Transplantation of skeletal myoblast in ischemic heart disease 2

The overall objective of heart cell therapy is to repopulate postinfarction scar tissue with contractile cells, which replace dead cardiomyocytes in sufficient numbers and restore functi

CHAPTER I INTRODUCTION

SECTION I ISCHEMIC HEART DISEASE

1.1.1 Introduction to Ischemic Heart Disease

Ischemic heart disease (IHD) is receiving a continuously growing interest because of the increased prevalence and incidence In USA, based on Heart Disease and Stroke Statistics Updates 2006, American Heart Association, the prevalence for IHD in 2003 was around 13,200,000 The incidence of IHD is around an estimated 1,200,000/year, with approximately 700,000 new and 500,000 recurrent cases It is estimated that an additional 175,000 cases of silent first heart attack occur each year In Singapore, IHD ranks the 3rd most prevalent cause of hospitalization (3.7% in total hospitalizations) according to statistics from Ministry of Health, Singapore The magnitude of the problem is expected to be even further amplified in the forthcoming years because of the aging population and paradoxically, the improved postinfarction survival rates resulting from recent pharmacological and interventional treatment

IHD is mainly caused by atherosclerosis, a process in which fatty deposits (atheroma) accumulate in the cells lining the wall of the coronary arteries When more than 50%

of the luminal diameter decreases (75% decrease of luminal area), it results in ischemia Complete occlusion of the blood vessel results in a myocardial infarction (MI), characterized by compromised coronary blood flow, massive cardiomyocyte death and impaired left ventricle contractile function Based on the 44 year follow-up

of the National Heart, Lung, and Blood Institute’s Framingham Heart Study, IHD

remains the leading cause of congestive heart failure (CHF).When patients with IHD also have an IHD-leading CHF, it is described as ischemic cardiomyopathy The pathophysiology that follows an IHD and eventually leads to ischemic cardiomyopathy is very profound Initially, there is a massive death of cardiomyocytes because of acute, severe ischemia In the acute setting, loss of cardiomyocytes reduces overall ventricular pump function This reduces blood pressure and cardiac output, which activates sympathetic nervous system as well as the rennin-angiotensin-aldosterone system In the short term, these factors attempt to restore cardiac output and blood pressure However, if sustained, neurohormonal activation and increased mechanical stresses conspire in a maladaptive process Within the healing infarct area, ischemia resistant fibroblasts are recruited and eventually replace the dead cardiomyocytes, leading to areas of fibrosis Unfortunately, this process does not improve any heart contract function In an attempt to compensate for the decrease of the heart function, a process so called cardiac remodeling occurs Importantly, in the post infarct heart, the remodeling process affects not only regions of infarction but also previous normally perfused myocardium The situation can be even worse in ischemic cardiomyopathy because non-infarcted regions can be supplied by stenosed coronary arteries so that active myocardial ischemia can influence the remodeling process In this process, compensatory mechanism in response to a loss of functioning contractile units includes cardiomyocyte hypertrophy and elongation, extracellular matrix exchanges, and subcellular remodeling etc However, none of them regenerates contractile tissue and further compensates for the heart performance

1.1.2 Current Status on IHD Treatment

Therapeutic interventions for IHD include behavioral and dietary modifications, pharmacological therapies (including anti-platelet agents, nitrates, β-blockers, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and calcium channel blockers etc), and invasive revascularization procedures such as coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) Although significant advances have reduced the mortality of IHD, the number

of cardiac interventions continues to grow In 2003, a total of 1.4 million inpatient cardiac revascularizations, 664,000 PCI procedures and 467,000 CABG procedures were performed in USA alone (Heart Disease and Stroke Statistics Updates 2006, American Heart Association) PCI is the most common treatment option for single vessel disease as well as simple multi-vessel disease However, high rate of restenosis

is still a major disadvantage and limits the application in selected cases Most recently, the development of drug-eluting stents seems to solve the problem of recurrent restenosis CABG is mostly recommended for patients with complex and life threatening IHD The in-hospital mortality rate for CABG is 1-3% at many institutes Despite the risk of vein graft failure, it remains the only form of therapy that has been shown to improve the life expectancy of patients with severe IHD However, though various effective treatments are available, IHD remains a leading cause of death worldwide IHD caused one of every five deaths in the United States in 2003 For those NYHA class 4 patients, the mortality is still unacceptably high at 60%/year (The Journal of the American Medical Association) In Singapore, IHD stands the

2nd contributing cause of death (18.1% in total deaths) The prevalence and the mortality of the disease are calling for new approaches in the treatment of IHD

1.1.3 No-option Patients: a Target Population for Cell Therapy

PCI and CABG are effective at relieving symptoms and improving outcomes in patients with IHD However, some patients with symptomatic IHD are no longer candidates for PCI or CABG, or have exhausted or failed these modalities A significant number of patients (5-21%) with ischemic heart disease are not optimal candidates for revascularization (PCI/CABG, McNeer et al.1974; Jones et al 1983; Atwood et al 1990; Feyter PJ 1992; Mukerjee et al 1999), and many have residual angina despite maximal medical therapy A recent study of 500 patients at Cleveland Clinic showed that about 12% patients (59 cases) were considered ineligible for revascularization (Mukherjee et al 2001) The different estimates of the magnitude of the problem may be attributed to wide regional and institutional variability in treatment patterns of coronary disease including more or less aggressive revascularization practices The conditions resulting in no-option status include recurrent in-stent restenosis, prohibitive expected failure, chronic total occlusion, poor targets for CABG/PCI, saphenous graft total occlusion, degenerated saphenous vein grafts, no conduits aorta, and comorbidities etc (McNeer et al.1974; Jones et al 1983; Atwood et al 1990; Feyter PJ 1992; Mukerjee et al 1999; Mukherjee et al 2001)

However, no-option patients are a heterogeneous group there are two different

amount of viable myocardium, in whom angina is the predominant symptom (Rizzello et al 2004; Di Carli et al 1998) On the other hand, the second group includes patients with limited or no myocardial viability, in whom heart failure symptoms predominate This group has a poorer response to increased myocardial perfusion (Rizzello V et al 2005) The current management strategies for these patients are limited The treatment of these patients is also a moving target since advances in interventional and surgical techniques have helped improve their quality

of life In addition, the development of various procedures such as endovascular cardiopulmonary bypass (Reichenspurner et al 1998), rotational atherectomy for calcified undilatable lesions (Madina et al 2003), and distal protection for vein graft interventions (Lev et al 2004) has made possible the treatment of many patients previous deemed to be no-option Thus before the definition of patients with no-option, consideration of advanced intervention is warranted If all these options are exhausted, then patients are deemed truly without any options and alternative treatment strategies are needed

1.1.4 Patients with End-stage Ischemic Cardiomyopathy: Another

Target Population for Cell Therapy

In the setting of ischemic cardiomyopathy, profound cardiac remodeling occurs, which affects not only regions of infarcted myocardium, but also normally perfused myocardium The impact is really broad, affecting molecular, biochemical, metabolic, cellular, extracellular matrix and ventricular structural characteristics This process is driven by increased mechanical stresses, in the form of increased preload and

afterload, as well as abnormally elevated levels of cytokines and neurohormones Different from mechanism of normal heart failure, ischemic cardiomyopathy has large quantity of cardiomyocyte loss and severely impaired heart function It is not surprising that treatment of these patients with medical management alone is dismal, especially those with advanced cardiac dysfunction (ejection fraction [EF] <30%-35%) Franciosa et al demonstrated 80% mortality over 3 years (Franciosa et al 1983) Until 1990’s, clinical experiment with CABG in advanced ischemic cardiomyopathy was developed Though EF after operation did increase to some extent, the hospital mortality is relatively high, from 5.3% to 20% (Luciani et al 1993; Kaul et al 1996; Elefteriades et al 1997) It seems that revascularization alone is not satisfied enough It was commonly held that the end-stage failing heart was irreversibly dilated, hypertrophic and dysfunctional Until the early 1990’s, after introduction of left ventricular assist devices (LVAD), it was appreciated that these abnormalities were not permanent, but could be reversed, at least to some extent (Barbone et al 2001) Now the goal of many ischemic cardiomyopathy treatments is

to slow or reverse this process In particular the concept of reverse remodeling is an important principle for new treatment of heart failure

1.1.5 The Challenges: Regenerate Contractile Tissue and Reverse

Remodeling by Cell Transplantation?

1.1.5.1 Rationale for Cell Transplantation

Cell transplantation for cardiac repair is based on two major assumptions: (a) cardiomyocytes that generate the contractile force are responsible for the cardiac

output, and when large quantities of cardiomyoctyes have been irreversibly lost, heart function deteriorates and (b) when the area of dysfunctional myocardium is repopulated with a new pool of contractile cells, heart performance could thus be improved The overall objective of heart cell therapy is to repopulate postinfarction scar tissue with contractile cells, which replace dead cardiomyocytes in sufficient numbers and restore functionality in these akinetic areas

Conceptually, this objective can be achieved through three distinct approaches The first approach is to stimulate the intrinsic regenerative capacity of heart Initially, heart has been considered as an organ composed of terminally differentiated myocytes incapable of regeneration However, the dogma is being challenged by recent studies, showing a certain degree of cardiomyocyte regeneration in various pathologic conditions, such as heart failure (Kajstura et al, 1998), orthotopic heart transplantation (Müller et al., 2002; Quanini, et al., 2002), or myocardial infarction (D Orlic, et al., 2001; Beltrami et al, 2001), and even surmising the existence of resident cardiac stem cells (Anversa et al., 2002), or cardiomyocyte progenitor cells (Laflamme et al., 2002) Nevertheless, although heart has some regenerative capacity,

it is by far too limited to compensate for the loss of cardiac cells (Packer et al., 1992; Mann et al 1999) Furthermore, the mechanism of cardiomyocyte proliferation and regeneration is obviously not well studied The second strategy is based on gene therapy and is targeted at the transformation of in-scar fibroblasts into contractile cells by transfection with the MyoD master gene which controls the skeletal muscle differentiation program Although this approach has yielded some successful experimental results (Tam et al 1995), it is fraught with the multiple issues associated

with gene therapy and is still of limited clinical relevance The third approach is to provide exogenously contractile cells as surrogate cardiomyocytes into the scar From

a clinical standpoint, this ‘transplantation’ strategy is likely the most realistic and, consequently, has been extensively investigated in preclinical and clinical settings

1.1.5.2 The Challenges for a Successful Cell-based Cardiac Repair

The path to develop successful cell therapy for cardiac repair is faced with many challenges (Figure 1.1) The challenge begins with choosing a suitable cell type Many different types are being investigated, each type having its own theoretical advantages and disadvantages (Caplice NM 2006) Up to date, there are no criteria of choosing suitable cells for cardiac repair The criteria need further preclinical and clinical trials and should be evidence-based Apart from cell type, cells need to be scaled up to a sufficient quantity (Pouzet et al, 2001; Tambara et al, 2003) The whole left ventricle have a cell population of about 4.5-5.8 billion (Olivetti G et al., 1995)

In patients with ischemic cardiomyopathy it is possible that as much as 30% of the original cardiomyocyte number is lost (Beltrami et al., 1994) Thus even if the goal were to replace a relatively small fraction of lost myocardium, there is a need to propagate large quantities of cells to get a beneficial effect, since a larger size of graft would probably lead to greater benefit for the treatment of damaged myocardium In order to get the best retention and cell survival, many delivery strategies have been tried in different models (Siepe M et al, 2005) Furthermore, in order to get better survival and suitable environment for differentiation, the cells need to be appropriately provided with nourishment via restored blood circulation In order to

contract most effectively, the grafted cells need to have an electrical or mechanical

link between grafted cells and native myocytes so that they could contract in

synchrony with native myocardium (Siepe M et al, 2005).Moreover, the contraction

benefit should be sufficient enough to produce a meaningfully influence on global

ventricular performance, and further to be translated into clinically beneficial effects

on patients In addition, it is ideal if the grafted cells could limit the scar formation

and induce beneficial effects on the ventricular structure so that the ventricular

remodeling could be reversed in the long run (Menasché P, 2007)

Figure 1.1 Challenges to a successful cell therapy for cardiac repair

Enhanced cardiac performance, Reverse cardiac remodeling

Identify and isolate suitable cell type

Large scale-up of selected cells

Cell retention and survival

Contraction apparatus / Angiogenesis

Electrical or Mechanical link

Beneficial effects on patient signs/symptoms/mortality

SECTION II: STEM CELL SOURCES AND DELIVERY

The concept of cellular transplantation to augment the function of the failing heart has been translated to clinic However, there are many questions left to be answered Here

we briefly review the choice of donor cells and cell delivery strategies

1.2.1 The Choice of Donor Cells

From a conceptual view, the ideal cell for transplantation has to meet several criteria: they should be easy to collect and propagate in vitro, relatively tolerant to ischemia so

as to survive in the ischemic and scar tissue, capable of proliferation and myogenic differentiation, electromechanically couple with the adjacent cells and they should not

be burdened with ethical and immunological problems (Siepe M et al, 2005) Unfortunately, no ideal cell type could satisfy all these demands so far

1.2.1.1 Fetal or Neonatal Cardiomyocytes

It seems logical that cardiomyocytes would be the best cell type to repair infarcted myocardium However, cardiomyocyte proliferation is strictly regulated by developmental control mechanisms, and most cells withdraw from the cell cycle and become post-mitotic after embryonic stage But recently some investigators have challenged the dogma and shown cardiomyocyte replication (Kajstura et al 1998; Müller et al., 2002; Quanini et al., 2002; Orlic et al., 2001; Beltrami et al., 2001; Anversa et al., 2002; Laflamme et al., 2002; Packer 1992; Mann et al., 1999) Unfortunately, the regenerative capacity of myocardium seems inadequate to

compensate for the tissue loss Due to poor proliferation of adult cardiomyocytes in vitro, cardiomyocytes from fetal or neonatal sources have to be used

Transplanting fetal or neonatal myocytes was initially regarded as the primary model

of cellular cardiomyoplasty When transplanted, suspension of cultured fetal cardiomyocytes in mice (Soonpaa et al., 1994), and fetal or neonatal cardiomyocytes

in rat hearts (Reinecke et al 1998) formed the viable grafts over 2 months in normal myocardium of syngeneic hosts Electron microscopy study showed the formation of intercalated disks and tight junctions between grafted fetal cardiomyocytes and host myocardium after transplantation (Soonpaa et al., 1994) Furthermore, in infarcted myocardium, viability of engrafted cells was demonstrated for up to 6–7 months after transplantation of fetal and neonatal cardiomyocytes in a permanent coronary-occlusion rat model (Müller et al., 2002) However, the initial enthusiasm generated

by the above studies died down when it was shown that fetal cardiomyocytes are highly sensitive to ischemia, and massive cell death prevents the formation of enough new myocardium to replace the infarct, and the therapeutic use of fetal cardiomyocytes might ultimately require additional interventions (Zhang et al., 2001; Muller et al., 2002; Reinecke et al 1999).Moreover, use of fetal cardiomyocytes also faces immunological problems as well as ethical difficulties in human application These problems prompted the search of alternative cell sources for cardiac repair

1.2.1.2 Myocardial Stem Cells

Despite the generally accepted dogma that adult cardiomyocytes are unable to regenerate, recent research suggests the existence of cardiac stem cells (CSCs, Table

1.1) Studies on human hearts shortly after myocardial infarction demonstrated a small proportion of cardiomyocytes (0.08%) apparently undergoing mitosis in the peri-infarct tissue (Beltrami et al., 2001) This paper challenged the dogma that the adult heart is a terminally differentiated organ and suggested that there may be a population of CSCs involved in myocardial regeneration Subsequently, from the adult rat heart, they isolated cells that expressed stem cell maker “c-kit” in 2003 (Beltrami et al., 2003) These cells had some characteristics of stem cells including self-renewing, clonogenicity, and multipotency When injected into an ischemic heart, the cells differentiated into cardiomyocytes, smooth muscle cells and vascular endothelium, producing new myocardium and new vessels that replaced the majority

of the infarcted tissue and contributed to improved ventricular contraction It is of translational interest that these cells traversed the vascular barrier and improved ventricular function in a rat infarct model when delivered via the intracoronary route, reported by Dawn et al recently (Dawn et al., 2005) In 2004, Messina E et al isolated and expanded adult CSCs from atrial or ventricular human specimens and from murine hearts CSCs were also shown to be present in patients with aortic stenosis and ischemic cardiomyopathy (Urbanek et al., 2003; 2005) Because CSCs are normal components of the adult heart and appear to be responsible for the physiologic and pathologic turnover of cardiac myocytes and non-myocytes, they may be particularly suitable for reconstituting dead myocardium

Table 1.1 Cardiac Progenitor Cells so far Identified and Their Characteristics

Cell type Species Phenotype Clonogenic Multipotent In vitro differentiation In vivo differentiation Cardiosphere formation

c-kit +

Beltrami et al

Dawn et al

Rat, dog c-kit

+ Lin

-CD34 - CD45 - Yes Yes Incomplete, into cardiomyocytes,

endothelial cells and smooth muscle cells

Cardiomyocytes, endothelial cells and smooth muscle cells organized in capillaries

Yes Yes Cardiomyocytes, endothelial cells Cardiomyocytes, endothelial

cells and smooth muscle cells organized in capillaries

cells and smooth muscle cells NA

Matsuura et al Mouse Sca-1+ c-kit+

Isl-1 + c-kit

UPCs

HC Ott et al Rat SSEA-1 + Yes Yes Crdiomyocytes, endothelial cells

and smooth muscle cells Cardiomyocytes, endothelial cells NA

UPCs: uncommitted cardiac precursor cells SSEA-1: stem cell marker stage-specific embryonic antigen 1 ABCG2+: ATP-binding cassette transporter Isl-1+: insulin gene enhancer binding protein c-kit: receptor for the stem cell factor Sca-1: stem cell antigen 1 MDR1+: P- glycoprotein Lin: Lineage markers KDR/Flk-1+: vascular endothelial growth factor receptor.

In addition to c-kit+ cells, several other populations of cardiac progenitor cells have been described with cardiomyogenic potential In 2003, Oh et al identified Sca-1+ cardiac progenitors that expressed CD31 but not c-kit or other markers of hematopoietic or endothelial progenitors (Oh et al., 2003) When these cells were subjected to DNA demethylation by 5-azacytidine treatment, they activated several cardiac-specific genes in vitro When injected intravenously into a mouse with myocardial infarction, the engrafted Sca-1+ cells expressing cardiac markers (sarcomeric, actin and troponin I) were found to generate cardiomyocytes, both with and without cell fusion These cells differed from those described by Beltrami et al in that they consistently express Sca-1 but not c-kit Subsequently, another type of Sca-1+ cardiac progenitor cells (Matsuura et al, 2004) was identified from adult murine hearts by a magnetic cell sorting system These cells co-expressed CD45 (40%) and CD34 (10%) When treated with oxytocin, Sca-1+ cells expressed genes of cardiac transcription factors and contractile proteins and showed sarcomeric structure and spontaneous beating These results suggest that Sca-1+ cells in the adult murine heart have potential as stem cells and may contribute to the regeneration of injured hearts Martin and colleagues isolated an Abcg2-expressing cardiac side population (SP) cells from embryonic as well as adult mouse hearts (Martin et al, 2004) They reported that, when co-cultured with unfractionated cardiac cells, some of the cardiac side population cells began to express the sarcomeric protein (actinin) Interestingly, these cardiac SP cells were negative for CD31 To our knowledge, in vivo transplantation studies with cardiac side population cells have not been reported

In yet another progenitor population is the cells expressing the LIM-homeodomain transcription factor islet-1 (isl1) During development, isl-1+ cells contributed substantially to the right ventricle, atria, outflow tracks and part of the left ventricle (Cai et al., 2003) Building on this work, later study has shown that isl-1+ cells were present in the neonatal heart, without expressing c-kit (Laugwitz et al, 2005) These cells exhibited a cardiomyocyte phenotype when cocultured with neonatal cardiomyocytes in vitro and showed electrical as well as contractile properties reminiscent of neonatal cardiomyocytes Interestingly, cardiomyocyte cultures fixed

in formaldehyde still induced cardiac differentiation of the isl1+ cells, effectively ruling out fusion as an explanation for activation of the cardiac program Despite these findings, however, it should be emphasized that the cells studied by Laugwitz et

al were isolated from neonatal hearts; it is unknown whether cells with the same properties can be isolated from the adult heart as well (a critical issue from the standpoint of therapeutic application)

The most recent candidate progenitor cell population is uncommitted cardiac precursor cells (UPCs, Ott et al., 2007) The cells were characterized by expression of the embryonic stem cell marker stage-specific embryonic antigen 1 (SSEA-1) When highly enriched populations of suspended UPCs were expanded over a cardiac-derived mesenchymal feeder layer, temporal maturation of the original suspended cells from an uncommitted SSEA-1+ was observed state to a mesodermally committed flk-1+ state, then to an flk-1+ and sca-1+ state and then to a cardiac committed nkx2.5+, GATA-4+, or isl-1+ state Ultimately, cells differentiated into mature cardiomyocytes, endothelial cells, and smooth muscle cells As compared to

the aforementioned sca-1+, c-kit+, isl-1+ or SP cardiac precursors, UPCs appeared

to resemble the more immature, common pool of embryonic lateral plate mesoderm progenitors that yielded both myocardial and endocardial cells during normal cardiac development Under controlled in vivo conditions, these cells could differentiate into endothelial, smooth muscle, and cardiomyocyte lineages Since these cells could be isolated and expanded to clinically relevant numbers from adult rat myocardial tissue, they would be a promising population to test in cardiac repair applications

At present, the relationship among the various cardiac progenitor cells outlined above

is unclear Nevertheless, the discovery of resident cardiac progenitors represents a momentous milestone in cardiac biology, for it refutes the long-held view of the heart

as a terminally differentiated organ Instead, it supports a new paradigm in which the heart is a self-renewing organ that undergoes a continuous turnover The cardiac progenitor cell revolution has shown that the heart is not different from other organs,

in which repositories of stem/progenitor cells have been reported for many years

1.2.1.3 Embryonic Stem (ES) cells

Derived from the inner cell mass of blastocysts, the ES cells have the pluripotent nature, capacity of self-regenerating, well-established protocols for their derivation, propagation and differentiation, which provides another potential source of cells for cardiomyoplasty (Evans et al., 1981; Martin 1981)

In 1988, Doetschman et al showed ES cells underwent spontaneous formation of three-dimensional “embryoid bodies" which included foci of beating myocardium (Doetschman et al 1988) However, cardiogenesis within embryoid bodies is

relatively inefficient Using genetic selection method, cardiomyocytes with >99% purity were obtained by Klug et al (1996) Later, this selection strategy has been adopted for large-scale production of highly purified ES cell-derived myocytes

(Zandstra et al 2003) Cardiomyocyte-like cells obtained by similar approaches were

characterized by immunohistology for troponin-T (Metzger et al., 1996), troponin I (Westfall et al 1996), gap junction protein connexin-43 (Oyamada et al., 1996), α-cardiac-myosin heavy chain (Maltsev et al., 1993; Kolossov et al., 2005), myosin light chain (Meyer et al., 2000), Nkx2.5 (Hidaka et al., 2003); and functionally with regard to Ca2+ signaling (Sauer et al., 2001) These findings demonstrated that ES cells provide an enormous potential for cell-based cardiac repair In 1996, Klug et al demonstrated that direct transplantation of mouse ES cell−derived cardiomyocytes into an immunocompatible recipient heart resulted in the successful formation of stable intracardiac grafts Subsequently, the transplantation efficacy of mouse ES cell have been confirmed by other investigators (Behfar et al., 2002; Etzion et al., 2001; Min et al., 2002; Min et al., 2003)

The generation of human ES lines with a stable developmental potential was firstly described by two groups (Thomson et al., 1998; Reubinoff et al., 2000) Subsequently, several studies have established spontaneous and directed differentiation systems from human ES cells into cardiac tissue (Kehat et al., 2001; Xu et al., 2002; Mummery et al., 2003; He et al., 2003) These human ES cell−derived cardiomyocytes showed the expected molecular, structural, electrophysiologic and contractile properties of early stage human cardiomyocytes In addition, the capability

of differentiation into endothelial cells was recently demonstrated (Levenberg et al.,

2002) As compared with mouse counterparts, in vivo transplantation of human ES

cell-derived cardiomyocytes is in infancy In 2004, Kehat et al described the generation of a reproducible cardiomyocyte differentiation system from human ES cells, and the generated myoctyes displayed functional and electromechanical integration with host myocardium (Kehat et al 2004) In 2005, Xue et al also demonstrated functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with recipient myocardium (Xue

et al 2005) These two studies used electrical mapping techniques to show an ectopic pacemaker site where human ES cell−derived cardiomyocytes were implanted, and unambiguously demonstrated host-graft electromechanical coupling These promising results have led to an exploding increase in this field of research

Despite of the development of the human ES cell technology, unresolved ethical and political issues, concerns about the tumorigenicity of the cells, and the need to use allogeneic cells for transplantation currently hamper their use in clinical application Furthermore, protocols for large-scale production of highly purified preparations of cardiomyocytes need to be optimized Human ES cell−derived myocytes should be up-scaled to yield clinically relevant number of cells, and should be highly purified to avoid electrophysiological disorders (He et al., 2003)

1.2.1.4 Bone Marrow Derived Stem Cells

Currently the vast majority of clinical studies using stem cells for cardiac repair are based on bone marrow derived stem cells (BMCs) It has been shown that adult bone marrow hosts heterologous populations of multipotent cells: the hematopoietic stem

cells (HSCs), endothelial progenitor cells (EPCs), CD133+ cells, and mesenchymal stem cells (MSCs)

The HSCs are precursors of blood and endothelial cells in adult bone marrow with expression of cell maker CD34 As compared to normal HSCs, bone marrow derived HSCs have a limited capacity for self-renewal and differentiation and can sustain hematopoiesis for a short-term period Previous studies have suggested transplantation of autologous bone-marrow-derived or circulating progenitor cells may be beneficial for post-infarction left ventricular contractile function (Orlic et al., 2001; Jackson et al., 2001; Reinlib et al., 2000) However, several subsequent preclinical studies using state-of-the-art genetic tools have seriously challenged the concept that stem cells truly trans-differentiate into functional cardiomyocytes capable of generating active force (Murry et al., 2004; Balsam et al., 2004; Nygren et al., 2004) Despite these divergent preclinical findings, several different types of cells have been used in clinical settings A small population of endothelial progenitor cells (EPCs) has originally been defined by their cell surface expression of the HSCs marker proteins CD133 and CD34 and the endothelial marker vascular endothelial growth factor receptor-2, and their capacity to incorporate into sites of neovascularization and to differentiate into endothelial cells in situ (Asahara et al., 2004) Increasing evidence suggests that when expanded in culture, EPCs also contain a CD14+/CD34–-mononuclear cell population with angiogenic effects by releasing paracrine factors (Rehman et al., 2003; Urbich et al., 2004) Notably, EPC numbers and their angiogenic capacity are impaired in patients with coronary artery disease, which may limit their therapeutic usefulness (Vasa et al., 2001; Hill et al.,

2003) It has been shown that CD133+ cells can integrate into sites of neovascularization and differentiate into mature endothelial cells (Asahara et al., 2004)

MSC is a rare population of CD34– and CD133– cells present in bone marrow stroma (0.001%–0.01%) and other mesenchymal tissues (Pittenger et al., 2004) However, there in no adequate cell marker to allow selection of purified cell populations, and thus their phenotypes remain unclear MSCs can readily differentiate into multiple lineages (Jiang et al., 2002) Differentiation of MSCs to cardiomyocyte-like cells has been observed in vitro under specific culture conditions and in vivo after injection

into healthy or infarcted myocardium in animals (Makino et al., 1999; Toma et al., 2002; Mangi et al., 2003) Transplantation of MSCs enhanced regional wall motion

and prevented remodeling of the remote, non-infarcted heart (Mangi et al., 2003; Shake et al., 2002) Moreover, cultured MSCs secreted angiogenic cytokines, which improved collateral blood flow recovery in a murine hind limb ischemic model (Kinnaird et al., 2004) As MSC clones have been reported of a low immunogenicity, they might be applied in allogeneic transplantation in future clinical studies (Pittenger

et al., 2004) Importantly, if the allogeneic acceptance of MSC is applied in clinical settings, it will greatly decrease the logistic concerns, cost and time for MSC isolation and propagation to allow the on-shelf availability of MSCs In addition, MSCs were demonstrated to home into the areas of injury which may provide the possibility of delivering the cells by intravenous route (Bittira et al., 2003)

To date, selected or non-selected BMCs have been widely applied in patients with acute MI (Strauer et al., 2002; Assmus et al., 2002; Britten et al 2003; Schachinger et

al 2004; Fernandez-Aviles et al 2004; Kuethe et al 2004; Wollert et al 2004), myocardial ischemia without revascularization (Hamano et al 2001; Tse et al 2003; Fuchs et al 2003; Perin et al 2003; Perin et al 2004), and ischemic cardiomyopathy (Stamm et al 2003; Stamm et al 2004; Assmus et al 2004) These studies have reported no significant short-term safety concerns However, long-term toxicity should be carefully observed due to the use of unselective BMCs The non-selected cells, after engraftment into myocardium, may generate severe calcification (Yoon et al., 2004) and other non-cardiac tissues, and further led to malignant ventricular arrhythmias Furthermore, since non-selected cells have good generative capacity, and may secrete angiogenic factors such as VEGF, it is possible to generate angiomas

or even tumors in the long run (Epstein et al., 2001) Thus, non-selected BMCs should be carefully used before enough information is available in this regard Additionally, although the previous studies have provided positive information on the transplantation efficacy, intermediate- sized, randomized, and well controlled trials need to be established to evaluate the real effectiveness of BMC transplantation

1.2.1.5 SkMs (please refer to SECTION III)

1.2.2 Cell Delivery Methods

There are various strategies for cell delivery in heart cell therapy The goal of any cell delivery strategy is to achieve the ideal concentration of viable cells for cardiac repair with the lowest risk to patients Therefore, cell-delivery strategies must take into

account the clinical setting and local milieu, because stem cells may perform differently according to local signaling The ideal delivery strategy should be safe, easy to use, applicable to a wide range of clinical scenarios, precise in target, and results in good cell retention Stem cells can be delivered through coronary arteries, coronary veins, or peripheral veins Alternatively, direct intramyocardial injection can

be performed using a surgical, transendocardial, or transvenous approach A delivery strategy may involve mobilization of stem cells from the bone marrow using cytokine therapy, with or without peripheral harvesting Each technique has its peculiarities, and so the choice of the modality should be based on the own clinical scenario

1.2.2.1 Stem Cell Mobilization

In humans, mobilization of bone marrow derived stem cells occurs after AMI, suggesting a natural attempt at cardiac repair (Shintani et al., 2001; Leone et al., 2005) Therapeutic mobilization of bone marrow progenitor cells after AMI would amplify the existing healing response Granulocyte-colony stimulating factor (G-CSF)

is one of the best-studied cytokines, with relative data published in small animals, primates, and early human trials Several investigators have demonstrated that G-CSF administration improves ventricular function of postinfarction in mice (Orlic et al., 2001), rats (Sugano et al., 2005) and pigs (Iwanaga et al., 2004) On the other hand,

no beneficial effect of G-CSF in mice and baboons has been observed (Norol et al., 2005; Deten et al., 2005) Despite these discrepancies, G-CSF treatment has already been applied in the clinical trials (Kang et al., 2004; Kuethe et al., 2004; Kuethe et al., 2005; Suárez et al., 2005; Jorgensen et al., 2005; Ince et al., 2005; Valgimigli et al.,

2005; Wang et al., 2005; Boyle et al., 2005) Stem cell mobilization is an attractive strategy to treat heart failure because it is simple and would obviate the need for invasive harvesting or delivery procedures However, dissimilar to the above studies, not all groups find G-CSF effective, particularly the two negative studies recently reported in treating chronic ischemia (Wang et al., 2005; Boyle et al., 2005) Further, safety concerns should be always attended because of concern about tumorigenesis (Boyle et al., 2005)

1.2.2.2 Direct Intramyocardial Injection

Intramyocardial injection, injected through the epicardium, endocardium, or coronary vein, has been performed in chronic myocardial ischemia (Herreros et al., 2003; Dib

et al., 2005; Siminiak et al., 2004; Pagani et al., 2003; Thompson et al., 2003; Smits et al., 2003) In this technique, stem cells are introduced into the myocardium under pressure using a hollow needle This is the preferred delivery route in patients with chronic total occlusion of coronary arteries and in clinical settings that involve weaker homing signals, such as chronic congestive heart failure In theory, it should

be the most suitable route for delivering larger cells, such as SkMs and mesenchymal stem cells, which can plug capillaries However, direct injection of cells into ischemic

or scarred myocardium creates islands of cells with limited blood supply and may lead to poor cell survival (Murry et al., 1996; Reinecke et al., 1999; Taylor et al., 1998; Suzuki et al., 2004; Fearon et al., 2004) Strategies that enhance graft survival are thus mandatory to optimize the benefits of this procedure

1.2.2.2.1 Transepicardial injection

Transepicardial delivery of stem cells is the most commonly used technique in cardiac stem cell therapy Cells are injected into infarct border zones or areas of infarcted/scarred myocardium under direct visualization (Herreros et al., 2003; Dib et al., 2005; Siminiak et al., 2004; Pagani et al., 2003), which performs as an adjunct to CABG This strategy requires thoracotomy or sternotomy and, is relatively invasive

as compared to other delivery approaches, associated with significant surgical morbidity In a planned open-heart surgery, the ancillary delivery of cell therapy in this fashion can be easily justified The main advantages of this method are its proven safety in several preclinical and human trials (Herreros et al., 2003; Dib et al., 2005; Siminiak et al., 2004; Pagani et al., 2003; Reinecke et al., 1999; Taylor et al., 1998; Suzuki et al., 2004) and its ease of use Another important advantage of this approach

is that it could provide a high level of cells per unit area injected However, in a direct external approach, visual assessment for infarcted border zone may be limited and not all areas of the myocardium (e.g the septum) can be readily reached (Thompson et al., 2003) Furthermore, the safety of direct injection in AMI has not been tested In addition, the invasiveness of this approach hampers its use as a stand-alone therapy Conversely, the efficiency of cell transplantation may be difficult to evaluate and ascertain if CABG is performed simultaneously Nevertheless, direct surgical injection might certainly have a role in the future of stem cell therapy One can easily envision the cardiac surgeon, during CABG , bypassing all feasible areas and then concomitantly injecting stem cells into those areas bypass grafting are not applicable

1.2.4.2.2 Transendocardial Injection

Transendocardial injection is performed using a percutaneous femoral approach Using an injection needle catheter advanced retrogradely across the aortic valve and positioned against the endocardial surface, cells can be directly injected into any area

of the left ventricular wall Up to date, four intramyocardial catheter-based delivery systems (including the HelixTM [Fearon et al., 2004], the MyoCathTM [Bioheart Inc., Sunrise, FL, USA], the MyostarTM [Vale et al., 2000], and the StilettoTM [Christman et al., 2004; Naimark et al., 2003]) have been used in clinical trials Among them, Helix and MyoCath systems are integrated systems As the tip of the device contacting the endocardium, the core catheter is advanced, forcing a straight needle to a controlled intramyocardial depth (3–8 mm) for cell injection The integrated design provides a relatively simple mechanism for navigation and repeated injections StilettoTM is guided fluoroscopically, usually in two planes MyostarTM is guided by a NOGA system (Cordis Corp.), a catheter-based electromechanical system, providing a 3-dimensional left ventricular electromechanical map, which represents the endocardial surface of the left ventricle These two systems are not integrated With the guidance, the system enables multiple, topographically distinct injections As compared with transepicardial injection, this method can be used as a stand-alone therapy and as an adjunct therapy to CABG/PCI with minimal invasiveness

1.2.4.2.3 Trans-coronary-vein Injection

Trans-coronary-vein injection uses a catheter system incorporating an ultrasound tip for guidance and an extendable needle for myocardial access To date, feasibility studies have shown a good safety profile for this technique in experimental animals as

well in clinical trials (Thompson et al., 2003; Siminiak et al 2004) Preliminary studies have suggested that the acute cell retention using trans-coronary-venous method with fluoroscopic guidance is more efficient as compared with endoventricular approaches guided by electromechanical mapping (Smits et al, 2002) This approach allows delivering cells parallel to the ventricular wall and deep into the injured myocardium However, positioning of the injection catheter in a specific coronary vein is technically more challenging as it has restrictions associated with the tortuosity of coronary veins and lack of site-specific targeting (Siminiak et al 2004)

1.2.2.3 Transvascular Approaches

Transvascular technique is mainly applied in the treatment of recently injured myocardium due to highly expressed chemoattractants and cell adhesion molecules (Barbash et al., 2003; Kawamoto et al., 2001)

1.2.2.3.1 Intravenous Infusion

Intravenous infusion of stem cells seems a promising approach for practical and economical reasons, since it is the least invasive way of delivering cells Intravenously infused stem cells gather at infarcted myocardium more intensely than infarction-free hearts (Barbash el al., 2003; Nagaya et al., 2004; Kawamoto et al., 2001), and when infused into the myocardium, human BMCs home to peri-infarct areas (Kocher et al., 2001) In preclinical animal models, intravenous delivery of EPCs or MSCs has been shown to improve cardiac function after AMI (Kawamoto et

al., 2001; Kocher et al., 2001; Pittenger et al., 2004) However, the homing process of

injected stem cells solitarily to the targeted organ needs to be investigated in detail The risk of side effects from homing to non-cardiac organs limits the clinical applicability of this approach (Barbash et al., 2003; Nagaya et al., 2004) Indeed, significant myocardial homing of unselected BMCs was observed only after intracoronary delivery but not after intravenous application in a recent study in post-AMI patients (Wollert et al., 2001) Additionally, since the technique relys on physiologic homing signals, it would be most applicable after AMI and less useful for treating chronic myocardial ischemia

1.2.2.3.2 Intracoronary Artery Infusion

Intracoronary injection allows homogeneous homing of cells into areas bordering the infarction zone It is the most popular mode of cell delivery in the clinical setting, especially after AMI (Fernández-Avilés et al., 2004; Siminiak et al., 2003; Assmus et al., 2002; Strauer et al., 2001) The technique is similar to that used for coronary angioplasty, which involves positioning of an angioplasty balloon in one of the coronary arteries The coronary blood flow is then stopped for approximately 2–4 min while the stem cells are infused under pressure This maximizes contact between stem cells and the microcirculation of the infarct-related artery Cells used for intracoronary infusion must be capable of transendothelial migration to perivascular spaces Because of the risk of microvascular obstruction and myocardial ischemia, cells that are viscous or whose diameters are large may not be suitable for intracoronary infusion (Vulliet et al 2004).Additionally, myocardial targets must be supplied by well-defined vascular channels readily accessed by delivery catheters.By

intracoronary approach, unselected BMCs, HSCs, and MSCs have been delivered to patients with AMI and ischemic cardiomyopathy (Fernández-Avilés et al., 2004; Siminiak et al., 2003; Strauer et al., 2001; Strauer et al., 2002; Assmus et al., 2002; Britten et al., 2003; Schachinger et al., 2004; Kuethe et al., 2004; Wollert et al., 2004;

Chen et al 2004; Vanderheyden et al 2004) However, cell quantity and infusion characteristics need to be precisely determined to avoid the risk of additional myonecrosis (Vulliet et al., 2004; Wollert et al., 2004)

SECTION III MYOBLAST-BASED CARDIAC REPAIR

1.3.1 The Rationale to Choose Myoblast Transplantation

Unlike cardiac muscle, skeletal muscle retains the ability to regenerate and repair after injury Mature skeletal muscle tissue originates from undifferentiated, mononucleated precursors that are termed myoblasts SkMs proliferate in response to local mitogens, such as fibroblast growth factor family members When local growth factors are depleted, SkMs withdraw from the cell cycle, and fuse to form multinucleated cells called myotubes Not all SkMs fuse into myotubes Some SkMs which are found on the surface of myofibers beneath the basal lamina, become quiescent stem cells After tissue injury, SkMs are mobilized, proliferate, and fuse, resulting in repair and regeneration of the damaged tissue SkMs have been maintained proliferating in culture for extended periods of time while maintaining their ability to fuse to form muscle cells (Alberts et al., 1994)

Table 1.2 Advantages of Using SkMs for Cardiac Repair

Autologous origin, which overcomes problems related to availability, ethics and

graft rejection

High proliferative potential and possibility of undifferentiated myoblast

amplification in vitro (Murry et al., 2004)

High resistant to ischemia (Murry, et al 1996)

As a well-differentiated myogenic lineage, thus eliminating the risk of

SkMs can be genetically modified (Suzuki et al 2001)

Of the various cell types used for transplantation, SkMs appear attractive for a number of reasons (Table 1.2) Firstly, as compared to non-autologous cell transplantation (neonatal cardiomyocytes [Soonpaa et al., 1994; Reinecke et al 1998]

or embryonic stem cells [Klug et al., 1996; Zandstra et al 2003]), the autologous origin of SkMs overcomes the problems related to availability and ethics More importantly, the transplantation of autologous SkMs would avoid the host immunological barrier to those stem cells of allogeneic origin Secondly, as compared with the other types of autologous adult stem cells which are relatively scarce (Muller-Ehmsen et al., 2002) and require ex vivo expansion in cell numbers prior to use in transplantation therapy, SkMs are relatively abundant, easily accessible, and easily cultured by less invasive biopsy techniques (Murry et al., 2004) Thirdly, it is also well-documented that skeletal myoblasts display much higher levels of ischemic tolerance (many hours of ischemia) and graft survival compared to other cell types (Murry, et al 1996), which definitely improves the efficacy of the whole

transplantation procedure Fourthly, SkMs are committed solely to the myogenic lineage Therefore, regardless of environmental influences, even if implanted into a scar build up mainly of fibroblasts, SkMs differentiate into muscle cells, thus eliminating the risk of tumorigenicity (H Reinecke, 2002) More importantly, the grafted myoblasts have not only been shown to develop into functioning muscle fibers (Murry et al., 1996; Ghostine et al 2002), but also can acquire resistance to fatigue by modifying themselves to a fatigue-resistant slow-twitch phenotype capable

of performing a cardiac–type workload (Kehat et al., 2001; Min et al., 2002), therefore, cellular grafts comprised of SkMs might offer long-lasting cardiac assistance Fifthly, the grafted SkMs may establish satellite reserves to counter subsequent injury (Sim et al., 2003) Finally, SkMs can be genetically modified in vitro to deliver angiogenic cytokines and growth factors to encourage angiomyogenesis (Suzuki et al., 2004)

1.3.2 Pre-clinical Assessment of SkMs for Cardiac Repair

Extensive preclinical data in a variety of animal models support the feasibility safety, and efficacy of SkM implantation into regions of myocardial infarction The preclinical trials are summarized in Table 1.3 Overall, preclinical trials demonstrate that transplanted cells survive and form well differentiated myofibers with a contractile apparatus as well as contribute to significant functional improvement in injured hearts However, the ability of SkMs to differentiate into cardiomyocytes or form cell-to-cell junctions with host cardiomyoctyes remains controversial

Table 1.3 Myoblast Transplantation for Cardiac Repair in Preclinical Studies

Study Species Model Cell type Delivery Duration Outcome

Marelli et al 1992 Dog Cryoinjury Autologous SkMs IM 8weeks Transdifferentitation into cardiomyocytes

Koh et al 1993 Mouse Normal heart C2C12 SkMs IM 30 days Differentiate multinucleated myofibers

Zibaitis et al 1994 Dog Cryoinjury Autologous SkMs IM 8weeks Intercalated discs, skeletal muscle, mosaic fibers Chiu et al.1995 Dog Cryoinjury Autologous SkMs IM 18weeks Striated muscle grafts

Koh et al 1995 Mouse Normal heart TGF-β1 expressing

C2C12 SkMs IM 30days Viable grafts till three months Yoon et al 1995 Dog Cryoinjury Autologous SkMs IM 6 weeks Viable satellite cells

Murry et al 1996 Rat Cryoinjury Neonatal SkMs IM 3months No coupling to graft, conversion to slow twitch fibers

after 2-7 weeks Robinson et al 1996 Mouse Normal heart C2C12 SkMs Artery

delivery 4months Fast twitch to slow twitch, Gap junction and cardiac protein expression Taylor et al 1997 Rabbit Normal heart Autologous SkMs IC 3weeks Intergration into cardiac environment

Taylor et al 1998 Rabbit Cryoinjury Autologous SkMs IM 6weeks striated cells that retained characteristics of both

skeletal and cardiac cells(gap junction); functional improved(7/12)

Dorfman et al.1998 Rat Normal heart Adult SkMs IM 4weeks 4/24 successfully transplanted

Atkins et al 1999 Rabbit cryoinjury Autologous SkMs IM 2 weeks two populations of striated cells

Atkins et al 1999 Rabbit cryoinjury Autologous SkMs IM 3weeks nine animals, diastolic properties improved

Atkins et al 1999 Rabbit cryoinjury Autologous SkMs IM 3 weeks improved diastolic properties; No coupling with

cardiac cells Reinecke et al 2000 Rat cryoinjury Neonatal SkMs IM 3months Myoblast express Cx43/N-cadherin, functionally

junction with cardiac cells Pouzet et al 2000 Rat LAD ligation Autologous SkMs IM 2months No connexin 43, functional imporvement

Suzuki et al 2000 Rat Normal heart L6 SkMs IC 4 weeks Heat shock improved engrafted myoblast survival Suzuki et al 2000 Rat Explanted

heart L6 SkMs IC 4 weeks Connexin 43 between myotubes and cardiac cells Lee et al 2000 mouse Normal heart VEGF- SkMs IM 16days successful implantation of myoblasts

Pouzet et al 2001 Rat LAD ligation Autologous SkMs IM 2month No connexin 43

Pouzet et al 2001 Rat LAD ligation Autologous SkMs IM 2months Stimulation before 48 hours increases cell harvest

Table 1.3 Myoblast Transplantation for Cardiac Repair in Preclinical Studies (continued)

Rajnoch et al 2001 Sheep Cardiotoxin Autologous SkMs IM 2months Increase of local and global EF

Suzuki et al 2001 Rat Doxorubicin

Suzuki et al 2001 Rat LAD ligation VEGF- SkMs IM 4 weeks Reduction and infarct size, increased angiogenesis,

improved heart function Jain et al 2001 Rat Occlusion with

reperfusion Neonatal SkMs IM 6 weeks Skeletal MHC myotubes Gulbins et al 2002 Rat Normal heart Syngeneic SkMs IM 28days myoblasts expressed n-cam, desmin, and a-actin Dib et al 2002 Pig LAD ligation Syngeneic SkMs PEI 10 days endoventricular injection was technically feasible Ghostine et al 2002 Sheep LAD ligation Autologous SkMs IM 1 year limits postinfarction EF deterioration and improves

systolic scar function Chedrawy et al 2002 Rats Normal heart Autologous SkMs IM 6weeks Integration between myoblasts with cardiomyocytes Reinecke et al 2002 Rats Normal heart Autologous SkMs IM 12weeks No transdifferentiation to cardiomyocytes

Chazaud et al 2003 Pig Normal heart Autologous SkMs PEI 10 days multinucleated myotubes in the infarcted region

Borenstein et al 2003 Sheep Normal heart Autologous SkMs IM 3 weeks Non-cultured skeletal muscle cells can successfully and

massively engraft in ovine myocardium Sugimoto et al 2003 Rat Normal heart VEGF expressing

H9C2 SkMs

IM 4weeks Decreased scar tissue diameter Leobon et al 2003 Rat Ligation Neonatal SkMs

IM 30 days Grafted myoblasts functionally isolated from heart tissue

Al Attar et al 2003 Rat Ligation Autologous SkMs IM 1year Increased heart function, the proportion of the slow and

composite (fast and slow) myosin isoforms expressed after 1 year was greater than that after 2 months Zhong et al 2003 Dog Ligation Autologous SkMs IM 8 weeks muscle fiber with striations intercalated discs

Tambara et al 2003 Rat Ligation Neonatal SkMs IM 4 weeks LV diastolic dimension decreased, fractional area

change increased with infarction area decreased Haider et al 2004 Pig Ligation Xeno- SkMs IM 7months Transient immunosuppressive

Suzuki et al 2004 Mouse Normal Allogeneic SkMs IM 72hours Inflammatory response may be important in acute graft

attrition

Table 1.3 Myoblast Transplantation for Cardiac Repair in Preclinical Studies (continued)

CX: circumflex coronary artery ICS: intra coronary sinus IC: introcoronary infusion PEI: percutaneous endoventricular injection LAD: left anterior

descending artery LV: left ventriluar SkMs: skeletal myoblasts EF: ejectional fraction

Study Species Model Cell type Delivery Duration Outcome

Murtuza et al 2004 Rat Ligation Syngeneic SkMs IM 3 weeks Increased LVED, decrease LV dilation

Suzuki et al 2004 Mouse Normal Allogeneic SkMs IM 72hours Il-1β is involve d in acute inflammation of graft death Rubart et al 2004 Mouse Normal heart Syngeneic SkMs IM 50days Fuse of myoblasts with cardiomyocytes engraftment of

SkMs generated spatial heterogeneity of [Ca2+]i signaling at the myocardial/skeletal muscle interface, Reinecke et al 2004 Mouse normal heart C2C12 SkMs IM 1 week Fusion between cardiac and SkMs

Mcconnell et al 2005 Sheep Microembolized

CX Autologous SkMs IM 6 weeks attenuation in the left ventricular dilatation after autologous SkM transplantation

He et al 2005 Dog Microembolized

LAD Autologous SkMs IM 10weeks mild improvements in hemodynamics and LV function and reduced LV remodeling Brasselet et al 2005 Sheep Microembolized

LAD Autologous SkMs ICS 2months LVEF was significantly higher in the transplanted group than in controls

Ye et al 2005 Rat Cryoinjury Xeno- SkMs IM 6 weeks Improved EF and fractional shortening

Ott et al 2005 Rat Ligation Autologous SkMs IM 6 weeks Improved EF

Tambara et al 2005 rats LAD ligation Neonatal SkMs IM 4 weeks Hepatocyte growth factor can greatly increase the graft

volume and vascularity and reduce fibrosis inside the graft, which enhances the efficacy of SM Tx to infarcted hearts

1.3.2.1 Retention, Distribution, and Survival of Transplanted SkMs

After transplantation into myocardium, SkMs will immediately encounter the problem of the retention, redistribution, and survival in the transplanted milieu, which will ultimately influence the engraftment rate of SkMs and further the efficacy of the transplantation Initial reports showed that when transplanted into muscle tissue, massive numbers of the transplanted SkMs were rapidly lost in the 48 hrs after implantation (Beauchamp et al., 1994; Huard et al., 1994; Fan et al., 1996; Stuart et al., 2000; Benoit et al., 1997) It was frequently claimed thereafter that a massive donor-cell death limits the success of SkM transplantation Later Suzuki et al also demonstrated the cell signal lost after myoblast transplantation into myocardium (Suzuki et al., 2004) He analyzed the early dynamics of SkMs used a novel dual system He used C14 to label the SkMs, and after transplantation C14 activity was measured to show the left cells from original transplanted SkMs At the same time, he performed sex-mismatched transplantation of male SkMs into a female heart model,

and used a male-specific gene, sry, to assess total SkMs, including both originally

transplanted SkMs and SkMs from proliferation, after transplantation In his study, at

10 minutes after transplantation only 39.2% of grafted cells were left and then the cells decreased steadily till 24 hours Previous studies attributed this massive cell loss

as multifactorial including mechanical injury, maladaptation, anoxic conditions, and theorigin and quality of the SkM preparation (Pouzet et al., 2000; Pavlath et al., 1994;

Qu et al., 2000) as well as host immune and inflammatory reactions (Guerette et al., 1997; et al., 1998)

However, cell death is only one possible cause of cell signal loss It could hardly explain such a fast and massive cell signal loss that even in 10 minutes after transplantation, only around 40% cells were left Other factors including poor cell retention and cell redistribution to other organs probably also contribute to the cell signal loss It is not surprising when cell leakage was observed form the site of injection because of continuous myocardial wall contraction (Muller-Ehmsen et al 2002) When microdepot injection system was used to deliver the same number of SkMs to the infarcted myocardium, a better SkM retention as well as better heart performance was observed as compared to standard technique (Grossman et al., 2002; Harald et al., 2005) Locally injected cells are taken up by the liver, spleen, and lungs providing unequivocal evidence for the existence of the phenomenon of multiorgan redistribution (Aicher et al., 2003) Similarly, different radiolabeled cell distribution was demonstrated after intramyocardial, intracoronary (IC), and interstitial retrograde coronary venous delivery (Hou et al., 2005) It was demonstrated that the majority of delivered cells is not retained in the heart for each delivery modality and significantly more cells were retained after IM injection (11+/-3%) as compared with IC (2.6+/-0.3%, P<0.05) delivery at 1 hour after cell transplantation

Despite all these findings, most studies have shown that the SkMs do survive and continue to proliferate after transplantation into myocardium Concerning the low engraftment rate, many strategies have been applied to enhance the SkM survival after transplantation Pretreatment of SkMs with heat shock (Suzuki et al., Circulation

102, 2000), basic fibroblast growth factor (Kinoshita et al., 1995), administration of hepatocyte growth factor (Tambara et al., 2005), genetic modification to over express

VEGF (Suzuki et al., Circulation 2001), insulin-like growth factor I (Mitchell et al., 2002), and anti-inflammatory strategy using interleukin-1 receptor antagonist (Qu et

al 1998), anti-LFA-1 (Guerette et al., 1997) and anti-IL-1beta antibody (Suzuki et al Circulation 2004) have been shown to stimulate proliferation and enhance survival of SkMs Similarly, anti-immune reactions such as depletion of host C3 complement (Hodgetts et al., 2001), depletion of CD4+, CD8+ cells or NK1.1+ cells (Hodgetts et al., 2000), and immunosuppression with monoclonal antibodies and CTLA4 IgG (Guerette et al., 1997) have also shown the efficacy of increasing myoblast survival

1.3.2.2 Fate of Transplanted SkM: Cardiomyocyte or Skeletal Myofiber

Kao et al hypothesized that SkMs injected into a recently damaged region of the myocardium would differentiate into myocardial cells once exposed to the new environment (Kao et al., 1989) The first experiment of SkM autotransplantation in dog heart muscle at 6-8 weeks showed the formation of striated muscle in the center

of the scar tissue formed by the cryo-damage (Marelli et al 1992) In fact, this was the first observation of a transdifferentiation of the injected SkMs into a cardiac milieu The milieu-dependent differentiation of SkMs into cardiac-like cells was also reported by other investigators (Robinson et al., 1996; Reinecke et al., 2000) However, the vast preponderance of later studies indicated that SkMs injected into ischemic or infarcted myocardium did not differentiate into cardiomyocytes (Murry et al., 1996; Ghostine et al 2002) After transplantation, the cells expressed skeletal myosin heavy chain (MHC)-fast isoform with some cells incorporated BrdU, indicating that some cells were dividing One week later, these cells demonstrated

conversion to slow-twitch fibers by expressing MHC-slow isoform, which was more resistant to the fatigue and more adaptable to cardiac pump function But none of the cells was stained for cardiac specific α-MHC, cardiac troponin I, or atrial natriuretic peptide at any time spot (Murry et al., 1996) Furthermore, Ghostine et al demonstrated that more than 30% of SkMs coexpressed the slow and fast MHC isoforms at 12 months after implantation into a sheep infarction model (Ghostine et al 2002) Apart from differentiation into skeletal muscle, a small portion of SkMs fused with the surrounding myocardium (Reinecke et al., 2004) Our own experience has also shown that transplanted human skeletal myoblasts (hSkMs) fused with the host cardimyocytes and form a mosaic formation (Ye et al., 2005)

Though electromechanical properties after SkM transplantation into myocardium have drawn much attention, yet it is not clear whether and how SkMs electrically integrated into surrounding myocardium In myocardium, gap junction and adhering proteins such as N-cadherin and connexin-43 are responsible for link-up of cardiomyocytes Although gap junctions with concurrent expression of N-cadherin and connexin-43 have been described during the early stages of skeletal muscle development, in adult skeletal muscle, N-cadherin and connexin-43 are down-regulated (Balogh et al., 1993) Therefore grafted mature SkMs should not be expected to link with surrounding myocardium through electromechanical gap junctions However, in 1994, Zibaitis et al reported intercalated discs between transplanted grafts and surrounding myocardium Later, similar finding have been also published by other groups (Chiu et al., 1995; Yoon et al., 1995; Murry et al., 1996; Taylor et al., 1998; Reinecke et al., 2000; Suzuki et al., 2000) On the contrary,

other groups have failed to find the evidence of electromechanical junction (Ghostine

et al., 2002; Leobon et al., 2003) Thus far, most studies indicated that transplanted SkMs do not form gap junctions with host cardiomyoctyes (Atkins et al., 1999; Hutcheson et al., 2000; Scorsin et al., 2000; Pouzet et al., 2000)

1.3.2.3 Efficacy of SkM Transplantation for Cardiac Repair

In 1998, Taylor et al transplanted autologous SkMs into cryoinjured myocardium of rabbits and firstly demonstrated the improved heart performance after SkM transplantation Subsequently, a number of pre-clinical studies have demonstrated that SkMs, transplanted into regions of injured myocardium, not only form stable grafts, but also improve cardiac function Using micromanometry and sonomicrometry, improvement in diastolic compliance and a reduction in diastolic creep was observed after SkM transplantation into cryoinjured rabbit hearts (Atkins et al., 1999) Evaluation of heart function using a Langendorff preparation system after SkM implantation into cardiomyopathic hamsters showed attenuated cardiac remodeling and preservation of global heart performance (Ohno et al., 2003) Using a coronary artery ligation model, Jain and colleagues (2001) assessed in vivo cardiac function by maximum exercise capacity testing after SkM transplantation Control animals demonstrated a gradual decline in exercise capacity, whereas animals that received SkMs did not Using tissue Doppler imaging, Ghostine et al (2002) demonstrated significant improvement in transplanted animals from 4 months after myoblast transplantation and persisting till 1 year

Recently, several preclinical studies have been performed to compare the beneficial efficacy of SkM transplantation with other cell types in cardiac repair (Table 1.4) SkM transplantation was more effective than transplantation of adult cardiomyoctyes, smooth muscle cells, and cardiac fibroblasts (Hutcheson et al., 2000; Ohno et al., 2003; Agbulut et al., 2004) This was of no surprise since SkMs were more regenerative than cardiomyocytes and smooth muscle cells so that they will survive better after grafting Additionally, this also reflects an active impact of myoblasts on cardiac systolic function as compared to a passive effect of fibroblasts on compliance Surprisingly, there was no significant functional difference between SkM transplantation and transplantation of fetal cardiomyocytes (Scorsin et al., 2000) As compared with BMCs, the results are relatively controversial Transplantation of BMCs increased regional systolic heart function to a similar degree of SkMs (Thompson et al., 2003) In this study, a portion of MSCs were shown to differentiate into muscle-like cells within the myocardium More surprising was the similar outcome in animals treated with SkMs or CD133+ stem cells, since SkMs had a direct contractile apparatus to enhance cardiac performance while CD 133+ cells mainly provided vascularization or angiogenesis (Agbulut et al., 2004) By contract, as compared to BM derived mononuclear cells (Ott et al., 2004), SkMs showed superior enhancement regarding ventricular function The controversial data may be caused

by different cell types as grafting into myocardium Of particular interest was the finding that a combined transplantation of bone marrow mononuclear cells with SkMs increased the efficacy of each, suggesting a potential synergistic effect between the two cell types (Ott et al., 2004)

Table 1.4 Experimental Studies Comparing Transplantation Efficacy of SkMs with Other Cell Types in Cardiac Repair

Hutcheson et al

2000

Rabbit Cryoinjury SkM vs Fb SkM superior to Fb, SkM improves systolic and

diastolic function while Fb only improves diastolic function

No difference regarding LV functional improvement

Ohno et al 2003 Hamsters Cardiomyopathy SkM vs smooth

muscle cells

SkM superior to smooth muscle cells in attenuation

of cardiac remodeling and preservation of global heart performance

SkM superior to BM cells regarding LV function, combination of these cells shows synergy