The Clinical Application of Mesenchymal and Cardiac Stem Cells as a
Therapy for Cardiovascular Disease
Jiyeon Kim, Linda Shapiro, Aidan Flynn
PII:
DOI:
Reference:

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

To appear in:

Pharmacology and Therapeutics

Received date:
Accepted date:

11 February 2015
11 February 2015

Please cite this article as: Kim, J., Shapiro, L. & Flynn, A., The Clinical Application of
Mesenchymal and Cardiac Stem Cells as a Therapy for Cardiovascular Disease, Pharmacology 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

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Cardiovascular Disease

Center for Vascular Biology, University of Connecticut Health

Center, Farmington, CT, 06030;

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Author Affiliations:

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

Department of Echocardiography, Division of

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

*:

Address

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of Connecticut Health Center, Farmington, CT, 06030

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: aidan.flynn@hhchealth.org.

Word Count: 4849

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ABSTRACT:

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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

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

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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

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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

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progenitor cells from bone marrow and the heart itself have been evaluated in
preclinical models, and have demonstrated some promise. Accordingly, several clinical

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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

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cell; pre-conditioning; clinical trial

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Table of Contents:

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1. Introduction

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2. Stem Cell Populations

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2.1 Bone Marrow Derived Mononuclear Cells (BMMNC)

2.2 Bone Marrow Derived Mesenchymal Stem Cells (BMMSC)

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2.3 Preconditioned Mesenchymal Stem Cells

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2.4 Cardiac Stem Cells

3. Clinical Trials of Stem Cell Populations

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3.1 Mesenchymal Stem Cells

3.2 Preconditioned Mesenchymal Stem Cells

3.3 Cardiac Stem Cells

4. Future Directions

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Abbreviations:

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BMMNC: Bone Marrow Derived Mononuclear Cells

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CSC: Cardiac stem cell

CVD: Cardiovascular disease

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MSC: Mesenchymal stem cell

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1. Introduction

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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

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

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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

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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

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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

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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

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

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therapies only. These early studies unanimously demonstrate the safety of using
patient-derived cells (Strauer and Steinhoff, 2011). Moreover, some show improvement

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

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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

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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

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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

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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.

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

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

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

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

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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

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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

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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

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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

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

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now considered more likely to be the preferred, or active, therapy. Indeed, in the TACHFT trial, direct comparison of BMMNC and MSC versus placebo demonstrated

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

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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

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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

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population will increase therapeutic potency. Of the different constituents that have
been described in the literature, there is increasing evidence that the mesenchymal

attention.

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

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

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

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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

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possibility of an off-the-shelf product.

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The advantages of using MSCs are widely recognized. Cell isolation from bone
marrow and subsequent transfusion has been clinically practiced safely for years to

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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

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

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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 5x10 5
allogeneic MSC to the border zone of a myocardial infarction in mice resulted in a

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

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volumes, despite transient engraftment and infrequent cellular fusion (Noiseux, et al.,
2006). Similarly, in a large animal model, Quevedo et al demonstrate that administration

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

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

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

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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

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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

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(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

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

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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

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cardioprotective effects, and include favorable effects on cardiac metabolism,

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contractility, regeneration and neovascularization.

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2.3 Preconditioned Mesenchymal Stem Cells

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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

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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

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

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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-

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

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

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

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(Lim et al., 2006; Mangi et al., 2003; Matsui et al., 2001; Noiseux et al., 2006). Akttransduced MSC secrete a number of proteins in response to hypoxia, including the

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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

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

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

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Preconditioning MSCs with physical and chemical manipulations avoids the use
of viral transfection and also may induce certain pathways that improve engraftment and

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

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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)

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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

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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

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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)

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

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continue to be evaluated. Adult mammalian myocardium harbors endogenous

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

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

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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.,

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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

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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%,

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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

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

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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

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

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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.,

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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

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

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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

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

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3. Clinical Trials of Stem Cell Populations

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3.1 Mesenchymal Stem Cells

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

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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

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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

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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

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

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

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

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

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autologous MSCs into akinetic or hypokinetic areas of the hearts that were unsuitable
for surgical revascularization during CABG in 6 patients. Cardiac MRI analysis

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demonstrated increased ejection fraction as well as scar reduction and contractile

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

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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

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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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3.2 Preconditioned Mesenchymal Stem Cells

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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

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

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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

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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-

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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

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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.

3.3 Cardiac Stem Cells

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
dySfunction

(CADUCEUS)

trial

evaluated

the

effectiveness

of

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ventricUlar

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

expressing endothelial progenitor markers grown from human heart biopsy subcultures

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

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only adult CSCs, which are capable of long-term self-renewal and cardiomyocyte
differentiation, but also vascular cells and differentiated progenitor cells. CADUCEUS

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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

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

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

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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

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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

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

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

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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

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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

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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

awaited.

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

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

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

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

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

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

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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

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(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-

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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

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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

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

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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

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

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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

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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

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

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

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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,

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and associated with important functional and perhaps mortality benefits.

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Acknowledgements: The authors would like to acknowledge the assistance of

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Amanda Zaleski and Katelyn Zaleski in preparing the illustrations.

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The authors declare that there are no conflicts of interest.

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Figure Legends:

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

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

responsible for repair of damaged myocardium.

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

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

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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;

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

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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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ACCEPTED MANUSCRIPT
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

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

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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

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

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

BMMNC

Cell number and

method of
administration
48-60x109
intracoronary infusion

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24.6x108 intracoronary
infusion
intracoronary infusion

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150x106 intracoronary
infusion
150x106 intracoronary
infusion
100x106
transendocardial
injection

MSC

Results

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Cell Type

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Classification.

100-200x106
transendocardial
injection
733x106
endoventricular
injection
20-200x106
intramyocardial
injection

Reference

Improved cardiac
contractility and
perfusion
Increased LVEF and
systolic function
Increased LVEF,
Reduced long term
negative outcomes
No significant
improvement at 6
months
No significant
improvement over
placebo
No significant
improvement at 6
months

Chen et al. 2004

Improvements in
multiple parameters
for both autologous
and allogeneic MSC
treatment groups
Improved LVEF, 6
minute walk
distance and NYHA
functional class
Reduced scar size,
Improved LVEF

Hare et al. 2012

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Wollert et al. 2004

Schachinger et al.
2006
Traverse et al. 2011

Traverse et al. 2012

Perin et al. 2012

Bartunek et al. 2013

Karantalis et al.
2014

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

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

Williams et al. 2013

12.5-25x106
intracoronary infusion

Reduced scar size,

no changes in LVEF

Malliaras et al. 2014

MA

TE

1x106 CSC+200x106
MSC, intramyocardial
injection

AC
CE
P

CDC

SC

Bolli et al. 2011

NU

Reduced scar size,

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

1x106 intracoronary
infusion
12.5-25x106
intracoronary infusion
1x106 intracoronary
infusion

CSC+
MSC

Suncion et al. 2014

RI

20-100x106
transendocardial
injection
CSC

Heldman et al. 2014

PT

transendocardial
injection

42

Makkar et al. 2012

Chugh et al. 2012

Figure 1

AC
CE
P

TE

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

43

AC
CE
P

TE

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 2

44

Figure 3

AC
CE
P

TE

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

45