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Eur J Cardiothorac Surg 2006;30:770-781
© 2006 Elsevier Science NL

Reviews

Cellular therapy and myocardial tissue engineering: the role of adult stem and progenitor cells

Kai Hong Wua, Ying Long Liua,*, Bin Zhoub, Zhong Chao Hanb

a Pediatric Cardiac Center, Department of Surgery, Cardiovascular Institute and Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 167 Beilishi Road, Beijing 100037, China
b State Key Laboratory of Experimental Hematology, National Research Center for Stem Cell Engineering and Technology, Institute of Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China

Received 11 February 2006; received in revised form 2 August 2006; accepted 7 August 2006.

* Corresponding author. Tel.: +86 10 88398188; fax: +86 10 68332747. (Email: pumcwu{at}yahoo.com.cn).


   Abstract
Top
 Abstract
1. Introduction
 2. Type of adult...
 3. Cellular therapy applications
 4. Tissue engineering...
 5. Conclusions and future...
 References
 
Acquired cardiovascular diseases and complex congenital heart diseases are leading causes of morbidity and mortality. Cellular therapy and tissue engineering are emerging as promising alternative approaches to treat cardiovascular diseases. Cellular therapy involves isolating cells and delivering the cells to the site of cardiac injury to restore blood flow and contractility to previously infarcted, scarred or dysfunctional heart. Myocardial tissue engineering, engineered heart tissue by seeding cells in three-dimensional matrices of biodegradable polymers or cell sheet engineering without artificial scaffolds to form new myocardial constructs. Questions are common to both these approaches, such as the best cell source and optimal conditions for therapeutic application. The capabilities of stem cells for pluripotency and long-term self-renewal make it an ideal source for myocardial tissue engineering and cell therapy. We review the current understanding of postnatal adult stem and progenitor cells in cellular therapy and myocardial tissue engineering from a surgical view point, and highlight the latest advances in these exciting fields.

Key Words: Tissue engineering • Stem cells • Cell therapy • Myocardial infarction • Congenital heart defects


   1. Introduction
Top
 Abstract
 1. Introduction
2. Type of adult...
 3. Cellular therapy applications
 4. Tissue engineering...
 5. Conclusions and future...
 References
 
Cardiovascular diseases like myocardial infarction, and subsequent heart failure are a leading cause of morbidity and mortality. Despite the enormous advances in the understanding and treatment of heart failure that have taken place during the past years, this condition remains a serious, and in fact, a growing problem in the United States and worldwide [1]. The human heart has a limited capacity for self-repair or regeneration after myocardial infarction, the irreversible loss of muscle, and accompanying contraction and fibrosis of myocardial scar can lead to progressive ventricular remodelling of nonischaemic myocardium, and the ventricular remodelling can result in progressive ventricular dilatation and heart failure. Heart transplantation has been a therapy for these severe cases for several decades, however, with an ever increasing shortage of donor organs for heart transplantation, its usage is much limited. There is a significant need for alternative therapies for cardiovascular diseases. Transplantation of stem cells to the injured heart can have a favourable impact on tissue regeneration and contractile performance of the infracted heart [2]. On the other hand, surgically significant congenital heart disease affects approximately 1% of live births in China and other parts of the world [3]. A very promising approach to repair congenital heart defects and large scar areas due to ischaemia may be the use of tissue engineering. In the past years, several excellent papers have addressed the issue of myocardial tissue engineering and shown the safety and feasibility of tissue-engineered myocardial grafts in paediatric cardiovascular surgery, in addition, surgical resection of nonviable myocardium after infarction and replacement with tissue-engineered cardiac grafts may improve cardiac function and prevent congestive heart failure [4–9].

In recent years, there has been a tremendous increase in the understanding of stem cell biology. There are two characteristics that distinguish stem cells from other types of cells. These are (1) stem cells can self-renew; and (2) they can differentiate into other types of cells. Stem cells are unspecialized cells that renew themselves for long periods through cell division, and given certain conditions, can be induced to become cells with special and unique functions [10]. Embryonic stem cells have been used experimentally in the context of myocardial tissue engineering and cellular therapy. However, unresolved ethical and legal issues, concerns about the tumourigenicity of the cells, and the need to use allogeneic cells for transplantation currently hamper their use in clinical studies and will not be reviewed here [11]. Human adult progenitor/stem cells, with the ability to proliferate and self-renew in vitro and the capacity to differentiate into specialized cell types, are an important potential cell source. This paper will review the current understanding of postnatal adult stem and progenitor cells in myocardial tissue engineering and cellular therapy from a surgical view point, and highlight the latest advances in these exciting fields.


   2. Type of adult stem/progenitor cells
Top
 Abstract
 1. Introduction
 2. Type of adult...
3. Cellular therapy applications
 4. Tissue engineering...
 5. Conclusions and future...
 References
 
Because of the unresolved ethical and legal issues of embryonic stem cells, the enthusiasm about adult stem cells in myocardial tissue engineering has been spurred by recent publications that demonstrated the versatility of these cells [12]. Postnatal adult stem cells have been isolated from a range of sources including bone marrow, peripheral blood, muscle, adipose, umbilical cord and local heart tissue (Table 1 ). Although stem cell populations isolated from the bone marrow are usually a heterogeneous mix of different subpopulations, cloned stem cell lines from any source also show a broad spectrum of differentiation potential. Their lack of specific tissue-related characteristics and ability to proliferate and differentiate have conjured immense interest in myocardial tissue engineering [13].


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  		Table 1. Sources of adult stem/progenitor cells for myocardial tissue engineering
 
2.1 Haemopoietic stem cells
Haematopoietic stem cells (HSCs) are routinely isolated from the bone marrow, umbilical cord and the peripheral blood based on the expression of the CD34 and/or CD133 antigens. HSCs have been primary targets for tissue engineering and cellular therapy owing to their capacity for differentiation and self-renewal, whereby multiple cell lineages can potentially be obtained for the lifetime of an individual. However, a true consensus about the characterisation and definition of HSCs has not been reached. It has been demonstrated that HSCs can significantly regenerate the infarcted myocardium. Orlic and colleagues [14] injected HSCs into the myocardium of rats, which were able to repair 60–70% of the ischaemia damaged tissue, by generating smooth muscle, endothelial and cardiomyocytic cells, but cardiomyocyte transdifferentiation is still controversial [15–17]. Recently, Kajstura and colleagues [18] provided further evidence that c-kit positive bone marrow cells, when properly administrated in the infarcted heart, efficiently differentiate into myocytes and coronary vessels with no detectable differentiation into haemopoietic lineages. However, even if an appropriate milieu-induced transdifferentiation phenomenon occurs in border zones it still remains to define the most appropriate cell population and to subsequently develop clinically usable expansion methods because the percentage of multipotent stem cells is extremely small in bone marrow and peripheral blood.

2.2 Endothelial progenitor cells
The discovery that endothelial progenitor cells (EPCs) are present in circulating adult human peripheral blood readily raises the possibility of EPCs-mediated therapeutic neovascularisation as a novel option for the treatment of ischaemic diseases [19]. EPCs are functionally and phenotypically distinct from mature endothelial cells (ECs), but have the potential to differentiate into mature ECs and contribute to the process of endothelium repair. Research over the past few years has established that EPCs exist in adult bone marrow, peripheral blood circulation and cord blood [20]. Primary studies indicated that impaired EPCs function is correlated with cardiovascular risk factors such as hypertension, atherosclerosis and diabetes mellitus [21]. Thus, EPCs injury might induce EC dysfunction, which further affects the progression of cardiovascular disease. Therefore, it is reasonable to believe that transplantation of EPCs might be a proper way to cure cardiovascular diseases due to injury and dysfunction of ECs [22].

There is growing evidence that EPCs from patients suffering from cardiovascular risk factors (hypertension, coronary artery disease) or healthy smokers have a reduced ability to proliferate and differentiate [23,24]. However, this impaired differentiation or proliferation ability is absent in EPCs isolated from the cord blood. Cord blood-derived EPCs show a higher proliferation capacity and express telomerase, a functional characteristic of stem cells that is very low or absent in other progenitor cell populations. These cells also demonstrate rapid self-renewal and low apoptosis, therefore, EPCs from umbilical cord blood are quite promising in future myocardial tissue engineering and cell therapies [25].

2.3 Mesenchymal stem cells
Mesenchymal stem cells (MSCs) have been isolated from a wide variety of tissues, including bone marrow, peripheral blood and adipose tissue. Bone marrow is an abundant and renewable tissue source for MSCs, they can be isolated and expanded with high efficiency, and its isolation is relatively safe and accessible. MSC clones can be expanded and reportedly have a low immunogenicity and high plasticity, interestingly, these cells can migrate to localized areas of injury following ischaemic insults [26–30]. In a canine chronic ischaemia model, Silva and colleagues [31] injected bone marrow-derived MSCs into the ischaemia myocardium and the MSCs differentiated into smooth muscle cells and endothelial cells, resulting in increased vascularity and improved cardiac function.

Recently, pluripotent stem cells termed multipotent adult progenitor cells (MAPCs), marrow-isolated adult multilineage inducible (MIAMI) cells and human BM-derived multipotent stem cells (hBMSCs) have been isolated from the bone marrow. These cells can be expanded in vitro without senescence [32–34]. They show clonal in vitro differentiation potential to cells of the three germ lineages. The relationship between these pluripotent stem cells is not clear, however, regardless of their origin, they are extremely valuable as a source of stem cells for cell therapy and tissue engineering applications.

2.4 Cardiac stem cells
Traditionally the heart has been viewed as a static organ incapable of repairing any form of damage. The discovery that the adult heart contains a pool of cardiac stem cells (CSCs) that can replenish the cardiomyocyte population and generate coronary vessels has dramatically changed the traditional view of the heart as a postmitotic organ [35]. Over the past few years, lineage negative c-kit positive cells, Sca-1 positive cells, cardiosphere-forming cells and Isl1 positive cells have been isolated from adult human and murine heart. In vitro, these cells grow as a monolayer when seeded in substrate-coated dishes or form spheroids when cultured in suspension. When injected into an ischaemic heart, these cells or their clonal progeny reconstitute a well-differentiated myocardial wall that encompasses up to 70% of the left ventricle [36–40]. Because of their natural role in regeneration of cardiac cells and the ability to proliferate and differentiate into cardiomyocytes, the endogenous CSCs appear to be the ideal cells for myocardial tissue engineering.

2.5 Muscle-derived stem cells
Muscle-derived stem cells also known as myoblasts or satellite cells are best defined as precursor rather than stem cells, which normally lie in a quiescent state under the basal membrane of skeletal muscle fibres. Following tissue injury, these cells are rapidly mobilized, proliferate and fuse, thereby regenerate the damaged fibres. They have a high proliferative potential under appropriate culture conditions allowing a substantial scale-up of the initial biopsy, and a high resistance to ischaemia which is expected to enhance cell survival following engraftment into poorly vascularized scars. Recent studies have shown that autologous skeletal myoblasts can differentiate into striated muscle cells within the damaged myocardium and that these cells can augment both diastolic and systolic myocardial performance after transplantation into the infarcted myocardium [41]. Pouly and colleagues’ [42] results demonstrated an additional proof that transplanted myoblasts could engraft into a nonischaemically diseased myocardium and contribute to improved left ventricular performance.

2.6 Adipose-derived stem cells
Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have suggested that stem cells are present within the adipose stromal compartment. Adipose-derived stem cells, like MSCs, have the ability to differentiate toward the osteogenic, adipogenic, myogenic, chondrogenic and neurogenic lineages [43,44]. The ease of access to fat and its abundance makes adipose tissue a potentially useful source of stem cells for clinical applications. Recently, Planat-Benard and colleagues demonstrated that beating cells with cardiomyocyte features could be identified after culture of adipose-derived stem cells. The cardiomyocyte phenotype was first identified by morphological observation, confirmed with expression of specific cardiac markers, immunocytochemistry staining and ultrastructural analysis, revealing the presence of ventricle- and atrial-like cells. Electrophysiological studies performed on early culture revealed a pacemaker activity of the cells [45]. Further, the same group indicated that adipose lineage cells can function as progenitors for endothelial cells. These cells participate in vascular-like structure formation in Matrigel plug and enhance the neovascularisation reaction in ischaemic tissue [46]. This opens new perspectives on angiogenic therapy based on the administration of adipose tissue-derived stem cells in the treatment of cardiovascular disease [47].

2.7 Umbilical cord-derived stem cells
The possibility that a decline in the numbers or plasticity of stem cell populations contributes to ageing and age-related disease is suggested by recent findings [48,49]. In addition, high degree of viral infection in patients and the ethical issues surrounding embryonic stem cells make it necessary to search for alternative sources of these cells for autologous and allogeneic use [50]. Recently, MSC-like cells were isolated from human umbilical cord tissues. These cells display a fibroblast-like morphology, express mesenchymal markers, under appropriate induction conditions, these cells can differentiate into osteogenic, adipogenic, cardiomyogenic and endothelial cells [50–53]. Umbilical cord-derived stem cells can be easily extracted and cryopreserved, allowing for individuals to store their own samples for possible future autologous use even if there were no immediate indication that stem cell therapy would be required. Some countries have now established private banks collecting umbilical cord/cord blood units for autologous transplantation. In the near future, cryopreserved autologous umbilical cord-derived stem cells for therapeutic medicine may become available. Recently, Kadner and colleagues [54] seeded umbilical cord-derived cells on bioabsorbable copolymer patches and grown in vitro in laminar flow for 14 days. These cells demonstrated excellent growth properties and tissue formation with mechanical properties approaching native tissue. It appears that umbilical cord-derived stem cells represent a promising alternative cell source for cardiovascular tissue engineering [55].


   3. Cellular therapy applications
Top
 Abstract
 1. Introduction
 2. Type of adult...
 3. Cellular therapy applications
4. Tissue engineering...
 5. Conclusions and future...
 References
 
Cellular therapy is the notion that if we can deliver stem or progenitor cells to the site of cardiac injury, we can restore blood flow and contractility to previously infarcted, scarred or dysfunctional heart. Cellular therapy may be attained in one of the following ways: (1) by transplanting cells through transepicardial, intracoronary or transendocardial delivery; (2) by mobilising resident stem cells to the site of injury with the use of cytokines, such as granulocyte colony-stimulating factor and stem cell factor; or (3) by administering local treatment with growth factors, such as insulin-like and hepatocyte growth factors, that induce the differentiation of cardiac progenitor cells into cardiomyocytes [56,57]. Here, we only discuss therapeutic effects of stem cells in myocardial regeneration at the time of surgical interventions.

3.1 Myocardial regeneration by stem/progenitor cells
The adult heart appears to contain a subpopulation of cardiomyocytes that are not terminally differentiated and re-enter the cell cycle and undergo nuclear mitotic division soon after myocardial infarction [35]. Despite this, myocardium does not substantially regenerate after myocardial infarction. This has encouraged new methods to replace scarred tissue with cells capable of augmenting systolic function. Towards this goal, investigators have transplanted various cells into infarcted myocardium of experimental animals, including HSCs [14,58], skeletal myoblasts [41,42], MSCs [59,60], endothelial precursors [61,62], resident CSCs [36], adipose-derived stem cells [63] and umbilical cord-derived stem cells [64]. However, the optimal cell type remains controversial, Table 2 shows the advantages and problems of different stem cells in cellular therapy. The results of these studies have generally suggested improved systolic performance, largely independent of cell type and demonstrated the feasibility and safety of cellular therapy. However, these results are still relatively preliminary and await additional preclinical studies.


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  		Table 2. Advantages and problems of adult stem/progenitor cells for in vivo myocardial tissue engineering
 
The mechanisms responsible for the beneficial effects of cellular therapy remain controversial. Elucidation of the exact mechanism of cell survival and exactly how the transplanted cells contribute to improvement in overall cardiac function remain the focus of intense investigation. Transdifferentiation of transplanted cells within infarcted myocardium has been presented by Orlic and colleagues [14–18], but it is still not clear whether the beneficial effect of these cells in regeneration can be explained by the transdifferentiation of transplanted cells. Recent advances indicate that stem cells can produce several angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF) and stem cell homing factor, increasing angiogenesis and cytoprotection, thus, attenuates left ventricular remodelling and improves cardiac performance [65,66]. Since the loss of cardiomyocytes is responsible for myocardial degeneration after acute myocardial infarction, preventing ongoing cell apoptosis is generally considered as one of the major mechanisms of cardiac regenerative therapy using stem cells. Kocher and colleagues demonstrated that transplanted cells can directly induce new blood vessel formation in the infarct-bed (vasculogenesis) and proliferation of pre-existing vasculature (angiogenesis) after experimental myocardial infarction. The neoangiogenesis resulted in decreased apoptosis of hypertrophied myocytes in the peri-infarct region, long-term salvage and survival of viable myocardium, reduction in collagen deposition and sustained improvement in cardiac function [67]. Thus, the favourable effects of cellular therapy are probably related to differentiation of transplanted cells within infarcted myocardium, secretion of angiogenic factors, prevention of apoptosis, angiogenesis and vasculogenesis.

3.2 Cell survival
Cellular therapy is confronted with the problem of donor cell survival after the cells are injected into ischaemic myocardium [69]. Transplanted stem cells must survive and integrate into the host myocardium in order to provide beneficial effects in ischaemic heart disease. An advantage of stem cells is their potential to proliferate and differentiate into large numbers of subpopulations that can be optimized so as to promote their survival and functional effects following transplantation. However, to date only limited survival of stem cells has been observed following transplantation. The molecular mechanism for stem cell death in ischaemic heart is in large part because of ischaemia; moreover, endogenous and environmental factors, such as ischaemia-reperfusion, inflammatory response and proapoptotic factors, play important roles [70]. Other mechanisms proposed for low cell survival include mechanical cell damage during grafting and cell leakage from the sites of needle puncture. In addition, for treatment to be successful, the cells need to migrate throughout the entire muscle after injection. Many factors have been tested to promote survival of stem cells and to enhance their survival following transplantation, including hypoxia-inducible factors, stem cell factors and antiapoptotic agents [71–74]. A reliable method for ensuring the survival of the desired numbers of transplanted stem cells would be of great value in developing a transplant strategy utilising stem cells.

3.3 Clinical trials
Despite uncertainty surrounding the mechanisms underlying adult stem cell plasticity, there is much speculation regarding potential clinical implications. Over the past few years, a large number of human studies showing the feasibility, safety and efficacy of postnatal adult stem cell therapy for ischaemic heart disease have been published, as shown in Table 3 . The first clinical application of cell transplantation as an adjunct to coronary artery bypass grafting (CABG) was performed by Menasche and colleagues using cultured autologous skeletal myoblasts in a 72-year-old male patient. The patient was in New York Heart Association (NYHA) class III with a mean left ventricular ejection fraction (LVEF) of 21 ± 2% by echocardiography. Follow-up at 5 months showed the patient was in NYHA class II with an improvement in the LVEF to 30 ± 1%. There were no substantial arrhythmias on 24 h Holter recordings and the procedure was performed without any complications. Consequently, this procedure has been repeated worldwide ever since. Recently, Dib and colleagues [92] published their 4-year experiences of autologous myoblast transplantation in patients with ischaemic cardiomyopathy and demonstrated the therapeutic efficacy of myoblast for end-stage heart disease.


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  		Table 3. Trials of adult stem/progenitor cells in patients during surgical intervention
 
Patel and colleagues reported the first randomized study of transplantation of autologous bone marrow stem cells as an adjuvant to off-pump coronary artery bypass grafting (OPCAB) in patients with congestive heart failure. All the patients in the OPCAB plus stem cell therapy group had significantly improved ejection fractions over those in the OPCAB-only group. There were no perioperative arrhythmias or neurologic or ischaemic myocardial events in either group [90]. Recently, Hendrikx and colleagues [96] reported their results of direct intramyocardial injection of autologous bone marrow cells (BMCs) during CABG. Twenty patients with a postinfarction nonviable scar were randomized to a control group (n = 10, CABG only) or a BMC group (CABG and injection of BMCs). During the 4-month follow-up, there was no significant difference in global LVEF between both groups, but a recovery of regional contractile function in previously nonviable scar was observed in the BMC group. Other clinical trials based on the same principle are currently being undertaken, but it is too early to make a definitive judgment about long-term functional efficacy and possible side effects.

3.4 Adverse events
Cellular therapy has drawn worldwide attention as a promising alternative strategy to treat end-stage heart failure [99]. Small-scale clinical trials suggested the feasibility and safety of stem cell transplantation into the injured myocardium and confirmed the improvement of global and regional left ventricular function, late after myocardial infarction. However, symptomatic ventricular arrhythmias occurred with an unexpected frequency within weeks following cell transfer. In the study by Menasche and colleagues, cultured autologous skeletal myoblasts were injected transepicardially into nonrevascularisable and nonviable scars at the time of CABG. Four patients had documented ventricular tachycardia at 11, 12, 13, and 22 days following surgery and cell implantation that was resistant to treatment by amiodarone and beta-blockers and necessitated the implantation of an automatic internal cardioverter/defibrillator [75,76].

Siminiak and colleagues also observed sustained ventricular tachycardia in two patients in the early postoperative period and in the other two patients after 2 weeks of follow-up. The aetiology of arrhythmia after myoblast transplantation is probably multifactorial and includes an inhomogeneous distribution of gap junctions, a difference in the isotypes of ion channels on skeletal muscle cells and cardiomyocytes, and the release of inflammatory mediators after needle puncture [86]. The presence of a nonmyogenic population in the myoblast cell population that is transplanted may further aggravate the situation. Another concern is the potential of undifferentiated cells to develop into undesired cell phenotypes with tumour formation (e.g., teratomas) and other unanticipated complications. Because the mechanisms of stem cell differentiation and proliferation have not been clearly elucidated, there remains the potential of risk of unregulated growth [100].


   4. Tissue engineering applications
Top
 Abstract
 1. Introduction
 2. Type of adult...
 3. Cellular therapy applications
 4. Tissue engineering...
5. Conclusions and future...
 References
 
Although clinical trials involving myocardial injection of autologous stem cells have produced favourable results, however, it is difficult to transplant a sufficient number of cells to restore cardiac function. Additionally, isolated cell transplantation is not enough for replacing congenital defects. A very promising approach to repair large scar areas and congenital heart defects may be the use of tissue engineering, in which cells are seeded in three-dimensional matrices of biodegradable polymers to form myocardial constructs. The ideal myocardial construct should display functional and morphological properties of native heart muscle and remain viable after implantation. Mechanical, electrical and functional integration into the organ architecture should result in improved systolic and diastolic function of diseased myocardium. Therefore, constructs should be (1) contractile; (2) electrophysiologically stable; (3) mechanically robust yet flexible; (4) vascularized or at least quickly vascularized after implantation; and (5) autologous [101,102].

4.1 Principles of myocardial constructs engineering
Tissue engineering is a multidisciplinary field combining biology, materials science and surgery to provide living tissue products to restore, maintain or improve tissue function. It aims at generating functional three-dimensional tissues outside of the body that can by tailored in size, shape and function according to the respective needs before implanting them into the body [103]. Fig. 1 shows the general principle of postnatal adult stem/progenitor cells in myocardial tissue engineering.


Figure 1
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  		Fig. 1. Basic steps of postnatal adult stem cells in myocardial tissue engineering: autologous stem cells or umbilical cord-derived stem cells are harvested, cultured, differentiated and expanded under precisely controlled culture conditions in the laboratory, and then seeded onto the scaffolds. Following additional culturing and development of the cell-scaffold constructs in the bioreactor, finally, transplantation of the constructs to the patient.

 
The biomaterial scaffolds play an important role in myocardial constructs engineering. The ideal scaffold should provide a suitable environment for tissue development. It should favour cell attachment, growth and differentiation, in vivo revascularisation, integration with the host tissues and the gradual replacement of the scaffold by newly formed tissues. At the same time, the materials and their degradation products must be nontoxic and nonimmunogenic, and their degradation rate should match the rate of new tissue formation. Polymers, such as polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA) are attractive as materials for reconstruction because of their great versatility and have been widely used to construct porous scaffolds [104]. Optimisation of cell seeding of polymer scaffolds is essential for the successful in vitro cultivation of functional tissue constructs. General seeding requirements to make implants for potential clinical use include: (a) high yield, to maximize the utilisation of donor cells; (b) high kinetic rate, to minimize the time in suspension for anchorage-dependent and shear-sensitive cells; and (c) high and spatially uniform distribution of attached cells, for rapid and uniform tissue regeneration and to enhance the rate of tissue development [105].

One approach that employs the use of a simulated biological environment is a bioreactor in which the in vivo biomechanical and biochemical conditions are created in vitro for functional tissue development [106]. Bioreactors combined with mechanical signals, such as under stretching or compression modes, improved the proliferation and distribution of the seeded cells throughout the scaffold volume and further stimulated the formation and organisation of extracellular matrix, all of which attributed to the improvement in the mechanical strength of the engineered grafts [107]. Vascularisation is one of the major obstacles in myocardial constructs tissue engineering. Local delivery of angiogenic growth factors, such as vascular endothelial growth factor and basic fibroblast growth factor provides an efficient means of stimulating localized vessel recruitment to the tissue-engineered grafts. However, blood vessels can still form too slowly, and the vessels generated are often not of a sufficient quality required for carrying blood [108].

4.2 Limitations of cardiomyocytes
Clearly, cardiomyocytes have to be the main cellular component of the heart. Three-dimensional cardiomyocyte cultures would thus be used for in vitro studies of cardiac tissue development and function and, if sufficiently large and functional, for in vivo cardiac repair. Several studies have shown the survival and function of tissue-engineered myocardial constructs using foetal or neonatal cardiomyocytes, but convincing evidence is lacking that heart tissues can be generated at a size and with contractile properties that would lend considerable support to failing hearts [4,6,7,9,101,109–113]. Recently, Zimmermann and colleagues reported that large (thickness/diameter, 1–4 mm/15 mm) heart tissue grafts could be created from neonatal rat heart cells in vitro. Engineered heart tissue formed thick cardiac muscle layers and showed undelayed electrical coupling to the native myocardium without evidence of arrhythmia induction when implanted on myocardial infarcts in immune-suppressed rats. Moreover, engineered heart tissue prevented further dilation, induced systolic wall thickening of infarcted myocardial segments and improved fractional area shortening of infarcted hearts compared to controls [114].

Several important questions remain in terms of using cardiomyocytes to form cardiac tissue. A major limitation of this approach is the inability or limited ability of cardiomyocytes to proliferate [115]. Moreover, mammalian heart is not composed purely from cardiomyocytes, but comprise almost all cell species that are normally found in the heart including cardiomyocytes, fibroblasts, smooth muscle cells, endothelial cells, macrophages and other cells of leukocytotic origin. Nonmyocytes play a pivotal role in heart and cardiac myocyte development, hypertrophy and function [116]. Accordingly, concepts have to be developed that take into consideration that the heart consists of multiple different cell types that most likely have to work in concert for an optimized function and thus would be required in tissue engineering of myocardial constructs.

4.3 Stem cell-based myocardial constructs engineering
The limited ability of cardiomyocytes to proliferate precludes in vitro propagation and therefore application of primary cardiomyocytes as an autologous cell source for myocardial tissue engineering. Thus, large-scale tissue engineering will require alternative cell sources. Recent advances have created the possibility of using stem cells as cell sources for myocardial tissue engineering, and this provide great hope for cardiac replacement therapy [117,118]. Previous studies have demonstrated the potential of postnatal stem cells to (trans-)differentiate into cardiomyocytes, smooth muscle cells and endothelial cells, which may fulfil the requirements of myocardial tissue engineering [27–30,40,43,119]. Hopefully, the engineered constructs would consist of viable and autologous tissue, which could theoretically function like a native biological structure with the potential to grow, to repair, and to remodel, and thus significantly improve the quality of patients’ lives. Preliminary experiments with human embryonic stem cells support this notion, offering a new perspective for the generation of cardiac muscle [120,121].

Krupnick and colleagues [122] demonstrated that MSCs, seeded on a three-dimensional matrix can engraft and differentiate within the left ventricle in a rat model. The newly constructed grafts resulted in minimal intracardiac inflammation without aneurysmal dilatation. No ventricular arrhythmias resulted from this surgical manipulation and echocardiography revealed both end systolic and diastolic volume augmentation with ventricular expansion. Moreover, MSCs can differentiate into muscle with cardiomyocytic potential using this scaffolding. Fuchs and colleagues [123] isolated myoblasts from skeletal muscle of foetal lambs, expanded in vitro, and then seeded onto collagen hydrogels. After birth, animals underwent autologous implantation of the engineered constructs onto the myocardium. Their results indicated that foetal skeletal myoblasts engraft in native myocardium up to 30 weeks after postnatal, autologous implantation as components of engineered onlay patches. Recently, Siepe and colleagues [124] provided further evidence that myoblast-seeded polyurethane scaffolds prevent postmyocardial infarction progression towards heart failure in a rat myocardial infarction model.

4.4 Cell sheet engineering
Shimizu and colleagues first proposed the concept of cell sheet engineering for myocardial tissue reconstruction without artificial scaffolds using temperature-responsive culture dishes. These cell sheets allow for cell-to-cell connections and maintain the presence of adhesion proteins because enzymatic digestion is not needed. Therefore, cell sheet transplantation may be a promising strategy for partial cardiac tissue reconstruction [125,126]. Implantation of engineered neonatal cardiomyocyte sheets to infarcted myocardium showed integration with impaired myocardium and improved cardiac performance. In addition, cultured cardiac cell sheets express angiogenesis-related genes, form endothelial cell networks and migrate to connect with the host vasculature after transplantation [127]. Furuta and colleagues tested the in vivo electrical communication which is essential for improving heart function between the host heart and the grafted cell sheet. They observed bidirectional smooth action potentials propagation between the host heart and the engineered tissue after cell sheet transplantation. There were neither spontaneous nor pacing-induced arrhythmias. This suggested functional integration of the engineered cell sheet with the host heart without serious arrhythmia [128].

Memon and colleagues [129] compared the therapeutic effects of myoblast transplantation and myoblast sheet implantation. Echocardiographic results indicated higher improvement of cardiac performance in the myoblast sheet group than myoblast transplantation group until 8 weeks after transplantation. Histologic comparison revealed greater cellularity and abundant widespread neocapillaries within the noticeable uniform thickened wall in myoblast sheet group hearts. Fibrosis was substantially reduced with skeletal myoblast sheet implantation compared with skeletal myoblast cell injection. Thus, they conclude that cell sheet implantation is better than cell transplantation because it repairs the infarcted myocardial wall and recruits supportive HSCs to attenuate cardiac remodelling and improve cardiac performance.

Recently, Miyahara and colleagues [130] transplanted the monolayered MSCs onto the scarred myocardium 4 weeks after coronary ligation and showed the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes after transplantation. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered MSCs reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered MSCs may be a new therapeutic strategy for cardiac tissue regeneration. There are several advantages to monolayered MSC transplantation. First, the self-propagating property of MSCs in situ leads to the formation of a thick stratum on the surface of the scarred myocardium. Second, the multipotency of MSCs and their ability to supply angiogenic cytokines allows neovascularisation in the MSC tissue. Third, the reconstruction of thick myocardial tissue reduces left ventricle wall stress and results in improvement of cardiac function after myocardial infarction. Finally, a substantial part of the transplanted tissue is composed of undifferentiated MSCs, and it is tempting to speculate that such cells may act against future progressive left ventricle remodelling [130].


   5. Conclusions and future directions
Top
 Abstract
 1. Introduction
 2. Type of adult...
 3. Cellular therapy applications
 4. Tissue engineering...
 5. Conclusions and future...
References
 
In summary, recent advances in the understanding of stem cell technology offers new possibilities for treatment of cardiovascular diseases. Encouraging clinical results have been achieved in cellular therapy, however, long-term observations and comparisons to control groups are required before this can be said to have been a proven success. Besides direct cell transplantation, transplantation together with the delivery of therapeutic genes and mobilisation of autologous stem cells for cardiac repair are attractive alternative strategy. Additional work is needed to elucidate the mechanisms involved in mobilisation, homing, integration and survival of stem/progenitor cells at the sites of implantation. This, in turn, will help define the optimal conditions for therapeutic application.

As to tissue-engineered myocardial constructs, although encouraging results have been achieved in donor cells research, biomaterial scaffolds, cell seeding conditions and cell sheet engineering, yet, there is still a long way to go before its clinical application. The results of the pioneering experiments raise hope for myocardial tissue engineering to repair or replace the infarcted myocardium. Theoretically, the bioengineered cardiac tissue could be used for surgical reconstruction of the infarcted myocardium or repair of congenital cardiac defects and eventually, solve the problem of organ donor shortage.


   Acknowledgments
 
This work is supported by the Specialized Research Fund for the Doctoral Program of Higher Education (20040023048) from the Ministry of Education of China.


   References
Top
 Abstract
 1. Introduction
 2. Type of adult...
 3. Cellular therapy applications
 4. Tissue engineering...
 5. Conclusions and future...
 References
 

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