SUPERVISOR: DR HELENA AZEVEDO MOHAMMED SHOWMICK HANNAN

110632760

April 1, 2014

Stem Cell Therapy for Acute Myocardial Infarction

DEN318: Third Year Project

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Stem Cell Therapy for Acute Myocardial Infarction

Degeneration of cardiac cells is a cause of major mortality in the western world without any current cure. Acute myocardial infarction is the biggest form of heart disease, accounting for 561,666 deaths in 2011 in the UK alone. Stem cell therapy for acute myocardial infarction (AMI) is a very promising approach in the cardiovascular field. Stem cells are undifferentiated cells that have the capability of giving rise to indefinitely more cells of the same type or from other kinds of cells by differentiation. Preliminary studies have indicated that stem cells have the capability to develop into functioning myocardial cells.

This report involves the investigation of many clinical trials that have been carried out with different forms of stem cells in order to explore the effects they have on the recovery of ischaemic heart disease through cell therapy. Main findings of this report indicated that: all stem cell types have shown the ability to differentiate into cardiomyocytes; optimal delivery of cells must be further investigated as the amount of cells reaching the host tissue is very small at less than 1%; skeletal myoblasts and bone marrow stem cells have both shown potential to be the ‘ideal cell’ and paracrine factors have a significant effect upon ventricular remodeling.

It is very difficult to claim a cell type is the ‘ideal cell’, as this is ultimately dependent upon the injury to be treated and the conditions upon transplantation. In conditions where reperfusion is the primary objective, the angiogenic potential of cells is of high priority; therefore the ideal cell in this condition will be bone marrow stem cells. However, in conditions such as the end-stage ischemic heart failure, priority is to transplant cells that are high in contractile potential. In such conditions, the ideal cell would be skeletal myoblasts.

Advanced studies are required to further explain the mechanisms that occur in stem cell therapy and which is the most effective. Large-scale clinical trials that are also randomised must be carried out in order to find out the optimum stem cell type, delivery method, number of cells to transplant and the time of delivery.

Abstract

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Table of Contents Nomenclature ........................................................................................................... 4

Introduction ............................................................................................................. 5 Historical Perspective of Heart Treatment ...................................................................... 5 The Failing Heart .............................................................................................................. 6 Acute Myocardial Infarction ........................................................................................... 7 Heart Transplant .............................................................................................................. 7 Mechanisms of Stem Cell Action in Human Heart .......................................................... 7 Potential of Stem Cells ...................................................................................................... 8

Stem Cell Types used for Transplantation .............................................................. 10 Embryonic Stem Cells ........................................................................................... 10

Adult Bone Marrow-Derived Stem Cells ....................................................................... 11 Mesenchymal Stem Cells ............................................................................................ 11 Endothelial Progenitor Cells ........................................................................................ 11 Haematopoietic Stem Cells ......................................................................................... 12

Skeletal Myoblasts .......................................................................................................... 12 Fetal Cardiomyocytes ..................................................................................................... 12 Cardiac Derived Stem Cells ............................................................................................ 13

Methods for Stem Cell Delivery ............................................................................. 14 Intramyocardial Injection ....................................................................................... 15 Intracoronary Transplantation ................................................................................. 15 Transendocardial Injection ...................................................................................... 16 Retrograde Administration ..................................................................................... 16

Stem Cell Therapy Mechanisms .............................................................................. 17

Methods .................................................................................................................. 18

Clinical Results and Indications ............................................................................. 20

Discussion ............................................................................................................... 21 Safety and Efficacy of Stem Cell Therapy ....................................................................... 27 The Ideal Cell .................................................................................................................. 28 Future Perspectives ........................................................................................................ 29

Conclusions ............................................................................................................. 30

References ............................................................................................................... 31

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N o m e n c l a t u r e

BMCs Bone Marrow Stem Cells

CABG Coronary Artery Bypass Graft

CDCs Cardiac Derived Stem Cells

CHD Coronary Heart Disease

CMCs Cardiomyocytes

CVD Cardiovascular Disease

ECM Extracellular Matrix

EPCs Endothelial Progenitor Cells

ESCs Embryonic Stem Cells

FCMCs Fetal Cardiomyocytes

HSCs Haematopoietic Stem Cells

ICD Implantable Cardioverter Defibrillator

IISC Intracoronary and Intramyocardial stem cell therapy

LVEF Left Ventricular Ejection Fraction

LVESV Left Ventricular End Systolic Volume

MRI Magnetic Resonance Imaging

MSCs Mesenchymal Stem Cells

PET Positron Emission Tomography

PTCA Percutaneous Transluminal Coronary Angioplasty

SCID Severe Combined Immunodeficiency

SkMB Skeletal Myoblast

SVI Stroke Volume Index

UPC Unmodified Primary Cells

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I n t r o d u c t i o nHeart failure is a leading cause for morbidity and mortality that affects a rapidly growing population of patients. It is estimated that over 23 million people worldwide suffer from heart failure, yet the question arises why treatments are not in place to tackle this worldwide issue (Vincenzo Lionetti C. V., 2013). As seen in figure 1, heart failure secondary to cardiovascular disease, is one of the leading causes of mortality. Over the last few decades, cardiac cell therapy has been studied and investigated in pursuit of a solution that would allow the regeneration of lost myocardium. Vincenzo Lionetti states, “heart failure has no cure”, a statement which currently stands true. As invasive approaches are associated with high risk, a less invasive therapy has come forward in the form of regenerative medicine that aims to find the solution of something that has no clear, successful treatment.

Figure 1: UK deaths by cause in Men (left) and in women (right) (N Townsend, 2012).

H i s t o r i c a l P e r s p e c t i v e o f H e a r t T r e a t m e n t In 2002, Dr. Milton Packer, a world-renowned expert in the field of cardiology, published an editorial with the title “The impossible task of developing a new treatment for heart failure,” (Packer, 2002). Historically, it seems that Dr. Packer can justify such a bold statement, with the development of over 1,000 new drugs and devices being introduced in the pursuit to overcome heart failure over the past 20 years without success (Vincenzo Lionetti F. A., 2010). Dr. Packer goes on to state, “Given these nearly impossible hurdles to the successful development of a new drug for heart failure, some investigators appear to have simply given up on pharmacologic interventions altogether… Understandably frustrated by recent experiences, such investigators have turned to the development of devices for the management of heart failure,” (Packer, 2002).

Dr. Packer highlights a big flaw within the treatment for heart failure, where it seems that conventional cell therapy has fallen behind the progressive development of devices that manage heart failure. Advancement of devices such as the pacemaker and implantable cardioverter

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defibrillators (ICD) that can be implanted in the chest of a patient and monitor the heart’s rhythm has taken the limelight in recent times. However, Dr. Packer failed to mention two state-of-the-art non-pharmacological approaches; stem cell therapy and cardiac gene therapy. Cardiac gene therapy was a massive break through for heart failure treatment and enjoyed its peak in 2002, but stem cell therapy has gained a great amount of interest in recent times and could be argued as the potential solution for heart failure.

T h e F a i l i n g H e a r t Cardiovascular disease (CVD) has been the biggest killer in the UK for many years. In 2011 alone, cardiovascular disease was responsible for 561,666 deaths in UK. Of these types of CVD, coronary heart disease (CHD) is the major cause of heart failure in humans. A primary defect in the coronary circulation initiates an anatomical condition known as ischaemic cardiomyopathy; this causes the loss of myocyte cells and ultimately ventricular failure (N Townsend, 2012).

Research has made it quite clear that the progressive steps towards heart failure is as a result of qualitative and quantitative changes to cardiomyocytes and extracellular matrix (ECM), where results have indicated that cardiac hypofunction is directly influenced by the number of cardiomyocytes within the heart, which ultimately affects the contractile power of the heart (Takemura, 2013).

Over the past few decades, many forms of treatment for heart disease have come to light, but currently stem cell therapy is grabbing attention of researchers in the cardiovascular field, but can this treatment be a complete solution? It is doubtful that a treatment for such a complex disease such as heart failure could come in the form of “one target fits all”, however as will be mentioned in this report, many studies show great potential of research heading towards this direction.

Table 1: Deaths by cause, sex and age (N Townsend, 2012).

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Generally, stem cells fall under four features: (1) plasticity; (2) multipotent differentiation ability; (3) being cultured ex vivo; and (4) self-renewal capacity, (Qi Zhang, 2005). Stem cells are expressed by their capacity for consistent self-renewal (proliferation) and their ability to increase functionality of a cell (differentiation). Their cell surface markers also define stem cells; this may include marker proteins such as CD34 and CD133, which induce vascular endothelial growth into sites of neovascularization (Wollert, 2008).

A c u t e M y o c a r d i a l I n f a r c t i o n Acute myocardial infarction (AMI) is characterised by scar formation, ventricular remodeling and the irreversible loss of cardiomyocytes which leads to functional deterioration (Tycho I.G. van der Spoel, 2011). The number of cardiomyocytes present in the heart is proportional to the contractile power of the heart (Takemura, 2013).

AMI causes the extracellular matrix (ECM) to become dysfunctional. In the ECM, the primary matrix component consists of fibrillar collagen network; this is essentially responsible for the alignment, myocyte shape and the transduction of cell shortening which results in effective ventricular ejection. AMI causes impairment of ECM, resulting in the collagen structure being damaged causing ventricular dilation (Fedak, 2008).

H e a r t T r a n s p l a n t In recent years, heart transplant has become a possible form of therapy for patients who suffer from complete heart failure. It is very intriguing, as a complete and functioning heart can be created from stem cells in an external environment, however this therapy faces severe limitations. There is poor availability of donor organs, high chance of immune rejection, infectious complications can occur as well as physical, rheologic and thrombotic issues (Qi Zhang, 2006). These severe limitations have led to investigative research in an alternative therapy, which could possibly find a solution to the biggest killer in the UK. A possible therapy that is being developed is cell transplantation which aims to regenerate damaged cardiac myocytes by transplanting cells of various types in order to promote vascularization in damaged tissue in the heart.

M e c h a n i s m s o f S t e m C e l l A c t i o n i n H u m a n H e a r t Cell transplantation for heart disease requires the following three steps; isolation of target cells, expansion, and implantation of the stem cell population into the injured myocardium (Fedak, 2008).

According to studies carried out by Strauer et al, four types of mechanisms exist within stem cell action in a diseased heart:

1. Process of bone marrow cells transdifferentiating into cardiac myocytes. 2. Cytokine factors inducing myocyte growth resulting in an increase of residual viable

myocytes. 3. Stimulation of intrinsic myocardial stem cells. 4. Stimulation of cell fusion between the resident myocytes and transplanted bone

marrow cells (Bodo-Eckehard Strauer M. G., 2011).

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Currently, one of the principal focuses of stem cell transplantation is on cellular and mechanical mechanisms which cause reversal maladaptive structural remodeling of the damaged myocardium, beneficial effects of cell therapy and the limitation factors of progressing heart failure (Fedak, 2008). These factors will be critically analysed and it is upon these factors that will allow stem cell therapy to reach an optimum viable option.

P o t e n t i a l o f S t e m C e l l s

Figure 2: Potential applications of stem cells (Qi Zhang, 2006).

As shown in figure 2, injured cardiac tissue can be regenerated with many types of pluripotent stem cell transplantation that are able to differentiate into functioning cardiomyocytes, smooth muscle cells and endothelial cells (Qi Zhang, 2006). Until a few years ago, heart failure was seen to be a problem without a solution, however the potential of stem cell therapy shows that the sequence of tissue damage leading to deterioration and eventually heart failure can be overcome by several candidate cells that include: endothelial progenitor cells, mesenchymal cells and cardiac derived cells. Despite the great potential of stem cell therapy, this form of therapy is still being optimized as there are many challenges remaining to resolve, such as: limited methods of delivery, uncertainty of optimal stem cell type, number of cells to transplant and little knowledge of homing. These factors also illustrated in figure 4, produce large obstacles to this therapy being a standardised clinical procedure (Vincenzo Lionetti F. A., 2010) (Fedak, 2008).

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Figure 3: The sequence of cardiac regeneration which could prevent tissue loss to heart failure (Vincenzo Lionetti F. A., 2010).

Cardiomyoplasty is a procedure that replaces deteriorating CMCs by supplying damaged areas with functional CMCs through cell-based therapies. Figure 3 illustrates the process of cell therapy that is able to improve vascular supply of the heart whilst augmenting the contracting function of the myocardium (Sheing-Tsung Kuo, 2009). The goals of cell therapy are revascularization and myocardial regeneration. This therapy ultimately aims to achieve improvement of cardiac function by improving the synchronous contractility and bioelectrical conductivity (Tycho I.G. van der Spoel, 2011). On the whole, cell therapy is making huge strides in the cardiovascular field and has massive potential to be ‘the’ treatment. A statement that is constantly heard in the cardiology community is one by Dr. Victor Dzau: “Cell and molecular therapies will soon enable the repair and regeneration of myocardium after myocardial infarction,” (VJ Dzau, 2004). It is such statements that give rise to this form of treatment, where 10 years down the line, evidence proves this to be quite accurate.

In this report, we will summarise the classifications of stem cell types and critically analyse what the ‘ideal’ stem cell is for stem cell therapy. We will then review the literature in which type of cell delivery is the most efficient according to the factors: potential of survival, growth, differentiation, integration into the host tissue and delivery methods. Finally, we will discuss current research and studies that have been conducted and consider future prospects for stem cell treatment in heart failure.

Figure 4: Cell therapy – regeneration vs. repair. Infarcted and remodeled myocardium lack in contractile elements, physiological excitability and tissue perfusion. Repair of injured myocardium rely on myogenesis to replace CMCs (red), vasculogenesis to regenerate perfusion (green) and newly formed CMCs by electrical reintegration (blue). Injured myocardium lack in these (Harald C. Ott, 2005).

S t e m C e l l T y p e s u s e d f o r T r a n s p l a n t a t i o n Regardless of their source, all stem cells have three characteristics in common: (1) The ability to differentiate into one or more cell type, (2) self-renewing ability to divide indefinitely, (3) to be able to produce an exact duplicate. There are many types of stem and progenitor cells that have been studied in cellular cardiomyoplasty. Many factors affect the suitability and effectiveness of the cells including: clinical ability, proliferating ability, differentiation and ethical considerations. Table 2 displays key properties of: availability, differentiation immunogenicity and oncogenicity, that determine how effective a type of stem cell is.

E m b r y o n i c S t e m C e l l s Embryonic stem cells (ESCs) are one of the classifications of stem cell origins. They are derived from the inner layer mass of the blastocyst. ESCs have been researched into very deeply due to it’s excellent differentiating ability, being able to differentiate into more than 200 different types of cells as well as tissues and organs. ESCs are harvested from 3 different sources: discarded embryos, cadaveric stem cells and research embryos. Although studies have shown that ESCs have the ability to differentiate into every tissue type for prolonged periods in culture, a major barrier of ethical issues has halted this research where many have argued against the process that includes the destruction of an embryo, (Qi Zhang, 2006).

In September 2001, the US government legalized the procedure for researchers to experiment with ESCs cells. This legalisation has been justified to an extent with animal studies carried out by Kehat et al demonstrating that ESCs can differentiate into cardiomyocytes in vitro (Kehat I, 2001). This has put forward that ESCs can actually be used as a form of therapeutic treatment. Alternatively, studies have shown that transplanting ESCs could cause problems. Thomas et al found the formation of teratomas upon transplanted ESCs, this included tumors that included a mix of undifferentiated totipotent cells in severe combined immunodeficiency (SCID) (Qi Zhang, 2006).

Table 2: Comparison of biological properties: Availability: present and ready for use; Differentiation potential: capacity of a cell to differentiate; Immunogenicity; the property of eliciting an immune response; and Oncogenicity; the likelihood to give rise to tumours.

Cell Type Availability Differentiation Potential

Immunogenicity Oncogenicity

Bone Marrow

Stem Cells

High Limited High Low

Skeletal Myoblasts

High Limited High Low

Embryonic Stem Cells

Limited High Low High

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A d u l t B o n e M a r r o w - D e r i v e d S t e m C e l l s Bone marrow-derived stem cells are the most popular and currently the most used cells in cell transplantation therapy. Adult bone marrow comprises of a number of different cell types that have proliferative capacity as well as the ability to differentiate into non-haematopoietic tissues. These include: haematopoietic, mesenchymal and stromal stem cells. These types of cells have the potential to make progenitor cells into cardiomyocytes whilst avoiding the ethical issues that embryonic stem cells face (Harald C. Ott, 2005).

M e s e n c h y m a l S t e m C e l l s Mesenchymal stem cells (MSCs), also known as bone marrow stromal cells are rare multipotent progenitor cells that have shown to have the ability to differentiate under specific culture conditions into osteocytes, chondrocytes, adipocytes and cardiomyocyte-like cells. A prerequisite to using MSCs in cell transplantation therapy is the delivery of the cells have to occur at an early stage of disease, as studies have shown that MSCs can only differentiate into CMCs whilst they are in contact with native CMCs (Harald C. Ott, 2005).

Transplanted MSCs have shown to have positive effects on the functionality of the heart, where it has been recorded that there is an improvement in increased neovascularization and improved regional contractility. Further studies have shown that MSCs enhance regional wall motion as well as preventing adverse remodeling (Wollert, 2008). However, it has also been reported that this cell type can cause micro-infarcts and promote damage of myocardium.

This cell type shows great promise in allogenic administration in vivo with the potential of this being developed as an off-the-shelf product. With very few negative issues being related to this cell, clinical studies have started in whether it would be possible to administer this allogenically.

E n d o t h e l i a l P r o g e n i t o r C e l l s Endothelial progenitor cells (EPCs) are defined by their ability to incorporate into neovascularization sites as well as their ability to differentiate into endothelial cells in situ (Wollert, 2008). Their cell surface expression contain the haematopoietic marker proteins CD133 and CD34 as well as the endothelial growth factor receptor-2. CD133 and EPCs have shown to collaborate in order to increase vascularization in ischaemic tissue. The release of paracrine factors has shown to have an effect on the EPC-capacity as it can mediate angiogenic effects. However, the functional capacity of the cells can be affected by many other factors such as the patient’s age, disease state and gender.

Studies have shown that the population of EPCs within a patient suffering from acute myocardial infarction increases, showing that EPCs tend to migrate towards ischaemic damage in the heart and induce neovascular repair. EPCs play a big role in the maintenance of vascular health as studies have found that the number of EPCs decrease with age, also increased risk of coronary artery disease affects the ability of EPCs to migrate towards damaged regions (Harald C. Ott, 2005).

It is clear that EPCs are critical in ischaemic cardiovascular events, but whether transplanting EPCs would have a large effect upon the recovery of damaged regions is still a question, where further study would be required.

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H a e m a t o p o i e t i c S t e m C e l l s Previously, haematopoietic stem cells (HSCs) were seen as cells that could only differentiate into red and white blood cells, however recent studies have shown that under strict laboratory conditions, HSCs have the ability to differentiate into many cell types including cardiomyocytes. Further research has been hindered, as HSCs have shown no potential in transdifferentiating into CMCs when transplanted into the infarcted myocardium.

A study carried out in mice revealed through the intramyocardial injection of HSCs, myocardium was regenerated. The delivered cells induced the production of myocytes, smooth muscle cells and endothelial cells. Furthermore, a gap junction protein which is usually present in CMCs called connexin 43 was expressed by the cells displaying a functional relationship between HSCs transplanted cells and host cells. However, a further study opposed these results where they found HMCs do not possess the phenotype required to produce CMCs but post transplant, develop into HMCs, which is the question that has been raised in terms of the long term prospects for this cell type (Sara D. Collins, 2007).

S k e l e t a l M y o b l a s t s Before cell therapy as a treatment for heart disease came into existence, skeletal myoblasts (SkMB) were being used to support the injured heart from as early as 1987 in dynamic cardiomyoplasty. This treatment has progressed rapidly since then where 𝐶!𝐶!! skeletal myoblast cell lines are implemented on a cellular level and have been successfully transplanted into normal, uninjured mouse hearts (Harald C. Ott, 2005).

Skeletal myoblasts, also known as satellite cells, are usually harvested from the patient, cultured and injected straight into the myocardium (Sara D. Collins, 2007). SkMB have an autologous cell origin, therefore accessibility is easy as it can be obtained from the patients themselves hence avoids immune rejection as well as ethical issues. It is safeguarded from tumour formation as it has a myogenic lineage restriction as well as high resistance to ischaemia (Menasche, 2011).

Benefits in transplanting SkMB cells are its resistance to ischaemia as well as its excellent proliferating ability. Conversely, a drawback for this cell is the arrhythmogenic potential where studies have shown that patients suffer from a high rate of arrhythmia upon transplantation. Animal studies have proved that SkMB can effectively home to the infarcted region of the heart, where striated muscle fibers were formed. Atkins et al found an enhancement in diastolic function and systolic function after myoblast transplantation. Additionally, Hagege et al found SkMB had high anti-ischaemic ability that increases the chances of survivability and post transplantation the ability to differentiate into myocyte-like cells (Qi Zhang, 2006).

F e t a l C a r d i o m y o c y t e s Given that the adult heart has a low regenerative capacity, fetal cardiomyocytes (FCMCs) have the prospect to be an ideal cell type due to its regenerative ability. Soopna et al carried out successful cell transplantation in mice where isolated FCMCs were injected into adult myocardium. A study carried out by Li et al showed the formation of new blood vessels around cell graft area indicating an improvement of heart function.

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Further studies have shown that FCMCs can be successful for long-term treatment; where cells have survived for 6 months and also displaying less dyskinesis in the infarcted region as well as improved ejection fractions (Sara D. Collins, 2007). However, a big ethical issue arises through the use of human fetal tissue; also lifelong immunosuppressive therapy may be required to avoid rejection (Qi Zhang, 2006).

C a r d i a c D e r i v e d S t e m C e l l s The existence of cardiac stem cells (CDCs) in the human heart has been questioned however in recent studies carried out, it has been found that stem and progenitor cells exist in the human heart. CDCs have shown great potential for cardiac repair as it has been suggested that these cells can be cloned in an autologous environment (Wollert, 2008). CDCs are derived from neonatal hearts through the transcription factor let-1 (isl1). CDCs are an attractive cell type as they have the ability to differentiate into functional cardiomyocytes (Harald C. Ott, 2005).

A reperfusion model carried out by Bradfute et al has shown that CDCs injected by an intravascular route would home to the damaged myocardium (Sara D. Collins, 2007). Postmortem analysis also found the existence of cardiac and myocyte progenitors highlighting the potential of this type of cell being used for cardiac repair.

Investigators have examined the level of plasticity of CDCs, i.e. the ability of a stem cell to differentiate into a specialised cell type of tissue, where they found that the plasticity of CDCs is close to the microenvironment of the heart. This ability can be further enhanced through the inclusion of growth factors that improves the communication pathways of cells as well as increasing levels of differentiation (Qi Zhang, 2006).

CDCs have a low chance of inducing arrhythmias upon transplantation in native cardiac cells, therefore ethical issues do not hinder this cell as a potential ideal stem cell. However, the differentiation level of this cell is still unknown, although CDCs have shown differentiating ability through the method of harvesting, expansion and in vitro growth, it is still unknown whether the functional capacity for cell proliferation and differentiation can be maintained within the infarct scar. Furthermore, CDCs have been found in large numbers only in neonatal human hearts and mammalian hearts, taking into consideration that the cell population rapidly declines with age and health, the long-term prospects of this type of stem cell is put into doubt (Alexander Lyon, 2007).

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M e t h o d s f o r S t e m C e l l D e l i v e r y There are currently various methods of delivery of stem cells that have been investigated. These include: endomyocardial, intracoronary, intramyocardial, retrograde coronary venous and transvenous intramyocardial (George, 2010). However, the optimum approach is not yet known.

Figure 5: Depicted and clinically used methods for myocardial cell delivery: (A) Intracoronary and (B) Intramyocardial transplantation methods for heart disease (Bodo-Eckehard Strauer G. S., 2011).

How the cells are delivered to the heart remains to be one of the most crucial factors of homing the stem cells to the infarcted cardiac ischaemic tissue. When stem cells are injected intravenously, a very small percentage of cells are able to reach the infarcted zone, assuming that the normal coronary blood flow per minute will be at 80ml/min/100 g intravenous weight, the amount of 160ml per left ventricle will flow per minute. This accounts for only 3% of cardiac output (assuming a cardiac output of 5,000 ml/min) (Bodo-Eckehard Strauer M. G., 2011). Therefore, for an intravenous approach, several circulation passages would be required to ensure that the stem cells would be in contact with the infarct-related artery. Homing of stem cells is a process that must be considered in terms of efficiency, many delivery routes are mentioned below as well as being depicted in figure 5.

Delivery of the engrafted cell as shown in figure 6, dramatically improves the cardiac structure and function of the damaged extracellular matrix. This diagram shows the composition of damaged ECM that is disrupted, in comparison to ECM that is restored after implantation of stem cells.

Figure 6: Diagram showing donor cell engraftment into damaged myocardium. The cardiac structure and function dramatically improves upon rebuilding damaged ECM. Matrix homeostasis of the failing heart is influenced by the release of paracrine factors and endogenous matrix protease inhibitors (TIMPs) from transplanted cells which results in the prevention of further ECM degradation as well as limiting apoptosis in cardiomyocytes (Fedak, 2008).

I n t r a m y o c a r d i a l I n j e c t i o n Epicardial intramyocardial application is a surgical procedure that is performed on well-exposed ischaemic areas, where a thin needle is used to carry out multiple injections around the infarct area. This method has been used successfully alongside coronary artery bypass graft therapy (CABG), where it has shown that this method overcomes many problems such as migration and insufficient vascularisation. This minimal invasive procedure increases the chances of homing the infused cells in the infarct area through high stem cell persistence in heart muscle (Kaminski A, 2008). Studies carried out by Klein et al have shown that this method improves patient’s myocardial perfusion and has also shown to reduce perioperative risks with stem cell surgical procedures (Klein HM, 2007).

I n t r a c o r o n a r y T r a n s p l a n t a t i o n Intracoronary cell administration is advantageous for cardiac tissue repair as it is able to supply the entire heart. It is better suited for the delivery of cells in a specific coronary territory (Emerson C. Perin, 2003). This is carried out by firstly reopening the occluded coronary artery that allows cells to flow through the infarcted and peri-infarct passages. Currently, this method is being developed clinically where a selective intracoronary delivery route is established that minimises cell loss through extraction towards organs of secondary interest by using the first pass-like effect. To assist migration of the cells into the infarct zone, cells are inserted through a pressure injection alongside ischaemic pre-conditioning.

This method places a balloon catheter-induced ischaemia in the infarct-related artery as shown in figure 6 and percutaneous transluminal coronary angioplasty (PTCA) is executed. During this infarct vessel occlusion, stem cells are infused intracoronarily by the balloon catheter, by 4 fractional high-pressure infusions of 5 ml cell suspensions, each suspension contains 6-10 million

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mononuclear cells (Bodo-Eckehard Strauer M. G., 2011). The key benefit to this delivery method is that it prevents backflow of cells as well encouraging the migration of cells in the infarcted zone by creating a stop flow beyond the balloon site. This stop flow allows additional contact time for the cells to migrate, where other methods are not able to prevent the washing away of cells. Studies have investigated the effects of stem cell specific adhesion molecules, where in this case is crucial to the effectiveness of cell homing.

T r a n s e n d o c a r d i a l I n j e c t i o n This interventional delivery route transplants the stem cells by using the transendocardial catheter injection; this is placed across the aortic valve and towards the targeted damaged area. This method is similar to the intramyocardial approach however it is less invasive. Clinical studies have shown that this method has excellent safety and feasibility, on the other hand, this method has also shown to be technically demanding due to the orientations being carried out via electromechanical mapping, as there are chances of wrong injection sites, cell loss and cardiac tamponade can happen (Bodo-Eckehard Strauer M. G., 2011). Nevertheless, this method also allows a greater accuracy for targeting as the electro mapping allows tissue of different degrees of viability to be seen in greater detail (Emerson C. Perin, 2003).

R e t r o g r a d e A d m i n i s t r a t i o n Amongst the more popular cell delivery techniques include: intramyocardial administration or transendocardial administration, however these forms of delivery suffer some drawbacks. Intramyocardial delivery is not suitable for patients with thinned myocardium hence this delivery method may cause perforation, where as transendocardial delivery of cells has an element of complexity as it involves electromagnetic mapping using NOGA devices. Retrograde administration involves temporary occlusion of the coronary circulation; this is done by the use of a balloon catheter with the administration being included against the outflowing blood (Leo Bockeria, 2013). This allows the solution to enter through the post-capillary venules into the myocardium. One could question why this particular route is chosen, this is due to the post-capillary venules having the smallest vessel diameter in comparison to arterioles or capillaries hence allows the largest transfer of material.

The coronary sinus plays a big role in cardiac contraction where it drains the anterior left ventricular wall. This form of administration into the coronary sinus is popular due the solution having the ability to administer cardioplegia throughout the myocardium (Leo Bockeria, 2013). Clinical studies have been carried out using retrograde administration where Suzuki et al carried out cell therapy using skeletal muscle progenitor cells in a rat infarct model (K, 2004). After 28 days, Suzuki et al reported an increase in cardiac function, high retention of administered stem cells and a decrease in fibrosis. George et al carried out a similar study where he reported a double in cell retention whilst carrying out a 90-minute occlusion ischaemia model (JC, 2008). Presently, there has only been one clinical trial using retrograde administration for cell therapy, Tuma et al carried this out. The trial included 14 patients whom suffered from chronic refractory angina using autologous bone marrow mononuclear cells. This study was very successful, cell delivery was effective in all patients with no complications recorded, furthermore the median base-line area of ischaemic myocardium reduced from 38.2% to 26.5% in the first year and to 23.5% in the second

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year (Tuma, 2011). As illustrated in figure 7, implantation of stem cells through cell therapy significantly increases the contractibility of the heart muscle.

Figure 7: Left: Postero-lateral view of electromechanical linear local shortening map. This shows severe cardiomyopathy as the red color indicates exceedingly low contractibility. Right: Similar map showing 4-month follow-up from cell therapy, green, blue, purple areas show dramatic increase of contractibility after stem cell injection (Emerson C. Perin, 2003).

S t e m C e l l T h e r a p y M e c h a n i s m s Numerous mechanisms have shown the beneficial effects of stem cell therapy, these include: cell fusion, transdifferentiation and paracrine effect.

Cell Fusion: Cell content is transferred from the fusion of host and transplanted cells including genetic material from the host cell (Sara D. Collins, 2007). This mechanism has given positive evidence for cell therapy as studies in an in vivo model demonstrated that BMCs were able to fuse with cardiac myocytes which induced tissue repair, (Sheing-Tsung Kuo, 2009). However, it is not possible for this mechanism to account for complete tissue regeneration due to its low frequency in co-cultured systems, which is estimated at 1:10000 cells, and less than 1% of cardiac myocytes in transplant models (QL Ying, 2002).

Transdifferentiation: This refers to differentiated stem cells adapting the cell phenotype of another cell lineage. This mechanism is slightly controversial with results showing benefits as well as drawbacks reported. Makino et al reported that bone marrow derived-stromal cells transformed into functional CMCs in vitro (S Makino, 1999). However, results reported in an animal model carried out by Murray et al showed that there is no clear evidence of transdifferentiation occurring (CE Murray, 2004).

Paracrine Effect: Proliferation and differentiation of host tissue is stimulated through the release of cytokines and growth factors from transplanted cells (Sara D. Collins, 2007). Myocardial repair is a complex process where cytokines act as mediators in the repair process. Heart failure is caused when the self-repairing capacity of tissue is outweighed by the tissue injury. Examples of growth factors that are secreted due to damage include: vascular endothelial growth factor (VEGF), stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF).

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This mechanism is seen to be more beneficial in comparison to the others mentioned, a study carried out by Iso et al showed that cell death can be minimized by growth factors secreted by bone marrow stromal cells. Figure 8, illustrates the process of cell transplantation and what effect paracrine factors have on cells (Y Iso, 2007).

Figure 8: Working hypothesis of stem cell transplantation. This figure indicates stem cells having a positive impact upon vascularization and myocyte formation. The contribution of transdifferentiation or cell fusion vs. paracrine effect is dependent upon the type of stem cell (Wollert, 2008).

M e t h o d s

Eligibility Criteria Models for acute myocardial infarction and ischaemic cardiomyopathy were screened. Strict criteria of each study being randomly controlled and the number of patients being greater than ten was implemented. The results of stem cell therapy were analysed according to the effect upon left ventricular ejection fraction (LVEF).

Search Strategy A ‘Science Direct’ search was carried out (January 1990 - March 2014) using the following terms: ‘(stem cells OR bone marrow cells OR myoblasts OR embryonic stem cells OR progenitor cells OR cardiac stem cells OR cardiac derived cells) AND (acute myocardial infarction OR heart disease OR heart failure OR coronary artery disease) AND (cell delivery OR

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cell therapy)’. The reports included were published and in English only. Each study was carefully reviewed so that no duplicate information would be included.

Data Abstraction Information was obtained from fully qualified studies which included the following characteristics: end-diastolic volume (EDV), end-systolic volume (ESV), mortality and left ventricular ejection fraction (LVEF). Data was also summarised and presented in a table format to illustrate findings and relationships.

Data Analysis The primary form of analysis in this review was the difference in left ventricular ejection fraction (reported in %) within a treated group in comparison to the control group. Secondary endpoints for analysis included the difference in EDV and ESV (reported as volume in millilitre) in a treated group in comparison to the control group.

Figure 9: Flowchart of trial search and selection for the pooled analysis.

278 articles identified by search

Search was carried out between the years

1990-2014

80 articles viewed in detail

Articles irrelevant to AMI excluded

15 studies included Studied in detail relevant to AMI

180 articles reviewed

Articles irrelevant to stem cell thearpy and

heart disease excluded

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C l i n i c a l R e s u l t s a n d I n d i c a t i o n s Table 3: Trials of several key clinical trials of stem cell therapy in acute myocardial infarction.

Authors/Studies Year n* Cell Application after AMI

Application Cell Type

Method Results

Li et al 1996 - - Fetal Cardiomyocytes

- Improvement in cardiac structure

and function

Boli et al 2011 14 4 months Autologous c-kit-positive, lineage-negative cardiac stem cells with coronary artery

bypass

- Increased LV ejection at 4

months

Hu et al 2011 31, randomised,

placebo-controlled

trial

6 months Coronary artery bypass graft and bone marrow

mononuclear cells

Graft vessel LV systolic function improved compared to the placebo group

Dib et al 2003 11 - Myoblast Cell transplantation

and CABG

Mean ejection improvement

from 22.7% to 35.9%

Strauer et al 2002 10 7 days BMCs Intracoronary Decreased in infarction region and LVESV and increase in SVI

Assmus et al TOPCARE -

AMI

2002 59 4-6 days BMCs and Circulating

Progenitor cells

Intracoronary Increase in LVEF and LVESV

Wollert et al (BOOST)

2004 30, randomised

5-7 days BMCs and coronary angioplasty

Intracoronary Improvement in LVEF +13% and

ESV – 2%

MAGIC 2004 97 2 days BMCs and G-CSF stimulating factor

Intracoronary Improvement in systolic function, increased rate of

in-stent restenosis

Menasch et al 2003 10 - Skeletal Myoblasts Transepicardial Improvement in LVEF and regional

thickening

STAR 2003

391 8.5±3.2 yrs

BMCs

Intracoronary

EF + 6.7%, ESV – 18ml, infarct

size – 4.5

* n - Number of patients involved in the clinical trial.

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D i s c u s s i o n This analysis involves the investigation of many clinical trials that have been carried out with different forms of stem cells in order to explore the effects they have on the recovery of ischaemic heart disease through cell therapy. The main findings are: (1) All stem cell types have shown the ability to differentiate into cardiomyocytes. (2) A vital factor that must be addressed is the optimal delivery of cells. (3) Skeletal myoblasts and bone marrow stem cells have shown angiogenic and myogenic potential in vitro to be a candidate for ideal stem cell. (4) Paracrine factors have a significant effect upon ventricular remodeling.

A vital point to be discussed regarding cell therapy is whether the transplanted cells directly affect the stimulation of newly formed CMCs. The number of transplanted cells that reach the host tissue is very small, sometimes less than 1%. This gives rise to the question that is it the transplanted cells which gives rise to cardiac remodeling or is it their initial presence which causes remodeling? This phenomenon contradicts the belief of cell therapy indicating the beneficial effects could be due to newly expressed bioactive mediators promoted by cell engraftment (George, 2010). A small number such as the 1% remaining in the host tissue could have a big effect upon the simulation of key mechanisms such as the ‘paracrine effect’.

Transplanted cells have the ability to secrete bioactive growth factors in a paracrine approach (Fig 12). Angiogenic biopeptides such as vascular endothelial growth factor (VGEF) and fibroblast growth factor (FGF) are released by transplanted cells which stimulate neovascularization (Fig 10). A study carried out by Yau et al has indicated that the release of paracrine factors initiates a sequence of events, Yau reported an up-regulation of the cell-surface receptor for VGEF in the border zone of fracture in CMCs (TM Yau, 2005). The stimulation of this led to the stimulation of the growth factor IGF-1. This indicates growth factors stimulating the secretion of other growth factors, a sequence of events which leads to multiple signaling pathways leading to beneficial effects of ventricular remodeling (Fig 11). These signals are able to develop a new blood supply to the damaged regions of the heart (Fedak, 2008).

Figure 10: Paracrine mechanisms limiting maladaptive ventricular remodeling (Fedak, 2008).

Figure 11: Effects of paracrine factors releasing growth factors that induce CMCs into cardiac regions and infarct zones (Vincenzo Lionetti C. V., 2013).

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Figure 12: Ventricular remodeling is influenced by cell transplantation through paracrine signaling (Fedak, 2008).

ESCs are termed pluripotent as they have the ability to differentiate into all three embryonic germ layers. Cardiomyocytes are obtained from all three types namely: embryonic stem (ES), embryonic germ (EG) and embryonic carcinoma (EC) cells (Eltyeb Abdelwahid, 2011). This section will focus on human ESCs (hESCs) due to their excellent differentiating ability.

ESCs have shown to have the ability to differentiate into cardiomyocytes due to its versatility, where hESCs have been differentiated into endothelial cells, cardiac tissue and pancreatic cells (Levenberg S, 2002).

Figure 13: hESC propagation and in vitro differentiation into cardiomyocytes. hESC can be continuously propagated when grown on the top of MEF feeder layer. hESC being plated onto gelatin coated plates after 2 weeks shows spontaneous contracting areas (Eltyeb Abdelwahid, 2011).

Although hESCs have shown to have the necessary attributes for a potential cell, many obstacles remain unresolved. The issue of whether these differentiated cells can be considered a mature CMC is questionable in accordance to its excitation-contraction coupling. Although studies have shown its electrophysiological properties in individual differentiation methods, this has not been entirely clarified due to minimally standardised procedures (Pekkanen-Matilla M, 2010).

A further limitation to using hESCs include the potential of a teratoma forming, as the implantation of an undifferentiated ESC could lead to the formation of teratomas. This is not a mechanism but a phenomenon that must be investigated further as to what procedure is required to reduce the likelihood of occurrence. Bjorklund et al demonstrated that teratoma formation could be prevented with his study of ESC cells being transplanted into rat brain at low numbers (Bjorklund LM, 2002).

Embryonic stem cells are available in small numbers and face high ethical concerns. Other factors such as regulatory dilemmas and risk of allograft immunologic reactions have allowed us to reach the conclusion that although ESCs have excellent differentiating capability, the limitations outweigh the benefits of this cell, therefore they are not the ideal stem cell type of cell therapy (Paolo Angelini, 2005).

Skeletal myoblasts (SkMB) has shown to be a promising type of cell both experimentally and clinically. Figure 14 shows that they have the ability to differentiate into skeletal muscle fibers within scarred tissue; however a limitation of this cell type is the differentiating ability into CMCs. The first clinical trial for cell therapy involved skeletal myoblasts, Menasch et al carried out an autologous transplantation of myoblasts via epicardial injection and CABG in a patient suffering from heart failure. There was a follow up of 52 months, results showed promising improvements, an increase of ejection fraction from 24.3%±4% to 31%±4.1% (P Menasch, 2003).

A study carried out by Souza et al showed that the transplantation of SkMB has led to increase in ventricular function and myogenesis. This study was compared to the transplantation of mesenchymal stem cells where there was no function effect recorded and it also resulted in neoangiogenesis (LC Souza, 2004).

Souza et al also carried out a study on a model of AMI that used the transplantation of both myoblast and mesenchymal cells. The results showed an increase in ejection fraction from 24.03±8.68% to 31.77±9.06%. This result was quite substantial when compared to the control group which compared from 31.77±9.06% against control result of 23.54±6.51%; this is further presented in figure 16 (LC Guarita-Souza, 2006). As figure 15 shows, this resulted in the formation of new blood vessels in the region of fibrosis where this was not shown in the control group.

Figure 14: New skeletal fibers (white arrows) and endothelial cells (black arrows) being formed (Eltyeb Abdelwahid, 2011).

Figure 15: Skeletal fibers (white arrow) formed in injured myocardium of Chagas disease (Eltyeb Abdelwahid, 2011).

Figure 16: Skeletal myoblast cell therapy carried out in two groups showing the difference in ejection fraction of left ventricle (Eltyeb Abdelwahid, 2011).

A study carried out by Dib et al in 2003 displayed that the implantation of myoblasts has a positive cardiovascular effect on the damaged myocardium (N Dib, 2003). Eleven patients took part in this study of myoblast transplantation alongside CABG. The key result that showed promise from this study was an improvement in the mean ejection fraction from 22.7% to 35.9%. Additionally PET and MRI scans showed that there was an increased viability in the damaged area of the myocardium tissue. This study demonstrated possible efficacy in performance of the left ventricle.

MAGIC clinical trial transplanted myoblast cells during CABG. This trial was carried out on 97 patients in a randomised, placebo controlled study. This study allows one to investigate the effectiveness of cell therapy alongside cardiac surgery. An advantage of such a procedure has

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been seen due to easy access and good visualisation of the target area of cell transplantation; however, this form of therapy can cause complications. These include a further risk to the surgery carried out as well as a limited view of the septal wall (Eltyeb Abdelwahid, 2011).

Bone marrow stem cells (BMCs) are a promising new perspective for myocardial regeneration. Past and ongoing clinical trials use predominantly BMCs as they have several populations with the ability to migrate, proliferate and differentiate. These cell populations include: mesenchymal stem cells, endothelial progenitor cells and hematopoetic cells (Bodo-Eckehard Strauer M. G., 2011).

The first non-randomised clinical trial involving BMCs took place in 2002 by Strauer et al. This trial involved intracoronary injection of BMCs into 10 patients 7 days after AMI (BE Strauer, 2002). Initial results from this trial was very promising, decrease in infarction region and left ventricular end systolic volume (LVESV) as well as an increase in stroke volume index (SVI) was reported after a 3 month follow up, indicating stem cell therapy has the potential of tissue regeneration. A similar clinical trial was carried out also in 2002, TOPCARE – AMI (Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction). This trial transplanted two groups of cells, BMCs and circulating progenitor cells using an intracoronary transplantation delivery method. Results showed no significant differences between the two cells, increases in LVEF and LVESV were reported for both groups after a 1-year follow up (Sheing-Tsung Kuo, 2009).

The first randomised controlled trial involving BMCs was carried out in 2004 on 60 patients. Namely BOOST (Bone Marrow Transfer to Enhance ST-Elevation Infarct Regeneration) trial. 30 patients received BMCs via intracoronary delivery whilst 30 patients in the control group received normal medical treatment (KC Wollert, 2004). However, a major limitation of cell therapy treatment was highlighted in this study, after an 18-month follow-up, the increase of LVEF was not consistent casting serious doubt over the long-term effects of this treatment. This could be due to a number of reasons such as ineffective cell homing strategies.

The biggest intracoronary autologous BMC transplantation heart study, STAR (acute and long-term effects of intracoronary stem cell transplantation in 191 patients with chronic heart failure) took place over a range of 3 months to 5 years (Bodo-Eckehard Strauer M. G., 2011). 391 patients with reduced LVEF due to heart disease were involved in this study. Two groups were involved, 191 patients underwent BMC therapy whilst the control group consisted of 200 patients. Results after 3 months for BMC therapy patients showed a considerable increase in exercise capability, improved haemodynamics where cardiac index at rest increased by 22.2% and oxygen uptake by 11%, an increase in LVEF from 29.4±12.7 to 36±13.3% and increased left ventricular contractibility behavior (Fig 18) (Bodo-Eckehard Strauer M. Y., 2010).

Figure 17: Effect of BMC therapy on mortality with patients suffering from ischaemic cardiomyopathy (Bodo-Eckehard Strauer M. Y., 2010).

The long-term effectiveness of stem cell therapy has been questioned over the years. STAR study has shown the effectiveness of this treatment, results 12 and 60 months after cell therapy revealed deterioration in left ventricular performance for the control group, in comparison to the BMC therapy group which maintained a high level of left ventricular performance after 5 years. Mortality was also significantly lower in the BMC therapy group with 7 patient deaths in contrast to 32 patient deaths in the control group as shown in figure 17. In general, this study proved that BMC therapy gives improvement to exercise capacity, haemodynamics, LVEF as well as left ventricular geometry. Most importantly, it shows that BMC treatment can increase a patient’s life span (Bodo-Eckehard Strauer M. Y., 2010). STAR study has justified BMC therapy can overcome effects patients suffer due to ischaemic cardiomyopathy and lower the rate of mortality. Further investigations must be carried out to evaluate what factors affect BMC therapy in regards to LVEF levels.

Although this study derived positive results, the question arises of what the optimal number of implanted cells are required to achieve greater effects. General studies carried out with doses of 10 to 40 x 106 show promising results, however studies must be carried out with higher doses. A study by Yuyama et al was carried out treating peripheral vascular disease with autologous bone marrow administering a large dose of 2.7 x 109 cells where results showed there was a decrease in inflammation as well as progressive results (E Tateishi-Yuyama, 2002).

Figure 18: Comparison of LVEF over time of BMC group to control group (Bodo-Eckehard Strauer M. Y., 2010).

Most of the clinical trials carried out have reported many benefits from cell therapy; however the mechanisms involved are still unclear, this is because the method used to track differentiation after transplantation is not very accurate, therefore differentiation could be due to factors such as paracrine effects or cell fusion (George, 2010). Studies that have been carried out with initiation of G-CSF therapy have shown to increase the myocardial homing of stem cells.

S a f e t y a n d E f f i c a c y o f S t e m C e l l T h e r a p y The safety of cell therapy is a vital factor in the effectiveness of cell delivery. Until now, the clinical procedure of intracoronary autologous bone marrow cell transplantation has been deemed safe, as there is no documented increase of malignant diseases or progression of coronary artery disease. Furthermore, there has been no documentation of inflammation after cell transplantation where white blood cell counts, creatine phosphokinase and serum levels of C-reactive protein are measured before, during and after treatment and have not shown this.

Transplanting a larger number of cells (>107) has shown to have a bigger impact in LVEF improvement.

Cell therapy has shown to have a lack of effect post 8 weeks after cell administration. This phenomenon is confirmed by many patient studies that have been carried out where initial improvements are not consistent over time. Such findings should indicate to researchers that new strategies must be implemented such as; genetic engineering of stem cells, multiple injections over time or slow release agents (Tycho I.G. van der Spoel, 2011).

There is a big variation in results from the studies included in this report. This could be due to the type of stem cell transplanted or the delivery method chosen. An evaluation of factors that could cause such variations include: variances in the number of cells transplanted, different methods of cell preparation, age range of patients, lack of standardised procedures and different times of cell application (Bodo-Eckehard Strauer M. G., 2011). To achieve a greater level of efficacy,

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standardised approaches to cell delivery, clinical patient selection and methodology of cell preparation is required.

T h e I d e a l C e l l Intense ongoing investigations and clinical trials are taking place to distinguish the ideal stem cell for cell-based therapy. Based upon the evidence derived from experimental data, many stem cell types could be considered such as bone marrow cells, skeletal myoblast cells and cardiac derived cells. Skeletal myoblasts was considered an option initially, however due to its transplantation causing increased frequency of arrhythmias and the cell not causing significant benefits, this was discarded as an ideal cell. Furthermore, cardiac derived stem cells were also discarded due to minimal clinical evidence and its inability to differentiate into CMCs in vitro. Embryonic stem cell and fetal cardiomyocytes have also been discarded, although they both have excellent differentiating ability, this is due to the ethical concerns that arise with the use of these cells. Based upon the studies considered in this report, it is quite clear that a relationship has formed between the extent of differentiation, effective engraftment and final effect of left ventricular function. In accordance to these factors, the ideal cell seems to be bone marrow stem cells. The question arises, which type of BMC is the most effective. The three most popular types of BMCs include: mesenchymal (MSCs), endothelial (EPCs) and haematopoetic (HSCs) cells.

MSCs proliferate in vitro, relatively easy to obtain and have high levels of transfection efficiency. Many studies have shown that MSCs initiate the following mechanisms: (1) The transplantation of MSCs inhibits the expression of inhibitory factors such as inerleukin-1 which reduces scar area as reported by Guo et al (J Guo, 2007). (2) MSCs expresses cardiac-specific contractile proteins that decreases scar tissue by differentiating into contractile CMCs. (3) Ventricular remodeling is reduced as well as improving diastolic function (Chaoquan Peng, 2013). However, a prerequisite to the transplantation of MSCs is that it must be transplanted at an early stage of the disease, this is because differentiation occurs when the cell is in contact with surviving CMCs.

HSCs have shown that they are able to differentiate into CMCs under strict laboratory conditions, although a big limitation to this cell type is that there is no evidence as of yet that it has the ability to differentiate into CMCs when transplanted into the infarcted myocardium, therefore due to this factor it is discarded as the ideal cell.

EPCs have excellent differentiating ability, alongside the fact that research has shown the number of EPCs present in the myocardium increases with AMI. Naturally, this cell migrates to areas of damage. Several paracrine roles have been linked to this cell. Taking into consideration the factors of long-term effects, successful homing and differentiating ability, EPCs have all the attributes to become the ideal cell.

In summary, it is very difficult to claim a cell type is the ‘ideal cell’, as this is ultimately dependent upon the injury to be treated and the conditions upon transplantation. In conditions where reperfusion is the primary objective, the angiogenic potential of cells is of high priority; therefore the ideal cell in this condition will be bone marrow stem cells. However, in conditions such as the end-stage ischaemic heart failure, priority is to transplant cells that are high in contractile potential. In such conditions, the ideal cell would be skeletal myoblasts (Harald C. Ott, 2005).

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F u t u r e P e r s p e c t i v e s Regarding clinical studies, it is very important that pre-clinical studies are carried out to very high standards which would include randomised study design, blind studies and a large amount of patients, whilst also including a detailed study pre-treatment and post-treatment.

Although there are many factors that remain to be solidified in terms of optimum cell type and delivery method, there are many obstacles stem cell therapy must overcome before it is implemented as a full clinical treatment, these include the following:

1. Optimum cell type for each individual that may suffer from different disease conditions. As discussed in this report, different cell types are more effective for certain treatments, further studies would need to be carried out to confirm which cell has the greatest ability for transdifferentiation and vascular improvement.

2. How to effectively track the effects of transplanted cells after homing. A more accurate form of tracking is needed to monitor the cells survival and mechanical properties. Although methods such as MRI and PET have been able to monitor cells in vivo, more study is essential for greater understanding post transplantation.

3. Optimum number of transplanted cells for best results. This issue has not reached a consensus amongst investigators. Studies carried out on a larger scale could investigate this issue whilst also investigating the optimal timing of cell delivery.

4. Developing a procedure that fits the majority of cases. Myocardial infarction is generally a disease that affects the older generation, the myocardium of the younger generation differs to the older generation therefore further studies must be carried out to investigate how to overcome the difference in interstitial space and messaging.

A possible form of treatment that could overcome many of these issues is a combination of cell and gene therapy. This therapy involves stem cells with genes that encode enzymes to stimulate growth factor and structural proteins. This results in an enhancement of regenerative potential, functional integration and cell homing (Vincenzo Lionetti F. A., 2010). Additionally, paracrine factors could be stimulated through the use of transgenes, which as stated earlier, is one of the mechanisms responsible for the effects of cell therapy (M Gnecchi, 2008).

Future prospects for stem cell therapy looks promising, as the biggest stem cell trial involving patients with AMI have begun in London. Bone acute myocardial infarction (BAMI) will involve 3,000 patients in 11 European countries in an attempt to overcome UK’s biggest killer. BAMI aims to cut the mortality rate by 25% in 15 years. One could say this target is very realistic, as stem cells cannot be patented, no profit is involved therefore this study and therapy is done solely for the health of patients as well as saving the NHS money (Walsh, 2014).

Furthermore, a recent discovery adds to the ever-growing case of stem cells being the future of heart therapy. A study carried out has shown that stem cells can be transformed by adding acid to blood cells. This breakthrough could make stem cell therapy cheaper, safer and more time efficient. This study currently has been carried out with mouse blood cells; many experts believe this revolutionary discovery can be achieved with human blood cells (Gallagher, 2014).

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C o n c l u s i o n s This report has used evidence contributed from both scientific and clinical arguments to indicate that stem cell based therapy has the potential to provide cardiac regeneration. Although complete cardiac regeneration is limited, there is no doubt that this therapy does limit the deterioration of the myocardium and stimulates mechanisms in vascular remodeling.

In summary, efforts must be made to carry out large-scale clinical trials that focus on finding the optimum stem cell type, delivery method, number of cells to transplant and the time of delivery.

Figure 19: Representative scheme of action by synthetic molecules on the failing heart (Vincenzo Lionetti C. V., 2013).

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