the clinical application of mesenchymal stem cells and cardiac stem cells as a therapy for...

The Clinical Application of Mesenchymal and Cardiac Stem Cells as aTherapy for Cardiovascular Disease

Jiyeon Kim, Linda Shapiro, Aidan Flynn

PII: S0163-7258(15)00047-9DOI: doi: 10.1016/j.pharmthera.2015.02.003Reference: JPT 6762

To appear in: Pharmacology and Therapeutics

Received date: 11 February 2015Accepted date: 11 February 2015

Please cite this article as: Kim, J., Shapiro, L. & Flynn, A., The Clinical Application ofMesenchymal and Cardiac Stem Cells as a Therapy for Cardiovascular Disease, Pharma-cology and Therapeutics (2015), doi: 10.1016/j.pharmthera.2015.02.003

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P&T #22675

Title: The Clinical Application of Mesenchymal and Cardiac Stem Cells as a Therapy for

Cardiovascular Disease

Authors: Jiyeon Kim Ph.D.1, Linda Shapiro Ph.D.1, Aidan Flynn M.B., Ph.D.2, 3,*

Author Affiliations: 1 Center for Vascular Biology, University of Connecticut Health

Center, Farmington, CT, 06030; 2 Department of Echocardiography, Division of

Cardiology, Hartford Hospital, Hartford, CT, 06102; 3 Department of Medicine, University

of Connecticut Health Center, Farmington, CT, 06030

*: Address for Correspondence: Aidan Flynn M.B., Ph.D., Department of

Echocardiography, S201, Hartford Hospital, Hartford, CT, 06102. Fax: 860-545-5631;

Tel: 860-972-2976; E-mail: [email protected].

Word Count: 4849

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ABSTRACT:

Cardiovascular disease (CVD) can be separated into two broad etiological categories,

based on the presence or absence of ischemia as a causative factor. In both ischemic

and non-ischemic heart disease, myocardial dysfunction or damage frequently results in

the development of heart failure, characterized by dyspnea, fatigue and reduced

survival. As one of the least regenerative organs in the human body, current standards

of care are limited to mitigating loss and preventing recurrence of damage, rather than

stimulating actual regeneration of functional heart tissue. Cell based therapies using

progenitor cells from bone marrow and the heart itself have been evaluated in

preclinical models, and have demonstrated some promise. Accordingly, several clinical

trials using autologous stem and progenitor cells have demonstrated that these cells

can be used safely in humans, and some studies suggest that they may improve

relevant clinical parameters in patients with heart disease. Two specific cell populations

that are particularly promising are the bone marrow derived mesenchymal stem cell

(MSC) and the heart muscle derived cardiac stem cell (CSC). This review will

summarize preclinical studies evaluating these stem cell populations and will discuss

the clinical application of these cells in contemporary clinical trials, and potential future

investigations.

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Key Words: Cardiovascular disease; therapy; mesenchymal stem cell; cardiac stem

cell; pre-conditioning; clinical trial

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Table of Contents:

1. Introduction

2. Stem Cell Populations

2.1 Bone Marrow Derived Mononuclear Cells (BMMNC)

2.2 Bone Marrow Derived Mesenchymal Stem Cells (BMMSC)

2.3 Preconditioned Mesenchymal Stem Cells

2.4 Cardiac Stem Cells

3. Clinical Trials of Stem Cell Populations

3.1 Mesenchymal Stem Cells



4. Future Directions

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Abbreviations:

BMMNC: Bone Marrow Derived Mononuclear Cells

CSC: Cardiac stem cell

CVD: Cardiovascular disease

MSC: Mesenchymal stem cell

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1. Introduction

Cardiovascular disease (CVD) is the leading cause of death in the United States and

worldwide (Heidenreich et al., 2011; Lopez et al., 2006). Currently affecting one in three

adults, or over 70 million people, in the United States alone, the projected prevalence of

CVD in the US by 2030 is over 40%, costing the nation over $1 trillion in direct and

indirect costs (Heidenreich et al., 2011; Thom et al., 2006). The most common

manifestations of CVD are hypertension, coronary artery disease, heart failure and

stroke. Heart disease and stroke account for almost 35% of the 2.4 million deaths in the

US in 2003, making them the first and third leading causes of mortality (Go et al., 2014).

Indeed, the morbidity and mortality associated with CVD is projected to worsen with the

current trends in obesity and aging of the population unless better preventative and

therapeutic modalities can be implemented (Hoyert et al., 2006).

Despite significant advances in the management of heart disease over the last two

decades, there remains an obviously unmet clinical need in treating this large and

expanding patient population. As part of a multi-faceted approach, the field of cell

therapy for advanced heart failure aims to provide an effective therapeutic option with

the goal of improving quality of life and perhaps reducing mortality. Cells such as bone

marrow derived mesenchymal stem cells (BMMSCs), autologous cardiac stem cells

(CSCs), embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells have

been used in experimental models and show improvement in cardiac function and/or

secretion of bioactive paracrine molecules that are pro-angiogenic and cardio-protective

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(Williams et al, 2014, Zhou et al., 2012, Yoshida and Yamanaka, 2011). While each of

these cell types have been extensively studied in animal models, until recently, most of

the phase I and II trials have tested the safety and efficacy of autologous adult stem cell

therapies only. These early studies unanimously demonstrate the safety of using

patient-derived cells (Strauer and Steinhoff, 2011). Moreover, some show improvement

in relevant clinical parameters as well as patient-described quality of life (Heldman, et

al. 2014). Further clinical investigation is underway to determine the ideal cell type or

combination of cell types as well as optimal dosage in larger scale phase II/III studies

based on these initial investigations. The purpose of this review is 1) to provide an

overview of the bone marrow derived mononuclear cell (BMMNC) and its use in clinical

trials, and 2) to describe the potential therapeutic option that is offered by the cell

populations for which there is the strongest evidence; namely the bone marrow derived

mesenchymal stem cell (MSC) and the heart muscle derived cardiac stem cell (CSC).

2. Stem Cell Populations

2.1 Bone Marrow Derived Mononuclear Cells (BMMNC)

Adult bone marrow harbors multiple cell populations including stem cells and various

lineage committed cell types. Unfractionated bone marrow derived mononuclear cells

(BMMNC) include a small number of stem cells, and a larger number of cells at different

levels of maturation. Early preclinical studies were very promising in demonstrating the

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effect of BMMNC in treating acute myocardial infarction (AMI), leading to the rapid

transition to phase I/II clinical trials (Orlic et al. 2001, Yoshioka et al. 2005, Chen et al.

2004, Wollert et al. 2004). Encouraging results were reported in two early studies, the

BOne marrOw transfer to enhance ST-elevation infarct regeneration (BOOST) trial

(Wollert et al., 2004), and the Reinfusion of Enriched Progenitor Cells and Infarct

Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial (Schachinger et al.,

2006). A significant improvement in left ventricular ejection fraction (LVEF) was

observed at 6 month and 4 month follow-up, respectively, with no signals of adverse

events. These findings provided a basis for continuing to investigate the efficacy and

safety of cell therapy, and led on to additional studies evaluating the optimal time of

delivery, the optimal dose of the cell product, and the optimal method for delivery.

Recently, the National Heart, Lung and Blood Institute (NHLBI) Cardiovascular Cell

Therapy Research Network (CCTRN) has coordinated trials evaluating the effect of

BMMNCs as an intracoronary therapy. In the Transplantation in Myocardial Infarction

Evaluation (TIME) trial (Traverse, et al., 2012) and LateTIME trial (Traverse, et al.,

2011), two timepoints were selected the first was 3-7 days after intervention for an

acute anterior wall ST-segment elevation myocardial infarction, and the second was 2-3

weeks after such intervention. In these trials, a significant improvement in LVEF was not

observed at 6 month follow-up in either study. As this is in contrast to the findings of

BOOST and REPAIR-AMI, it has caused some re-evaluation of the efficacy of this

particular cell population. One particular consideration is that using LVEF as a measure

of effectiveness may not be the most prudent approach. As the cell product generates

its greatest effect in the border zone of the infarct, anticipating a global improvement

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may not be realistic, nor may it be necessary for a benefit to be derived. Perhaps more

importantly, BMMNC represent a broad selection of cell types, and this unfractionated

cell population may not represent the optimal cell therapy. Individual constituents are

now considered more likely to be the preferred, or active, therapy. Indeed, in the TAC-

HFT trial, direct comparison of BMMNC and MSC versus placebo demonstrated

improvement in infarct size, regional myocardial function, and 6 minute walk distance

with MSC treatment only, even though treatment with either cell type reduced patient

reported Minnesota Living with Heart Failure scores (Heldman, et al. 2014). Although

the authors acknowledge limitations in sample size due to the multiple parameters being

compared, this study implies that administration of specific cell types within the BMMNC

population will increase therapeutic potency. Of the different constituents that have

been described in the literature, there is increasing evidence that the mesenchymal

stem cell (MSC) is most associated with efficacy, and thus has become a major focus of

attention.

2.2 Bone Marrow Derived Mesenchymal Stem Cells (BMMSC)

The MSC is a cell population that was originally isolated from the bone marrow, but

has since been identified in many tissues, including adipose tissue and umbilical cord

blood (da Silva Meirelles et al., 2006). The characteristics which define MSCs are the

ability to adhere to plastic, the expression of surface antigens CD73 and CD90 (and the

absence of CD34 and CD45), and the ability, under appropriate conditions, to

differentiate into osteoblasts, chondrocytes and adipocytes. It has been shown that

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MSCs may differentiate into cardiomyocytes in vitro and in vivo (Amado et al., 2005;

Miyahara et al., 2006; Shake et al., 2002), albeit with inconsistent rates of differentiation

and engraftment (Shake et al., 2002; Toma et al., 2002). MSC are immune-privileged,

as they do not express MHC class II molecules or Fas ligand and other co-stimulatory

molecules. This allows allogeneic use (Pittenger et al., 1999), with the encouraging

possibility of an off-the-shelf product.

The advantages of using MSCs are widely recognized. Cell isolation from bone

marrow and subsequent transfusion has been clinically practiced safely for years to

treat other conditions. MSCs survive and differentiate in allograft and xenograft animal

models without immune suppression (Amado et al., 2005; Atoui and Chiu, 2012; Le

Blanc and Ringden, 2007; Le Blanc et al., 2003; Nauta and Fibbe, 2007), allowing an

allogeneic donor if the patients bone marrow is compromised by age or other co-

morbidities. Unlike other stem cell-derived therapies, MSCs do not have to be

differentiated into a mature cell type prior to administration, and also have powerful

homing capabilities to sites of injury after intravenous administration (Chamberlain et al.,

2007). Indeed, a number of studies have demonstrated the ability of intravenously

administered MSCs to migrate specifically to areas of inflammation from ischemic injury

to provide measurable benefit (Kawada et al., 2004; Price et al., 2006; Wu et al., 2003).

Thus, the ease of isolation and administration coupled with that fact that these cells are

immune-privileged and have been used safely in patients for years make MSC-based

regenerative approaches a very appealing therapeutic.

A large number of preclinical investigations have been performed using MSC, and

nearly uniformly demonstrate a significant beneficial effect on cardiac structure and

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function. For example, Noiseux et al demonstrated that administration of 5x105

allogeneic MSC to the border zone of a myocardial infarction in mice resulted in a

significant reduction in infarct size, and significant improvements in left ventricular

volumes, despite transient engraftment and infrequent cellular fusion (Noiseux, et al.,

2006). Similarly, in a large animal model, Quevedo et al demonstrate that administration

of allogeneic MSC to a swine model of chronically infarcted myocardium resulted in

improvements in regional contractility and myocardial blood flow, as well as evidence

supporting engraftment, differentiation and enhanced survival (Quevedo, et al., 2009).

In a similarly well-designed and rigorously performed large animal study, Williams et al

show significant reductions in scar size, improvements in ejection fraction and absence

of further adverse LV chamber remodeling (all quantified by serial cardiac MRI) in

animals receiving IM injections of MSC at 3 months post-MI as compared to controls

(Williams, et al. 2013). These studies, and many others, demonstrate that the functional

benefit of MSC therapy is considerable. A consistent observation is that the

considerable functional benefit appears to be out of proportion to what would be

expected from the rates of engraftment and differentiation. This observation has led to

the theory that cell therapy produces factors that act locally or systemically to favorably

impact recovery the paracrine hypothesis.

A wide variety of cytokines, chemokines and growth factors are produced by MSC,

and many are involved in restoring cardiac function or regenerating myocardial tissue.

Administration of conditioned medium from MSC generates similar beneficial effects to

the administration of the cell product itself (Gnecchi, et al., 2005). Factors such as basic

fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and insulin-like growth

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factor (IGF)-1 have been used to pre-condition MSC, and enhance their reparative

effect (Bartunek, et al., 2007). Additional paracrine factors that are secreted, and have

beneficial effects are vascular endothelial growth factor (VEGF), transforming growth

factor (TGF)-, secreted frizzled-related protein (SFRP)-1 and SFRP-2 (Gnecchi, et al.,

2008). The effects of these, and other paracrine factors, extend beyond their

cardioprotective effects, and include favorable effects on cardiac metabolism,

contractility, regeneration and neovascularization.


As the safety and efficacy of MSCs has clearly been demonstrated by preclinical

work for a variety of organ systems, there has been an increasing focus on enhancing

the benefit of MSC therapy. Combining MSC and pharmacotherapy (Yang et al., 2009),

genetically modifying MSCs (Li et al., 2007; Noiseux et al., 2006; Tang et al., 2010) and

pre-conditioning MSCs (Wu et al., 2011; Mylotte et al, 2008) are approaches that are

being explored to augment MSC-mediated cardiac repair. In the realm of combination

MSCs/pharmacologic therapy, a few groups are reporting mixed results. For instance,

co-administration of MSCs and simvastatin improves systolic wall thickening and MSC

engraftment (Yang et al., 2009). On the other hand, supplementing MSC infusion with

hepatocyte growth factor (HGF) does not seem to improve any measure of outcome

over cell therapy alone (Yang et al., 2006); however, this may not be surprising given

that the effective synergy between a cell and a drug may be dose and time dependent,

and may require more than one factor.

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Separate from pharmacologic modification is another process, whereby MSCs

are genetically modified with viruses to express certain enzymes, cytokines or cell

surface molecules that can affect their survival, engraftment or function (Figure 1). The

genes being studied in animal models include anti-apoptotic factors such as Bcl2 and

Akt, angiogenic factors such as VEGF and Ang1, and the stem cell homing factor SDF-

1 (Li et al., 2007; Noiseux et al., 2006; Tang et al., 2010). For instance, MSCs

transfected to overexpress Akt, a kinase involved in cellular activities including

apoptosis and cell proliferation, seem to confer a possible myocardial protective function

(Lim et al., 2006; Mangi et al., 2003; Matsui et al., 2001; Noiseux et al., 2006). Akt-

transduced MSC secrete a number of proteins in response to hypoxia, including the

recently described Hypoxia and Akt induced Stem cell Factor (HASF). This has been

shown to be an important mediator of cardioprotection following ischemic injury (Huang

J, et al., 2014). These studies indicate improved ejection fraction and reduced infarct

size with the administration of the Akt-overexpressing MSCs over that seen with

injection of control MSCs. Furthermore, MSCs engineered to express combinations of

gene products such as Akt and Ang1 are also showing promise in animal models

(Shujia et al., 2008). Interestingly, the combined overexpression of VEGF and SDF-1 in

MSCs also seem to work via Akt activation (Tang et al., 2010). MSCs transfected to

express heme-oxygenase 1 (HO-1), an enzyme that improves MSC tolerance to

hypoxia, and subsequently infused into a cardiac ischemia-reperfusion model

demonstrated improved ejection fraction and lower end systolic volume than plasmid

transfected controls (Tang et al., 2005). Further histologic and molecular analyses of

hearts treated with HO-1 overexpressing MSCs demonstrated increased capillary and

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arteriolar density as well as a cytokine profile that is anti-inflammatory and pro-

angiogenic (Tang et al., 2005). As a whole, these data appear promising, but the safety

of these cells must be carefully and thoroughly addressed before use in humans.

Preconditioning MSCs with physical and chemical manipulations avoids the use

of viral transfection and also may induce certain pathways that improve engraftment and

survival of transplanted cells (Figure 2). Common approaches include heat shock,

hypoxia, anoxia and treatment with pharmacologics (Wu et al., 2011; Mylotte et al,

2008). Brief treatment with hypoxia is known to induce the hypoxia-inducible factor (HIF)

1 and SDF1 pathways which both limit infarct size and improve angiogenesis in the

heart, but interestingly, hypoxia treated MSCs also demonstrate increased survival and

engraftment in the heart (Chacko et al., 2010; Hu et al., 2008; Tang et al., 2009). Brief

periods of anoxia, or ischemia, is also known to reduce apoptosis through activation of

Akt and HIF1 pathways (Fenton et al., 2005; Kim et al., 2009). Pretreatment of MSCs

with trimetazidine, a fatty acid oxidation inhibitor commonly used to treat angina, seems

to improve myocardial recovery and decrease tissue fibrosis again by stimulating HIF1,

Akt and Bcl-2 (Wisel et al., 2009). Culturing MSCs with growth factors, such as basic

fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-1 and bone

morphogenetic protein 2 (BMP2) can also improve myocardial repair in rat models of MI

(Hahn et al., 2008). Finally, heat-shocking cells has been shown to increase cellular

fortitude by activating certain heat shock proteins; however, the precise pathways that

get activated in MSCs and how they converge to enhance MSC-mediated therapy have

yet to be determined.

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2.4 Cardiac Stem Cells (CSC)

In addition to BMMSC being a promising cell therapeutic, other cell populations

continue to be evaluated. Adult mammalian myocardium harbors endogenous

populations of cells that can be stimulated in certain circumstances to generate new

cardiomyocytes. One hypothesis is that these are cardiac stem cells (CSC) that have

the ability to differentiate to cardiomyocytes as well as other supporting cell types such

as endothelium and vascular smooth muscle (Beltrami et al., 2003; Laugwitz et al.,

2005; Martin et al., 2004; Smart et al., 2011). A second suggestion is that these cells

are existing cardiomyocytes re-entering the cell cycle, and that they replicate, thus

generating new cardiomyocytes (Senyo et al., 2013). These cells are understood to

maintain normal homeostasis in the heart. With an annual rate of turnover under 1%,

endogenous CSCs are unable to completely remedy the massive loss of tissue after

myocardial infarction (Malliaras et al., 2013; Mollova et al., 2013). Because these cells

are already located in the heart and are primed for cardiac repair, protocols to enhance

the endogenous activity of these cells or expand these cells in vitro before re-implanting

them in the heart are currently being tested. Indeed a number of animal studies indicate

that the administration of CSCs can slow left ventricular remodeling and cardiac

improve function after ischemic injury (Beltrami et al., 2003; Linke et al., 2005).

Various groups have identified CSCs with c-kit, Sca1 or Isl1 expression, the

ability to efflux Hoechst dye, or even as a side population upon flow cytometric analysis

(Beltrami et al., 2003; Breitbach et al., 2007; Laugwitz et al., 2005; Martin et al., 2004;

Oh et al., 2003; Pfister et al., 2005). All of these putative cardiac stem cells have been

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isolated from rodent hearts and expanded in vitro under varying culture conditions

before assessing cardiac differentiation via protein expression or rescue of animals with

myocardial infarction and the results have been encouraging. Isl1 positive cells have

been shown to form not only myocardial cells, but also endothelial, endocardial, and

smooth muscle lineages in the embryonic heart, but appear to be restricted to the right

atrium in the adult heart (Laugwitz et al., 2005). Their location in the atrium limits the

ability to exploit them endogenously; however, ex vivo expansion or generation of

similar Isl1 lineage cells from pluripotent stem cells still hold promise (Moretti et al.,

2006). Nkx2.5 expressing cells have also been isolated from the heart, but they seem to

be bipotent progenitors, producing only cardiomyocytes and smooth muscle cells, that

are downstream of the Isl1 population (Yi et al., 2010). The epicardium has also been

found to harbor a quiescent population of Wilms tumor 1 (Wt1) positive cardiac stem

cells (Smart et al., 2011). In mice, these cells seemed to increase cardiomyocyte

generation after activation with thymosin 4 and myocardial injury, but later studies

seem to favor a pro-angiogenic mechanism rather than replacement of actual muscle

(Zhou et al., 2012). Further studies are being conducted to elucidate this phenomenon.

While many groups are working to delineate individual cardiac lineages that

reconstitute the heart, other groups have chosen to use cardiospheres, heterogeneous

cell aggregates that can be grown as spheroids in suspension cultures after isolation

from heart tissue. This amalgam of cells has been demonstrated to express markers of

stem-ness such as c-kit and to improve cardiac performance in animal models, which

has prompted human clinical trials using autologous cardiosphere derived cells (Makkar

et al., 2012; Messina et al., 2004). Although the exact types of cells and the correct

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ratios to use are not precisely known, researchers and clinicians are actively pursuing

this line of inquiry not only in animal models, but also in early human trials.

3. Clinical Trials of Stem Cell Populations

3.1 Mesenchymal Stem Cells

As discussed earlier, contemporary clinical trials of BMMNC have produced results

which suggest that unfractionated marrow may not generate the effect necessary for

transition to widespread clinical use. Several studies have evaluated the BMMSC, in the

anticipation that they will have a greater clinical benefit. Chen et al. administered 48-60

billion bone marrow derived MSCs by intracoronary injection into 34 patients and

reported a 14% higher ejection fraction compared to placebo-treated controls (Chen et

al., 2004). These patients exhibited a 10% improvement between 3-6 months after

treatment. More recently, the PercutaneOus StEm Cell Injection Delivery Effects On

Neo-myogenesis (POSEIDON) and Prospective Randomized Study of Mesenchymal

Stem Cell Therapy in Patients Undergoing Cardiac Surgery (PROMETHEUS) trials

have utilized a transendocardial or intramyocardial method of delivering MSCs directly

to sites of injury (Karantalis et al., 2014; Suncion et al., 2014). The POSEIDON trial

tested the ability of autologous and allogeneic MSCs to promote cardiac recovery

following transendocardial stem cell injection (TESI) (Suncion et al., 2014). Using

multidetector computed tomography (MDCT) and biplane left ventriculography, this

study reports scar size reductions of approximately 44% in treated groups versus only

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25% in untreated groups with the greatest improvement seen in those who received 20

million autologous MSCs. Furthermore, MSC-treated myocardial segments

demonstrated an over 40% improvement in segmental ejection fraction whereas no

improvement was detected in untreated segments. Thus, this study clearly

demonstrates the importance of the location and delivery of MSCs and also indicates

the safety of using allogeneic MSCs. The smaller PROMETHEUS trial injected

autologous MSCs into akinetic or hypokinetic areas of the hearts that were unsuitable

for surgical revascularization during CABG in 6 patients. Cardiac MRI analysis

demonstrated increased ejection fraction as well as scar reduction and contractile

improvement in areas that received MSC injection over those areas that were surgically

reperfused (Karantalis et al., 2014). While the lack of a placebo control group and the

very small number of recruited patients prevent the establishment of definitive

improvement with this treatment, this trial also seems to indicate potential benefits of

MSCs injected directly into non-revascularized myocardium.

Cumulatively, these studies demonstrate the safety and efficacy of using BMDCs

and MSCs for cell therapy. In fact, a meta-analysis of the 16 largest randomized

controlled studies shows variable decreases in infarct size and an average increase in

left ventricular ejection fraction of 11.3% (Strauer and Steinhoff, 2011). Similarly, a

meta-analysis by Jeevanantham et al demonstrated that bone marrow derived cells

were associated with improved ejection fraction (an absolute improvement of almost

4%) and smaller infarct size, as well as reductions in mortality and the incidence of

recurrent myocardial infarction (Jeevanantham et al., 2012). A summary of clinical trials

and their results is presented in Table 1.

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A further development in MSC therapy is the pretreatment of MSCs with certain growth

factors to enhance cardioprotective functions. The Cardiopoietic stem Cell therapy in

heart failure (C-CURE) trial has tested the ability of a cardiogenic cocktail to enhance

the therapeutic benefits to the heart rendered by autologous MSCs (Bartunek et al.,

2013). The rationale for this study stems from the fact that CSCs from heart failure

patients may be impaired and the MSCs from the bone marrow can be coaxed to adopt

a cardiopoietic lineage, which improves therapeutic benefit (Behfar et al., 2010). The C-

CURE trial treated 21 patients suffering from heart failure with an average number of

over 700 million cells in 9-26 electromechanically guided endomyocardial injections. No

adverse events or systemic toxicity was observed. Moreover, significant improvements

in left ventricular ejection fraction, end-systolic volume and 6-minute walking test were

reported. The Safety and Efficacy of Autologous Cardiopoietic Cells for Treatment of

Ischemic Heart Failure (CHART-1) trial is powered to evaluate the efficacy of this

therapy, and is currently enrolling patients.


In line with the promising preclinical work on CSC, this cell type has already been

investigated in clinical studies. There are three clinical trials using cardiac stem cells for

treatment of ischemic cardiomyopathy and even though they use slightly different

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approaches, all report significant improvements in certain measures of cardiac function

(Oldroyd et al., 2012). The CArdiosphere-Derived aUtologous stem CElls to reverse

ventricUlar dySfunction (CADUCEUS) trial evaluated the effectiveness of

cardiospheres, which, as described above, are clusters of undifferentiated cells

expressing endothelial progenitor markers grown from human heart biopsy subcultures

(Messina et al., 2004). Cardiospheres are heterogenous groups of cells that contain not

only adult CSCs, which are capable of long-term self-renewal and cardiomyocyte

differentiation, but also vascular cells and differentiated progenitor cells. CADUCEUS

analyzed cardiac MRI scans of 25 patients who were given 12.5-25 million autologous

cardiosphere derived cells (Makkar et al., 2012) after successful percutaneous coronary

intervention. The cardiospheres were expanded approximately 36 days in culture from

right ventricular endomyocardial biopsies taken 2-4 weeks after acute myocardial

infarction and injected into the previously stented coronary artery between 6-12 weeks

after the heart attack. Despite the lack of improvement in left ventricular ejection fraction

or patient reported outcomes, the scar mass was 7.7% and 12.3% lower at 6 and 12

months respectively and regional wall motion was significantly improved in treated

patients. Serious adverse events were also reported to be three times higher in the

treated group, but the relatively small number of patients prohibited the use of this trial

in ascertaining safety. Furthermore, this study was not blinded due to ethical

considerations surrounding the harvest of cardiac tissue from the control group, but

additional investigations are necessary to determine the safety and potency of

cardiospheres as this initial inquiry seems promising.

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In a slightly different approach, the Stem Cell Infusion in Patients with Ischemic

cardiOmyopathy (SCIPIO) trial isolated autologous CSCs during coronary artery bypass

grafting (CABG) procedures (Bolli et al., 2011). SCIPIO enlisted 23 randomized patients

who had experienced myocardial infarction in the remote past and exhibited an ejection

fraction of under 40%. One million cKit+ lineage- cardiac stem cells (CSCs) were

isolated with magnetic beads from cultures of patient-specific right atrial appendage

tissue and administered via intracoronary infusion one month after CABG. Four months

after this treatment, 14 out of 16 treated patients saw a 24% relative decrease in infarct

size, an 8.2% absolute improvement in left ventricular ejection fraction and reported

improvements in New York Heart Association functional class. The benefits of treatment

were sustained and even increased over time - after one year, eight of these patients

demonstrated an 8% improvement in ejection fraction, which became 12% after 2 years

(Chugh et al., 2012). Publication of the complete findings of the two-year follow-up is

awaited.

Analogous to the SCIPIO procedure, the AutoLogous human CArdiac-Derived

stem cell to Treat Ischemic cArdiomyopathy (ALCADIA) trial also harvests patient

cardiac tissue during CABG (Yacoub and Terrovitis, 2013); however, like the

CADUCEUS trial, endomyocardial tissue served as the source of CSCs. With only six

subjects, ALCADIA is the smallest trial and combines the use of stem cells,

bioengineered scaffolds and biologics to create a hybrid therapy. Cells from these

patients were cultured for one month before intramyocardial injection of half a million

cells per kilogram distributed in 20 injection sites, followed by placement of a

biodegradable hydrogel scaffold containing basic fibroblast growth factor (bFGF) over

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those sites. At the 6 month time point, cardiac MRI indicated an increase in ejection

fraction of 12.1%, a 3.3% reduction in infarct size and significant improvement in wall

motion as well as maximum aerobic exercise capacity. This was a small study and

these results will need to be confirmed in a larger cohort.

Lastly, combining the use of MSC and CSC in post-MI treatment may further

enhance the therapeutic effects of each cell type. Indeed, recent work by Williams et al

demonstrate that the combined use of one million human CSCs and 200 million human

MSCs provide greater recovery, almost to baseline, in swine models of anterior wall MI

(Williams AR, et al. 2013). While all stem cell treated animals demonstrated improved

LVEF compared to placebo controls, notably, animals receiving dual cell therapy had 2-

fold greater reductions in scar size (21.1% for CSC/MSC versus 10.4% for CSC alone

or 9.9% for MSC alone) and improved rates of pressure change during diastole. Overall

left ventricular chamber dynamics were improved in both the dual therapy and CSC or

MSC alone treated groups. Interestingly, CSC alone treated animals demonstrated

better isovolumic relaxation as compared to controls, while MSC alone treated animals

exhibited improved diastolic compliance, indicating that the enhanced effect of dual

therapy on both systolic and diastolic function may be due to a synergistic effect

between CSC and MSC targeted mechanisms.

4. Future Directions

The heart is one of the most important, but ironically, one of the least

regenerative organs of the body. With the rising trend in heart disease, developing

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methods to enhance cardiac repair has become one of the most important areas of

translational research. Cell therapy continues to be well positioned to fill the void that

currently exists in heart failure management. Before cell therapy can be considered a

widely accepted therapeutic option however, continued research on a variety of fronts is

required (Figure 3). At the forefront of this is an emphasis on safety. To date, no

adverse signals have been identified in the many clinical investigations of cell therapy in

humans. The field must remain vigilant however, as the consequences of unreported or

underreported adverse events would be profound. In light of the promising safety profile

experienced thus far, a particular emphasis is being focused on the optimal cell type. It

appears that CSC and BMMSC are prime candidates, and are certainly worthy of

continued investigation. Indeed, a combination of both cell populations may be more

effective than either one alone, and this consideration remains under investigation.

Similarly, pre-conditioning MSC is a hugely promising approach, and further

investigation is eagerly anticipated. Identifying the optimal dose and method of delivery,

as well as the optimal time for delivery are important variables that are being studied.

Perhaps one of the most important advances in the field has been the collaborative

approach that has recently been engendered by CCTRN. This multi-institutional group

of highly respected researchers is ideally positioned to have a very formative role in the

future of cell therapy trials, and aims to address the many remaining questions in an

objective and definitive manner. The evolution of cell therapy for heart disease has

resulted in a refinement of a number of variables that were initially being broadly

investigated particularly the cell population. We will watch with intense interest how

the field will progress over the coming years, with the anticipation that cell therapy will

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become a mainstream treatment for heart disease, free of significant safety concerns,

and associated with important functional and perhaps mortality benefits.

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Acknowledgements: The authors would like to acknowledge the assistance of

Amanda Zaleski and Katelyn Zaleski in preparing the illustrations.

The authors declare that there are no conflicts of interest.

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Figure Legends:

Figure 1: Pre-Conditioned MSC receive stimuli from various external factors, which

either act directly, or via intermediaries to upregulate genes which act in a protective or

anti-apoptotic manner. These factors result in production of proteins that are

responsible for repair of damaged myocardium.

bFGF: basic fibroblast growth factor; HGF: hepatocyte growth factor; BMP2: bone

morphogenetic protein 2; IGF-1: insulin-like growth factor-1; HIF-1: hypoxia induced

factor-1; SDF-1: stem cell derived factor-1; VEGF: vascular endothelial growth factor;

TGF-: transforming growth factor-; Sfrp: secreted frizzled related peptide.

Figure 2: Genetic Modification of MSC. Various factors have been transfected into

MSC, including anti-apoptotic, angiogenic and stem cell homing factors, as well as Akt.

A potentially important factor, secreted frizzled related peptide is produced, as are many

other intermediaries, which result in the secretion of Hypoxia and Akt induced Stem cell

Factor, an important mediator of the reparative process.

HASF: hypoxia and Akt induced stem cell factor; Sfrp: secreted frizzled related peptide;

VEGF: vascular endothelial growth factor; SDF-1: stem cell derived factor-1.

Figure 3: Cardiac regenerative processes are multi-faceted, and approaches to cardiac

repair have included MSC, pre-conditioned MSC, genetically modified MSC and

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CSC/CDC. Questions remain to be answered as to which (or combinations of which) will

be the optimal approach. Some of the remaining questions are highlighted, with safety

and efficacy being the primary factors in identifying the optimal approach.

CSC/CDC: cardiac stem cell / cardiospheres; MSC: mesenchymal stem cells

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Table 1: Comparison of the results of selected clinical trials of cell therapy for cardiac

disease sorted by cell populations. BMMNC: Bone marrow derived mononuclear

cells; CDC: Cardiospheres; CSC: Cardiac stem cells; LVEF: Left ventricular

ejection fraction; MSC: Mesenchymal stem cells; NYHA: New York Heart

Classification.

Cell Type Cell number and method of administration

Results Reference

BMMNC 48-60x109 intracoronary infusion

Improved cardiac contractility and perfusion

Chen et al. 2004

24.6x108 intracoronary infusion

Increased LVEF and systolic function

Wollert et al. 2004

intracoronary infusion Increased LVEF, Reduced long term negative outcomes

Schachinger et al. 2006

150x106 intracoronary infusion

No significant improvement at 6 months

Traverse et al. 2011


No significant improvement over placebo

Traverse et al. 2012

100x106 transendocardial injection

No significant improvement at 6 months

Perin et al. 2012

MSC 100-200x106 transendocardial injection

Improvements in multiple parameters for both autologous and allogeneic MSC treatment groups

Hare et al. 2012

733x106 endoventricular injection

Improved LVEF, 6 minute walk distance and NYHA functional class

Bartunek et al. 2013

20-200x106 intramyocardial injection

Reduced scar size, Improved LVEF

Karantalis et al. 2014

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transendocardial injection

Reduced scar size, Improved regional wall dynamics, improved 6 minute walk distance and Minnesota Living with Heart Failure score

Heldman et al. 2014

20-100x106 transendocardial injection

Reduced scar size, Improved segmental EF

Suncion et al. 2014

CSC 1x106 intracoronary infusion


Bolli et al. 2011

12.5-25x106 intracoronary infusion

Reduced scar size, greater regional contractility. No changes in LVEF.

Makkar et al. 2012



Chugh et al. 2012

CSC+ MSC

1x106 CSC+200x106 MSC, intramyocardial injection

2-fold higher reduction in scar size, improved systolic and diastolic measured of function with combination

Williams et al. 2013

CDC 12.5-25x106 intracoronary infusion

Reduced scar size, no changes in LVEF

Malliaras et al. 2014

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Figure 1

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Figure 2

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Figure 3

the clinical application of mesenchymal stem cells and cardiac stem cells as a therapy for...

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