASED CARDIAC THERAPY Conceptually, stem cell-based therapy aims to regenerate new myocardium, restore blood flow, and improve contractility by delivering stem or progenitor cells to the injured region of the heart (121). In general, there are two strategies for the treatment of IHD using a cell-based approach: cellular cardiomyoplasty (cell transplantation) and cardiac tissue or organ engineering. Cellular Cardiomyoplasty Over recent years, the repair and regeneration of damaged myocardium in patients with AMI and postinfarct HF has been investigated with cellular cardiomyoplasty involving the implantation of cells via either direct intramyocardial (IM) (transepicardial or transendocardial) injection or coronary infusion by intracoronary (IC), retrograde coronary sinus (RCS), or systemic intravenous (IV) administration (33,39,119). Each of these cell delivery methods aimed to transfer sufficient numbers of cells to the target site in the infarcted heart and to maximize cell retention, thereby achieving robust therapeutic effects (119). However, regardless of the cell type and the route of cell delivery, low retention and engraftment occurred, and this remains one of the key issues limiting the therapeutic outcome after cell transplantation (109). It has been reported that, within the myocardium, less than 10% of the cells were retained 1 h after cell administration by either IC or RCS (IM: 11 ± 3%; IC: 2.6 ± 0.3%; RCS: 3.2 ± 1%) (39). Acute loss of delivered cells from the heart occurs because they are often washed out via coronary blood flow or mechanically ejected at the injection site shortly following transplantation (59). Terrovitis et al. reported that cell retention rates in beating hearts are significantly lower than in nonbeating hearts after intramyocardial cell delivery (109). Additionally, the postinfarct heart is a hostile milieu, which results in poor survival when cells are transplanted into the damaged myocardium because of localized hypoxia, acidosis, lack of nutrients, and accumulation of toxic waste (101). It was suggested that the survival rate of transplanted cells was estimated at between 0.1% and 10% following intramyocardial injection (124,131). A variety of pretreatments, including upregulation of AKT (60), overexpression of B-cell lymphoma 2 (Bcl-2) (55), statins (100), and endothelial nitric oxide synthase (eNOS)-enhancing substances (91), and hypoxic preconditioning (40), have been shown to improve the survival of transplanted cells through activating

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antiapoptotic and/or nitric oxide (NO)-related signaling pathways (87,101). Theoretically, cellular cardiomyoplasty, especially via intracoronary administration for the treatment of heart disease, is an exceptionally attractive treatment as it is minimally invasive and widely available worldwide (44). Additionally, in the past decade, this method has been used in a large number of clinical trials, which have demonstrated safety, feasibility, and efficacy in human beings. However, in addition to the limitations of low retention and poor survival, there are still many questions and issues that need to be addressed, such as patient selection, ideal cell type, sufficient cell dosage, route of cell delivery, and optimal timing for cell administration (43,101). Importantly, the underlying mechanism by which stem or progenitor cells could improve cardiac function is not fully ascertained. However, it has been widely suggested that the major mechanism for the beneficial effects of transplanted cells, especially bone marrow-derived cells, may be largely due to paracrine effects rather than to the generation of new myocardium (116). In other words, the observed beneficial effects resulted from the secretion of growth factors, cytokines, and other local signaling molecules, which lead to angiogenesis, anti-remodeling, anti-inflammation, antiapoptosis, and possibly to activation of resident CSCs (37) and proliferation of residual cardiomyocytes, all eventually improving cardiac function (33,74,94). Cardiac Tissue Engineering Various approaches have been proposed to fabricate engineered tissue constructs for the treatment of CVDs through the use of different biomaterials and assembling technologies. These approaches can be broadly categorized into two forms: in vitro tissue engineering, such as biomaterial-based constructs (porous scaffolds or extracellular matrix-based hydrogels), scaffold-less systems (cell sheets), and decellularized matrix and biological patches, generally designed to replace a fibrous scar tissue after attachment to an injured part of the myocardium, and in situ tissue engineering (injectable alginate or selfassembling peptide nanofibers) (43,64,116). In Vitro Tissue Engineering In vitro tissue engineering can produce artificial tissue constructs, typically known as scaffolds, which involve the combination of cells and biomaterials. A scaffold can act as a vehicle providing cells with a suitable environment to populate and work before implantation to the injured area and integration into the host tissue. Cardiac tissue engineering has been considered as a potential alternative to repair and regenerate the diseased heart, especially for postinfarct HF (43). In contrast to cell transplantation, even though the application of bioengineered cardiac tissue for reconstruction of injured myocardium involves


surgical intervention, it could also be applied to repair of congenital and acquired heart defects and even radical regeneration of end-stage failing heart in the future (121). A variety of biodegradable and biocompatible materials have been employed to develop three-dimensional biomaterial constructs. Natural and synthetic polymeric biomaterials, and combinations thereof, have been suggested for use in cardiac tissue engineering, including collagen, alginate, gelatin, polylactic acid (PLA), and polyglycolic acid (PGA). An ideal biomaterial is required to enhance cell attachment, proliferation, and differentiation, and therefore may solve the problems of low engraftment and survival following cell implantation (42,64). Zimmermann and colleagues developed an engineered heart tissue (EHT), a heart muscle model system, by combining neonatal cardiomyocytes and collagen type I under mechanical strain (30). Furthermore, the EHT was reported to improve the heart function and increase systolic wall thickness of infarction after implantation (133). Leor and colleagues prepared 3D porous alginate scaffolds seeded with foetal rat cardiac cells using a freeze-drying technique. After the scaffolds were implanted onto the injured myocardium as a pericardial patch in a rat model of MI, it was reported that the biografts enhanced neovascularization, attenuated left ventricular dilation, and improved heart function (51). However, although they are biocompatible and biodegradable, inflammation and immunogenicity may occur in the process of scaffold degradation in the body (3). The production of decellularized patches involves the removal of cellular materials from tissue using detergent, which gets rid of any potentially immunogenic components but retains the underlying extracellular matrix (ECM). Various decellularized cardiovascular tissues, such as pericardium, vascular wall, and valve leaflets, have been engineered using immersion decellularization (21,35,90). An acellular ECM, prepared from porcine small intestine submucosa, was seeded with rabbit MSCs and implanted onto the infarcted region of the rabbit heart. In addition to an improvement in heart function, differentiation of MSCs into cardiac and vascular cells was observed in the injured region (106). In 2002, cell sheet engineering was first proposed by Shimizu and colleagues (96). Unlike other in vitro engineering techniques, the cell sheet engineering is a scaffoldless system. Briefly, this technology involves the use of novel cell culture surfaces, coated with a specific polymer, called poly(N-isopropylacrylamide), or PIPAAm, which is temperature-responsive. Importantly, the polymer is slightly hydrophobic and cell adhesive at 37oC, but becomes very hydrophilic and loses cell adhesion below 32oC, so cells can be harvested simply by reducing the temperature. Furthermore, without the necessity for trypsinization, the confluent monolayered

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cells with the underlying ECM are harvested together as a cell sheet, which can be directly attached to other cell sheets or naturally implanted onto tissue surfaces in the body (65,96). In 2006, Miyahara and colleagues reported that the monolayered MSC sheet reversed wall thinning in the scar area and improved heart function after transplantation onto the infarcted area of the rat heart, suggesting that transplantation of monolayered MSC sheet may be a potential therapeutic means for cardiac regeneration (72). One of the major challenges of in vitro engineering is to overcome the limited thickness of the construct, as the maximum oxygen diffusion is limited to approximately 200 µm (73,78). Channelled cardiac extracellular matrix scaffold, oxygen carriers, and stacked cell sheets have been employed to increase the construct thickness by improving oxygen supply (32,129). In Situ Tissue Engineering In contrast to in vitro approach, in situ tissue engineering, characterized by a scaffold-free approach, involves the direct injection of the biomaterial mixed with cells into the injured site (43). A variety of biomaterials have been employed for in situ engineering, including fibrin glue, collagen, Matrigel, alginate, and self-assembling peptides (19,22,25,47,52). For example, Davis et al. employed self-assembling peptides, naturally forming nanofibers when injected with cells into the myocardium, to produce a microenvironment of scaffold in situ, which is advantageous for cell growth and vessel development (25). Leor et al. demonstrated that intracoronary injection of alginate hydrogel formed in situ could reverse ventricular remodeling in a swine model of AMI, which resulted from increased scar thickness and physical support by the injectable implant (53). Finally, it is probable that different therapeutic strategies should be adopted depending on the extent of damaged vasculature and myocardium in the ischemic heart. For example, cardiac organ engineering might be a better therapy for terminal HF than single cell transplantation or myocardial patches. In the regenerative medicine field, therefore, generating a replacement organ is the ultimate goal to treat the patient in the end-stage of disease. However, this is challenging, as the heart is a complex anisotropic helix in architecture composed of uniquely organized muscular bands (13,64). Myocardial patches hold promise to replace focal noncontractile fibrous scar tissue for postinfarct HF, but are not adequate for replacement of the whole organ in terminal HF. In 2008, Taylor and coworkers developed a perfusable rat heart matrix with preserved vascular network, competent valves, and intact chamber geometry using a perfusion decellularization method. After reseeding with neonatal rat cardiomyocytes and endothelial cells, the recellularized heart construct generated pumping work

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consistent with about 2% of adult and 25% of fetal heart function under electrical and mechanical stimulation (83). The next steps are to repopulate the decellularized matrix with autologous cells from either stem or differentiated cells and optimize construct growth at different stage of maturity (116). CONCLUSIONS Cardiovascular disease, in particular IHD, represents the major medical health care burden in the world. To date, the treatment for postinfarct HF remains limited to heart transplantation. With the advance of stem cell research, cell-based therapy has been attracting increased attention because of the enormous potential for cardiac regenerative medicine. However, the data from a number of clinical trials using cell transplantation strategies showed encouraging, but marginal, effects partly due to inefficient engraftment and low survival rate of cells. There is still a long way to go before stem cell-based cardiac therapy can be routinely used in clinical service. Further basic research and well-designed clinical trials are needed to elucidate unanswered questions, such as the ideal cell type, cell dosing, and optimal timing for delivery. On the other hand, cardiac tissue engineering has made rapid progress and produced inspiring findings. By using different strategies, such as myocardial patches and whole organ decellularization-recellularization, stemcell based cardiac therapy could be expanded to treat nonischemic heart disease, such as congenital heart diseases and acquired heart defects. ACKNOWLEDGMENTS: This work was supported by the British Heart Foundation (grant number PG/07/059/23259). The authors declare no conflict of interest.

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