msc circ res 2011 williams

Review

This Review is part of a thematic series on Stem Cells, which includes the following articles:

Stem Cells Review Series: An Introduction [Circ Res. 2011;109:907–909]Biomaterials to Enhance Stem Cell Function in the Heart [Circ Res. 2011;109:910–922]

Mesenchymal Stem Cells: Biology, Pathophysiology, Translational Findings, and Therapeutic Implications forCardiac Disease

Role of Cardiac Stem Cells in Cardiac Pathophysiology: A Paradigm Shift in Human Myocardial Biology

Imaging: Guiding the Clinical Translation of Stem Cell Therapy

Side Population Cells

Endothelial Progenitor Cells

Genetic and Pharmacologic Modulation of Stem Cell Function for Cardiac Repair

MicroRNAs and Stem Cells

Epigenetic Modifications of Stem Cells: A Paradigm for the Control of Cardiac Progenitor Cells

Chemical Regulation of Stem Cells

Cell Therapy of Acute Myocardial Infarction and Chronic Myocardial Ischemia: From Experimental Findings to Clinical Trials

Cell Therapy of Heart Failure: From Experimental Findings to Clinical Trials

Cell Therapy of Peripheral Vascular Disease: From Experimental Findings to Clinical Trials

Methodological Issues in Cell Therapy Studies: What Have We Learned From Clinical Studies

Stefanie Dimmeler, Douglas Losordo, Editors

Mesenchymal Stem CellsBiology, Pathophysiology, Translational Findings, and Therapeutic

Implications for Cardiac DiseaseAdam R. Williams, Joshua M. Hare

Abstract: Mesenchymal stem cells (MSCs) are a prototypical adult stem cell with capacity for self-renewal and differenti-ation with a broad tissue distribution. Initially described in bone marrow, MSCs have the capacity to differentiate intomesoderm- and nonmesoderm-derived tissues. The endogenous role for MSCs is maintenance of stem cell niches (classicallythe hematopoietic), and as such, MSCs participate in organ homeostasis, wound healing, and successful aging. From atherapeutic perspective, and facilitated by the ease of preparation and immunologic privilege, MSCs are emerging as anextremely promising therapeutic agent for tissue regeneration. Studies in animal models of myocardial infarction havedemonstrated the ability of transplanted MSCs to engraft and differentiate into cardiomyocytes and vasculature cells,recruit endogenous cardiac stem cells, and secrete a wide array of paracrine factors. Together, these properties can beharnessed to both prevent and reverse remodeling in the ischemically injured ventricle. In proof-of-concept and phase Iclinical trials, MSC therapy improved left ventricular function, induced reverse remodeling, and decreased scar size. Thisarticle reviews the current understanding of MSC biology, mechanism of action in cardiac repair, translational findings, andearly clinical trial data of MSC therapy for cardiac disease. (Circ Res. 2011;109:923-940.)

Key Words: stem cells � regeneration � cell differentiation � hematopoietic stem cells

Original received June 17, 2011; revision received August 3, 2011; accepted August 11, 2011.From the Interdisciplinary Stem Cell Institute (A.R.W., J.M.H.), Department of Surgery (A.R.W.), and Department of Medicine (J.M.H.), University

of Miami Miller School of Medicine, Miami, FL.Correspondence to Joshua M. Hare, MD, FACC, FAHA, Louis Lemberg Professor of Medicine, Director, Interdisciplinary Stem Cell Institute, University of Miami

Miller School of Medicine, Biomedical Research Bldg/Room 824, PO Box 016960 (R-125), Miami, FL 33101. E-mail [email protected]© 2011 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.111.243147

923 at University of Texas Medical Branch--Galveston on February 29, 2016http://circres.ahajournals.org/Downloaded from

http://circres.ahajournals.org/

Ischemic heart disease is the leading cause of death indeveloped countries and carries significant morbidity.1

After an acute myocardial infarction (MI), the heart haslimited capacity for self-renewal and undergoes remodelingwith resulting depressed left ventricular (LV) function.2 Overthe past decade, there has been tremendous enthusiasm in thequest to find a stem cell capable of regenerating lost myo-cardium and restoring cardiac function.

Mesenchymal stem cells (MSCs) were first identified andisolated from bone marrow (BM) more than 40 years ago3

and have emerged as one of the leading candidates in cellularcardiomyoplasty (Figure 1). The unique properties of MSCs(easily isolated and amplified from the BM,4 immunologi-cally tolerated as an allogeneic transplant,5 and their multi-lineage potential6) have led to their intense investigation as acell-based therapeutic strategy for cardiac repair. In thisreview, we describe the biology of MSCs and discuss the datasupporting the translation of MSC therapy to clinical trials forcardiac disease.

Historical OverviewIn 1970, Friedenstein and colleagues3 demonstrated that BMcontains a population of hematopoietic stem cells (HSCs) anda rare population of plastic-adherent stromal cells (1 in10 000 nucleated cells in BM). These plastic adherent cells,initially referred to as stromal cells and now commonly calledMSCs, were capable of forming single-cell colonies. As theplastic-adherent BM cells were expanded in culture, roundcolonies resembling fibroblastoid cells formed and weregiven the name colony forming unit–fibroblasts. Friedensteinwas the first investigator to demonstrate the ability of MSCsto differentiate into mesoderm-derived tissue and to identifytheir importance in controlling the hematopoietic niche.7

Control of stem cell niches (functional and structural units thatspatiotemporally regulate stem cell division and differentia-tion8,9) is emerging as a key role played by MSCs in a broadarray of tissues, including hair follicles and the gut, and recently,MSC ablation was shown to disrupt hematopoiesis.10

During the 1980s, MSCs were shown to differentiate intoosteoblasts, chondrocytes, and adipocytes.11,12 Caplan13 dem-onstrated that bone and cartilage turnover was mediated byMSCs, and the surrounding conditions were critical to induc-ing MSC differentiation. In the 1990s, MSCs were shown todifferentiate into a myogenic phenotype,14 and Pittenger andcolleagues6 demonstrated that individual adult human MSCswere capable of being expanded to colonies while retainingtheir multilineage potential. Also during the late 1990s,Kopen et al15 described the capacity of MSCs to transdiffer-entiate into ectoderm-derived tissue.

During the early 21st century, in vivo studies demonstratedthat human MSCs transdifferentiate into endoderm-derived

Non-standard Abbreviations and Acronyms

BM bone marrow

CSC cardiac stem cell

DE-MRI delayed-enhancement cardiac magnetic resonance imaging

HGF hepatocyte growth factor

HSC hematopoietic stem cells

MSC mesenchymal stem cell

Figure 1. Delivery and potential effects of MSC therapy in cardiac disease.

924 Circulation Research September 30, 2011

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cells and cardiomyocytes,16,17 and in vitro coculturing ofventricular myocytes with MSCs induced transdifferentiationinto a cardiomyocyte phenotype.18 It was also during this timethat MSCs were demonstrated to suppress T-lymphocyteproliferation, which paved the way for the application of MSCtherapy for allogeneic transplantation and as a potential immu-nomodulatory therapy.19 Large-animal preclinical studies ofMSC administration in post-MI hearts demonstrated the abilityof MSCs to engraft, differentiate, and produce substantial func-tional recovery.20–22 Recently, MSC therapy has been translatedto clinical trials for ischemic heart disease.23–25

Definition of an MSCNo unique cell surface marker unequivocally distinguishesMSCs from other HSCs, which makes a uniform definitiondifficult. The International Society for Cell Therapy proposedcriteria26 that comprise (1) adherence to plastic in standardculture conditions; (2) expression of the surface moleculesCD73, CD90, and CD105 in the absence of CD34, CD45,HLA-DR, CD14 or CD11b, CD79a, or CD19 surface mole-cules as assessed by fluorescence-activated cell sorter analy-sis; and (3) a capacity for differentiation to osteoblasts,adipocytes, and chondroblasts in vitro. These criteria wereestablished to standardize human MSC isolation but may notapply uniformly to other species. For example, murine MSCshave been shown to differ in marker expression and behaviorcompared with human MSCs.27 As MSCs are isolated andexpanded in culture, it has also been proposed that certain invivo surface markers may no longer be expressed afterexplantation, although new markers are acquired duringexpansion. For example, an MSC line was isolated thatuniformly expressed HLA-DR (a marker that should not beexpressed on MSCs by the above definition) while alsoexpressing CD90 and CD105, adhering to plastic in culture,and being capable of differentiating into osteoblasts, adi-pocytes, and chondroblasts.28 Indeed, MSCs possess species-specific characteristics that make an unequivocal definitiondifficult to apply to both human and animal models.

Stem cells have classically been defined by their self-renewal capability and multipotency. Some have questionedwhether MSCs are true stem cells or rather multipotentprecursor cells. MSCs isolated from various adult tissuesources express significantly different morphology, differen-tiation capabilities, and gene expression.29 These observa-tions suggest that MSCs from different tissues may not bebiologically equivalent and exhibit variable self-renewal andmultipotential capabilities. As described above, MSCs ex-press diverse surface markers and are likely a heterogeneouspopulation of cells, which has also raised concern regardingtheir “stemness.” Further work is required to fully understandthe biological differences in MSCs derived from varioustissue sources and to characterize tissue-specific MSC self-renewal and multipotency. In view of these considerations,we and others refer to BM-derived cultured MSCs fromhumans as stem cells, because they have proven capacity forself-renewal and multilineage differentiation.

Sources, Isolation, and Types of MSCsMSCs isolated from BM,6 adipose tissue,30 synovial tissue,31

lung tissue,32 umbilical cord blood,33 and peripheral blood34

are heterogeneous, with variable growth potential, but allhave similar surface markers and mesodermal differentiationpotential.35 Furthermore, MSCs have been isolated fromnearly every tissue type (brain, spleen, liver, kidney, lung,BM, muscle, thymus, aorta, vena cava, pancreas) of adultmice, which suggests that MSCs may reside in all postnatalorgans.36 MSCs can be derived from many tissue sources,consistent with their broad, possibly ubiquitous distribution.From a translational perspective, because most studies ofMSC therapy for cardiac repair have used BM-derived MSCs,we will focus the present review on BM-MSCs.

The BM is the major source of HSCs that continuallyrenew red blood cells, platelets, monocytes, and granulocytes.The HSCs are housed within the BM by non-HSCs thatsupport the microenvironment necessary for development anddifferentiation of HSCs,37 and the MSC is one of the mostimportant cells that supports the BM microenvironment,termed the “hematopoietic niche.”38 Physiologically, MSCsdo not migrate easily in the peripheral blood, and availableprotocols are not very successful in inducing the translocationof this cell pool from the BM to the periphery. Therefore,isolation and culture expansion of MSCs is usually necessaryfor therapeutic purposes.

MSCs have been isolated from numerous species,6,39,40 andthe 3 critical steps that allow MSCs to be isolated from otherBM cells are as follows: (1) the use of density gradientcentrifugation (Ficoll or Percoll) to separate nonnucleated redblood cells from nucleated cells; (2) the ability of MSCs toadhere to plastic; and (3) the ability of monocytes to beseparated from MSCs by trypsinization.4 Protocols for isola-tion and expansion are similar among various species, asdepicted in Figure 2.

Expansion of plastic adherence MSCs is the most widelyused method of obtaining MSCs and represents the mostcommonly used source of MSCs for cardiac repair. Asknowledge evolves regarding MSC biology, attempts havebeen made to identify the endogenous MSC precursor basedon cell surface antigen expression. A subset of MSCs ex-pressing the marker neurotrophic growth factor (CD271)have been shown to have similar differentiation capacity astraditional MSCs, but they secrete higher levels of cytokinesand have greater immunosuppressive properties.41 AnotherMSC subpopulation is the Stro-3–positive mesenchymal pre-cursor cell, which has extensive capacity for proliferation anddifferentiation.42

Immunology of MSCsHuman MSCs express moderate levels of human leukocyteantigen (HLA) major histocompatibility complex class I, lackmajor histocompatibility complex class II expression, and donot express costimulatory molecules B7 and CD40 li-gand.43–45 Tolerance of MSCs as an allogeneic transplant isdue to this unique immunophenotype coupled with powerfulimmunosuppressive activity via cell-cell contact with targetimmune cells and secretion of soluble factors, such as nitricoxide, indoleamine 2,3-dioxygnease, and heme oxygenase-1.46–49 MSCs produce an immunomodulatory effect by inter-acting with both innate and adaptive immune cells (Figure 3).

Williams and Hare Mesenchymal Stem Cell Therapy for Cardiac Disease 925



The innate immune cells (neutrophils, dendritic cells,natural killer cells, eosinophils, mast cells, and macrophages)are responsible for a nonspecific defense to infection, andMSCs have been shown to suppress most of these inflamma-tory cells. Neutrophils are one of the first cells to respond toan infection, and an important process in their response toinflammatory mediators is the respiratory burst, characterizedby large oxygen consumption and production of reactiveoxygen species.5 MSCs have shown to dampen the respira-tory burst process by releasing interleukin (IL)-6.50 Dendriticcells play an important role in antigen presentation to naïveT cells, and MSCs have been shown to inhibit the differen-tiation of immature monocytes into dendritic cells.51 Addi-tionally, cocultures of MSCs and dendritic cells inhibit theproduction of tumor necrosis factor-�, a potent inflammatorymolecule.52 Natural killer cells are important innate cells inthe defense against viral organisms and in tumor defense by

their secretion of cytokines and cytolysis.5 MSCs culturedwith freshly isolated resting natural killer cells have beenshown to inhibit IL-2–induced proliferation and decreasesecretion of interferon (IFN-�) by 80%; however, IL-2–acti-vated natural killer cells can lyse autologous and allogeneicMSCs.52,53

The adaptive immune system, composed of T and B lympho-cytes, is capable of generating specific immune responses topathogens with the production of memory cells. Once a T cell isactivated by a foreign antigen binding to a specific T-cellreceptor, the T cell proliferates and releases cytokines.5 T cellsexist as CD8� cytotoxic T cells that induce death of targetcells or CD4� helper T cells that modulate the other immunecells, and MSCs have been shown to suppress T-cell prolif-eration in a mixed lymphocyte culture.19,52 Proliferation ofcytotoxic and helper T cells are suppressed via soluble factorsreleased by MSCs, such as hepatocyte growth factor (HGF)

Figure 2. Isolation of mesenchymal stem cells from a bone marrow biopsy. Many stem cells and maturing cells exist in the bonemarrow cavity. The mononuclear cells are isolated from red blood cells by Ficoll density centrifugation, and the MSCs are separatedfrom the mononuclear cells by plastic adherence in culture.

Figure 3. MSC interactions with immune cells. MSCs are immunoprivileged cells that inhibit both innate (neutrophils, dendritic cells,natural killer cells) and adaptive (T cells and B cells) immune cells. Illustration credit: Cosmocyte/Ben Smith. INF-� indicatesinterferon-�; TNF, tumor necrosis factor. (Illustration credit: Cosmocyte/Ben Smith.)




and transforming growth factor (TGF)-�1.19 When MSCs arepresent during naïve T-cell differentiation to CD4� T-helpercells, there is a marked decrease in the production ofinterferon-� and an increase in the production of IL-4,52

which suggests that MSCs alter naïve T cells from a proin-flammatory state (heavy production of interferon-�) to anantiinflammatory state (greater production of IL-4). TheB cell, which produces antibodies, is highly dependent onT cells, and MSC inhibition of T cells likely contributes to theinteractions of MSCs with B cells.5 Conflicting data haveshown that MSCs can inhibit and promote proliferation ofB cells.54,55

In vivo studies have demonstrated that MSCs may play animportant role in immune-mediated diseases and rejection oftransplanted tissue. Skin allograft transplants survive longerwith concomitant intravenous administration of MSCs in ababoon model,56 and systemic infusion of MSCs results inpreferential homing to areas of injury.57 Infusion of MSCs ingraft-versus-host disease, a life-threatening complication ofallogeneic HSC transplantation, has demonstrated encourag-ing results in steroid-resistant graft-versus-host disease.58

Although MSCs have been shown to suppress innate andadaptive immune cells, in vitro experiments with MSCsinduced to acquire a cardiac or vascular phenotype haveincreased myosin heavy chain Ia and II (immunogenic)expression and decreased myosin heavy chain Ib (immuno-tolerant) expression.59 Furthermore, when allogeneic MSCswere transplanted into post-MI rats, the cells were eliminatedfrom the heart and did not improve cardiac function.59 Thesedata from rats suggest that MSCs may lose their immuno-privileged properties as they differentiate; however, alloge-neic MSCs transplanted into pigs were shown to engraft to alarge extent as undifferentiated MSCs (�75% of cells iden-tified), with few MSCs differentiated (�25%), and they didnot induce an immune response.21 In addition, engraftment ofthese undifferentiated MSCs in post-MI hearts can stimulateendogenous repair mechanisms.40

Multilineage Potential of MSCsStem cells are characterized by their ability to self-renew,clone, and differentiate into multiple tissues.60 The firstMSCs isolated more than 40 years ago were observed inculture to form bone and cartilage deposits,3,61 and thismultilineage capability has become a defining feature ofMSCs.62 Subsequent experiments in the 1970s using autolo-gous transplantation of pellets of BM-MSCs to the subcap-sular region of a rabbit kidney demonstrated the in vivodifferentiation capabilities of MSCs to osteocytes.7 Caplan13

expanded on this pioneering work by demonstrating thatMSC differentiation into a distinct phenotype (eg, chondro-cytes or osteoblasts) is dependent on surrounding conditions.For example, MSC differentiation into osteoblasts is depen-dent on close proximity to vasculature; however, differenti-ation to the chondrocyte requires no vasculature.63 Ulti-mately, these early observations paved the way forexperiments that have defined specific conditions to promoteMSC differentiation.6

In Vitro StudiesMSCs in the presence of dexamethasone, �-glycerol phos-phate, and ascorbic acid express alkaline phosphatase andcalcium accumulation, a morphology consistent with osteo-genic differentiation.6,64–66 These osteogenic cells will reactwith antiosteogenic antibodies and form a mineralized extra-cellular matrix. The osteogenic potential of MSCs is con-served through numerous passages by their increased alkalinephosphatase activity.65 To induce chondrocyte differentiation,MSCs are cultured in a medium that contains dexamethasoneand TGF-�3.67 Subsequently, the cells begin secreting anextracellular matrix, which includes type II collagen, aggre-can, and anionic proteoglycans, consistent with articularcartilage formation.6 Glucocorticoids play an important rolein the differentiation of MSCs to the chondrocyte lineage bypromoting TGF-�–mediated upregulation of collagen type IIand inducing the matrix components aggrecan, dermatopon-tin, and collagen type XI.68 MSC differentiation into anadipogenic lineage can be induced by culturing cells in thepresence of dexamethasone, insulin, indomethacin, and1-methyl-3-isobutylxanthine.6 Cells express markers consis-tent with adipocytes, such as peroxisome proliferation-acti-vated receptor-�2, lipoprotein lipase, and fatty acid bindingprotein aP2.6 These adipocytes accumulate lipid-rich vacu-oles and eventually coalesce.

BM-MSCs also have a capacity for differentiation intoother mesoderm-derived tissue, notably myocytes, as shownin some but not all studies.14,69 Rat and human BM-MSCscultured with 5-azacytidine differentiate into multinucleatedmyotubes, consistent with a myocyte lineage.14,18 Theseinduced myocytes express �-myosin heavy chain, desmin,and �-cardiac actin and display spontaneous rhythmic cal-cium fluxes and potassium-induced calcium fluxes.18 Furtherexperiments have also shown that both human and porcineMSCs can be induced to skeletal myogenic differentia-tion.17,70 Murine BM-MSCs exposed to 5-azacytidine havebeen shown to spontaneously beat alone, connect to adjoiningcells, and after 2 to 3 weeks beat in synchrony.71 Interest-ingly, electrophysiological evaluation of these cells showed 2types of morphological action potentials, sinus node–likepotentials and ventricular myocyte–like potentials. Reverse-transcription–polymerase chain reaction demonstrated atrialnatriuretic peptide, brain natriuretic peptide, �- and [�-myosin heavy chain, Nkx2.5, and GATA4 genes. The authorsconcluded that MSCs are capable of adopting a cardiomyo-cyte phenotype in vitro.71

Several coculture experiments with cardiac myocytes haveshown the ability of MSCs to transdifferentiate into a cardiacphenotype.69,72 When mouse MSCs and rat ventricular myo-cytes were cocultured, MSCs became �-actin positive andformed gap junctions with native myocytes, and these differ-entiating MSCs exhibited synchronous contractions withnative myocytes. However, MSCs did not become �-actinpositive when separated by a semipermeable membrane frommyocytes, which suggests that transdifferentiation requirescell-cell contact.72 Contrary to these findings, rat BM-MSCscocultured with neonatal rat ventricular myocytes separatedby semipermeable membrane were shown to transdifferenti-ate into cardiomyocytes.69 After 1 week, these MSCs were




observed to be contracting, expressed SERCA2 and ryano-dine receptor 2 by reverse transcription–polymerase chainreaction, and were positive for cardiac troponin T, sarcomeric�-actinin, and desmin by immunohistochemistry. These stud-ies suggest that physical cell-cell contact, the local cardiacmilieu, and factors secreted by myocytes play important rolesin MSC transdifferentiation to cardiomyocytes. Functionalcardiac differentiation has been described by some to notoccur without coculturing of MSCs.73 These interestingfindings require further investigation but speak to the conceptthat factors traverse from cell to cell to facilitate stem celldifferentiation, which clearly has an important role in vivo.40

Another key factor is mechanical loading, as shown elegantlyby Pijnappels et al.74 This factor may also help explain theincreased propensity for MSC differentiation to occur in vivoor in coculture situations that add mechanical forces. MSCstransdifferentiate into nonmesoderm–derived tissue, such asneurons, when exposed to �-mercaptoethanol.75,76 As early as30 minutes in culture, MSCs express the neuronal markersNSE (neuron-specific enolase) and neurofilament-M (NF-M).75 Importantly, these MSCs that appeared to adopt aneuronal phenotype were not shown to produce an actionpotential.

Regulation of DifferentiationThe molecular regulation of MSC differentiation has mainlyfocused on 2 pathways, the Wnt canonical pathway and theTGF-� superfamily pathway.62 Wnt glycoproteins are solubleglycoproteins that engage receptor complexes composed ofLrp5/6 proteins to induce a cascade of intracellular eventsthat regulate cell proliferation and differentiation (Figure 4).77

The Wnt pathway has been shown to be critical in skeleto-genesis by promoting osteoblast proliferation77 and by sup-pression of chondrocyte formation via �-catenin, an essentialcomponent in transducing Wnt signaling to the nucleus.78

Numerous Wnt ligands and receptors are expressed on MSCs,and regulation of Wnt proteins controls MSC differentia-tion.79 For example, exposure to Wnt3a inhibited MSCdifferentiation into osteoblasts while promoting undifferenti-ated MSC proliferation.80

The other molecular pathway that regulates MSC differen-tiation is the TGF-� pathway, a family of proteins involved inskeletal tissue growth and regulation of MSC differentiationinto chondrocytes.6,62,67 TGF-�3 upregulates gene expression inMSCs to promote chondrogenic differentiation via several intra-cellular cascades, including extracellular-signal regulated kinase(ERK1/2), SMAD proteins, mitogen-activated protein (MAP)kinases, p38, and JNK (Figure 4).62,81 Several other moleculeshave been shown to regulate MSC differentiation, such asepidermal growth factor (EGF), platelet-derived growth factor(PDGF), and fibroblast growth factor (FGF).82,83 Indeed, manygrowth factors likely interact with the Wnt and TGF-� pathwaysto control MSC differentiation.62

In Vivo Mechanism of Action inCardiac Repair

Engraftment and Differentiation: Cardiomyocytesand VasculatureNumerous in vivo rodent and swine studies have demonstratedthe ability of MSCs to engraft and differentiate within theheart.17,20,21,84–87 In 2002, Toma and colleagues17 reported theresults of human BM-MSCs transfected with �-galactosidasereporter gene injected into immunodeficient adult mouse hearts.The majority of human MSCs were found in the spleen, lung,and liver at 4 days after injection. Only 0.44% of injectedhuman MSCs were identified in the myocardium, and overtime, they adopted a morphology indistinguishable from hostcardiomyocytes. Immunofluorescence staining with anti-�-galactosidase identified engrafted MSCs in the myocardium,

Figure 4. Molecular regulation of MSC differentiation. Wnt signaling and TGF-� induce intracellular signaling and regulate differentia-tion of MSCs. (Illustration credit: Cosmocyte/Ben Smith).




which colocalized with markers of cardiomyocyte lineage(desmin, �-actin, and cardiac troponin T) as early as 14 daysafter injection. Shake and colleagues20 expanded on theseearly murine studies by surgically injecting autologous por-cine DiI-labeled MSCs directly into post-MI swine myocar-dium. Successful engraftment was demonstrated by observingthe labeled MSC engrafting into scarred myocardium, as wellas by expression of the cardiomyocyte markers �-actin,tropomyosin, troponin T, myosin heavy chain, and phospho-lamban within 2 weeks after injection.

In a swine model of chronic ischemic cardiomyopathy, ourgroup reported the capacity of allogeneic MSCs to engraftand differentiate into cardiomyocytes, smooth muscle cells,and endothelium.21 Male BM-MSCs were injected into fe-male swine and identified by colocalization withY-chromosome fluorescence in situ hybridization. Cardiomy-ocyte differentiation was present in approximately 14% ofY-positive MSCs as identified by costaining for the cardiacstructural proteins �-sarcomeric actinin and tropomyosin andthe transcriptional factors GATA-4 and Nkx2.5. Additionally,differentiated myocytes exhibited the capacity for couplingwith host myocytes via connexin-43. MSCs also were shownto participate in coronary angiogenesis (actinin, calponin, andsmooth muscle protein 22-� expression) and accounted forapproximately 10% of Y-positive cells identified, with theremaining 76% of engrafted cells identified as clusters ofimmature cells in the interstitial compartment. In anotherswine study, Makkar and colleagues87 also demonstrated theability of allogeneic MSCs to engraft and differentiate intovascular cells and cardiomyocytes that formed connexin-43gap junctions with adjacent host cardiomyocytes. Yang andcolleagues84 found that combining a statin with intramyocar-dial injections of BM-MSCs improved the efficiency ofcardiomyocyte differentiation by �4-fold in a swine model ofacute myocardial infarction. In a dog model of chronicischemic cardiomyopathy, Silva and colleagues85 showed thatcanine MSCs engraft and differentiate into cells with a vascularphenotype, but they were unable to show MSC differentiationinto cardiomyocytes using troponin I colocalization techniques,which suggests important species differences.

In contrast to numerous reports of engraftment, Dixon andcolleagues88 transplanted male mesenchymal precursor cells(a subpopulation of MSCs that express STRO-3) into post-MIfemale sheep and were unable to demonstrate engraftment. At1 hour after injection, successful implantation was confirmedby immunolocalization for the Y chromosome, but at 8 weeksafter transplantation, staining for the Y chromosome was notdetected, which suggests failure of prolonged engraftment.Furthermore, polymerase chain reaction confirmed the lack ofengraftment by the inability to detect sex-determining regiongenes in any female swine hearts at 8 weeks after injection.

Paracrine SignalingThe frequency of MSC engraftment and differentiation in theheart is low compared with the robust functional recoveryobserved after cell transplantation, which has raised questionsas to whether MSC engraftment and differentiation is thepredominant mechanism of action. MSCs are known tosecrete soluble paracrine factors that have been postulated to

contribute to endogenous cardiomyogenesis and angiogene-sis.89–94 MSCs secrete a wide array of cytokines and growthfactors that can suppress the immune system, inhibit fibrosisand apoptosis, enhance angiogenesis, and stimulate differen-tiation of tissue-specific stem cells (Table 1).92,95–97

In vitro studies have shown that conditioned medium fromhypoxic genetically modified MSCs overexpressing Akt

Table 1. Paracrine Factors Secreted by MSCs

Secreted Factor Function

Proangiogenesis

Fibroblast growth factor-2(FGF-2)90,96

Induces endothelial and smoothmuscle cell proliferation

Fibroblast growth factor-7(FGF-7)96

Induces endothelial cell proliferation

Monocyte chemoattractantprotein-1 (MCP-1)92

Induces angiogenesis; recruitsmonocytes

Platelet-derived growth factor(PDGF)96

Induces smooth muscle cellproliferation

Placental growth factor (PlGF)92,96 Promotes angiogenesis

Transforming growth factor-�(TGF-�)96

Promotes vessel maturation

Vascular endothelial growth factor(VEGF)90,92

Induces endothelial cell proliferation,migration, tube formation

Remodeling of extracellular matrix

Metalloproteinase-1 (MMP1)96 Loosens matrix; tubule formation

Metalloproteinase-2 (MMP2)96 Loosens matrix; tubule formation

Metalloproteinase-9 (MMP9)96 Loosens matrix

Plasminogen activator (PA)96 Degrades matrix molecules

Tumor necrosis factor-� (TNF-�)96 Degrades matrix molecules; cellproliferation

Stem cell proliferation, recruitment,and survival

Basic fibroblast growth factor(bFGF)92

Enhances proliferation of endothelialand smooth muscle cells

Granulocyte colony stimulatingfactor (G-CSF)97

Increases proliferation anddifferentiation of neutrophils

Insulin-like growth factor-1(IGF-1)90

Regulates cell growth andproliferation; inhibits apoptosis

Macrophage colony stimulatingfactor (M-CSF)97

Increases proliferation anddifferentiation of monocytes

Thymosin-�4 (T�4)90 Promotes cell migration

Stem cell-derived factor (SDF)96 Progenitor cell homing

Secreted frizzled-related protein-1(SFRP1)135

Enhances cell development

Secreted frizzled-related protein-2(SFRP2)93

Inhibits apoptosis; enhances celldevelopment

Immunomodulatory

Heme oxygenase-1(HO1)48 Inhibits T-cell proliferation

Hepatocyte growth factor (HGF)90 Inhibits CD4� T-cell proliferation

Indoleamine 2,3-dioxygenase(IDO)46

Inhibits innate and adaptiveimmune cell proliferation

Inducible nitric oxide synthase(iNOS)49

Inhibits inflammation

Interleukin-6 (IL-6)97 Regulates inflammation; VEGFinduction

Prostaglandin E2 (PGE2)53 Inhibits inflammation




(Akt-MSCs) inhibits apoptosis and can trigger spontaneouscontractions of rat cardiomyocytes.90 When this conditionedmedium from Akt-MSCs was injected into post-MI rat hearts,infarct size was reduced and LV function improved.90 Severalgenes coding for vascular endothelial growth factor, fibro-blast growth factor-2, HGF, and insulin-like growth factor-1are upregulated in hypoxic Akt-MSCs, and these factorscould mediate the functional improvements observed.90 Fur-thermore, these investigators suggest that these improvementsare seen within 72 hours, and meaningful engraftment isunlikely to provide such rapid improvements, but secretedparacrine mediators are able to immediately affect the mi-lieu.91 Additionally, Akt-MSCs secrete frizzled-related pro-tein (Sfrp2), a paracrine factor that exerts a prosurvival effecton ischemic myocardium through modulation of Wntsignaling.93

Murine BM-MSC concentrated conditioned media hasbeen shown to consist of vascular endothelial growth factor,basic fibroblast growth factor, placental growth factor, andmonocyte chemoattractant protein-1, which enhance prolifer-ation of endothelial and smooth muscle cells.92 Comparedwith controls, increased local levels of vascular endothelialgrowth factor and fibroblast growth factor proteins weredetected when �-galactosidase–labeled MSCs were injectedinto ischemic murine peripheral muscles. These MSCs weredetected in decreasing quantities from days 7 to 28 aftertransplantation and failed to incorporate into vessels. Admin-istration of the factors secreted by MSCs alone may not havethe same impact as administration of the cells. In a swinemodel of MI, intramyocardial injection of MSCs reducedinfarct size by recruiting endogenous c-kit� cardiac stemcells (CSCs), whereas a single application of concentratedconditioned media did not.40

Cell-Cell Interactions and Niche ReconstitutionOur group has shown the ability of MSCs to stimulateproliferation of endogenous c-kit� CSCs and enhance cardio-myocyte cell cycling.40 Post-MI female swine were injectedwith green fluorescent protein (GFP)–labeled allogeneicMSCs to the infarct border zone and on histological exami-nation were shown to exhibit chimeric clusters that containedimmature MSCs and c-kit� CSCs. These niches containedadult cardiomyocytes, GFP� MSCs, and c-kit� CSCs thatexpressed connexin-43–mediated gap junctions andN-cadherin mechanical connections between cells. A 20-foldincrease in the endogenous c-kit� CSCs was observed inMSC-treated animals compared with those given placebo.Additional coculture experiments with c-kit� CSCs andMSCs demonstrated a 10-fold increase in c-kit� CSC prolif-eration compared with c-kit� CSC cultures without MSCs.These data suggest that MSCs induce endogenous c-kit� CSCproliferation, and cell-cell interactions play an important rolein MSC-based cardiac repair mechanisms.

Using genetically engineered mice that permanently ex-press GFP in all cardiomyocytes after tamoxifen therapy,Loffredo and colleagues99 compared the ability of BM-derived c-kit� stem cells and BM-MSCs to induce prolifer-ation of endogenous CSCs. Mice were treated with tamoxifento induce GFP expression in cardiomyocytes, underwent MI,

and were given intramyocardial injections of stem cells in theinfarct border zone. At 8 weeks, BM c-kit� stem cells led toa significant reduction in the GFP� cardiomyocyte pool andparallel increases in �-galactosidase–positive cardiomyocytescompared with control, a finding consistent with increasedprogenitor activity induced by BM c-kit� stem cells. How-ever, BM-MSCs did not lead to a reduction in the GFP�

cardiomyocyte pool or increase �-galactosidase–positive car-diomyocytes, which suggests that MSCs may not stimulateendogenous progenitors. These findings in a murine modelare clearly different from the body of work conducted withporcine models and additionally reflect species-specific func-tions of MSCs.

MSC and Cardiomyocyte FusionFusion of BM stem cells with adult cells, including cardio-myocytes, has been proposed as a potential mechanism ofaction.100,101 BM-MSCs expressing Cre recombinase wereinjected into Cre reporter gene mice hearts, which can detectfusion of MSCs with mouse cells by LacZ gene expression.102

As early as 3 days after transplantation, rare cellular fusionwas detected at injection sites by hematoxylin-and-eosinstaining with LacZ gene expressing distinct blue cells, whichpersisted at 28 days after transplantation. These findings lendsupport to the potential for MSC fusion with cardiomyocytes,but the paucity of fused cells detected makes it unlikely to bea predominant mechanism of action considering the degree offunctional recovery.

Localization of MSCs to In Vivo NichesOne of the key roles of endogenous MSCs is to participate inthe regulation of niches, which are clusters of cells thatregulate stem cell proliferation and differentiation.103 Nichesare found prototypically in the BM,8 hair follicles bulge,104

and the intestinal epithelium.105 In addition, MSCs are locatedendogenously in mesoderm-derived bone, adipose tissue, andvascular epithelium, which are tissues also affected in Prog-eria syndrome.106 The presence of MSCs in these tissues andthe association with Progeria suggest a role for MSCs inregulating successful aging. Thus, an emerging concept isthat MSCs both serve as precursors for certain lineages andregulate the function of other lineages through participationin niches. The presence of MSCs or stromal cells in the heartsupports the concept that this principle may be operative thereas well.36

Preclinical Trials of MSC Therapy: Effects onCardiac Structure and Function

Several large-animal species, including swine, sheep, anddogs, have been used to investigate the effects of MSCtherapy in models of chronic and acute post-MI left ventric-ular dysfunction (summarized in Table 2). Stem cells can bedelivered to the heart via peripheral intravenous infusion, viadirect surgical injection during open heart surgery, or viacatheter-based intracoronary infusion, retrograde coronaryvenous infusion, or transendocardial injection.114,115 Usingradiolabeled BM stem cells, �-emission counting of harvestedorgans 1 hour after stem cell delivery demonstrated thatintramyocardial injection had the highest retention rate of




cells.115 Interestingly, most cells delivered to the heart by anymethod are not retained in the myocardium and commonlyare found in the lungs and spleen.115 Positron emissiontomographic tracking of MSCs delivered by catheter-basedtransendocardial injection showed retention of approximately6% of injected cells in the myocardium at 10 days afterinjection, with increased uptake in the pericardium andpleura.112 Although stem cell retention in the myocardiumappears to be low by any delivery route, preclinical data ofMSC therapy for cardiac disease have shown highly promis-ing results. Here, we will review the preclinical trials usingallogeneic and autologous MSCs delivered to the heart via the4 most common techniques: peripheral intravenous infusion,intracoronary infusion, catheter-based transendocardial injec-tion, and direct surgical injection.

Intravenous MSC TherapyIntravenous infusion of MSCs is the easiest and most practicalmethod for delivery because it only requires peripheral venousaccess; however, for the cells to reach the myocardium, theymust travel through the pulmonary circulation, where entrap-ment of cells is a concern.116 Intravenous infusion of MSCs ina swine model of acute MI was conducted at 15 minutes afterleft anterior descending (LAD) coronary artery occlusionwith swine randomized to vehicle or various doses (1, 3, or 10million cells/kg) of allogeneic BM-MSCs.109 At 12 weeksafter MI, LV ventriculography showed no difference inejection fraction (EF) between MSC-treated animals versusplacebo. Pressure-volume loop analysis demonstrated a sig-nificant improvement in the end-systolic pressure-volumerelationship and preload recruitable stroke work in MSC-

treated animals compared with placebo. Histological analysisof postmortem hearts showed significantly greater density ofvon Willebrand factor–positive blood vessels and vascularendothelial growth factor expression in MSC-treated animals.Steady state coronary blood flow reserve was similar amonggroups, but adenosine-recruited coronary blood flow reservewas improved in MSC-treated animals.

In a swine model of acute MI, hemodynamic and electro-physiological effects of intravenous infusion of allogeneicMSCs were studied.108 Using transthoracic echocardiogra-phy, EF improved, and less eccentric hypertrophy in MSC-treated animals was detected at 3-month follow-up than withplacebo. Confocal spectral imaging of explanted organsconfirmed DiI fluorescence in the lungs and rarely in thehearts of MSC-treated animals. Electrophysiological studiesat 3 months after MI showed shortened epicardial effectiverefractory periods in MSC-treated animals compared withplacebo. Shortened effective refractory periods may induceventricular tachycardia,117 which raises the possibility thatMSCs may induce proarrhythmic remodeling.

Intracoronary Infusion of MSCsIntracoronary infusion of stem cells is delivered with astandard over-the-wire balloon angioplasty catheter placedinto the target coronary artery.118 After the angioplastycatheter is positioned, the balloon is inflated at a low pressureto block blood flow (which allows adhesion and potentialtransmigration) while the cells are infused through the distallumen.113,118 Concern about inducing ischemia during coro-nary artery occlusion and the lack of vessels in chronicallyoccluded areas of scar tissue may not allow effective cell

Table 2. Results of MSC Therapy in Large-Animal Models

Source Species Model Type of BM-MSCRoute ofDelivery Follow-Up Imaging Effects of MSC Therapy

Shake et al20 Swine Subacute MI Autologous Surgical Sonomicrometry crystals 1Systolic wall thickening

Quevedo et al21 Swine Chronic MI Allogeneic TESI CMR 2Scar size and circumferential extent;1EF; 1regional contractility;1myocardial perfusion

Schuleri et al22 Swine Chronic MI Autologous Surgical CMR 2Scar size; 1EF; 1regionalcontractility; 1myocardial perfusion

Hatzistergos et al40 Swine Acute MI Allogeneic TESI CMR 1EF; 2scar size

Hamamoto et al42 Sheep Acute MI Allogeneic Surgical Echocardiography 1EF; 2EDV

Silva et al85 Canine Chronic ischemia Allogeneic Surgical Echocardiography Attenuation of 2EF

Amado et al86 Swine Acute MI Allogeneic TESI CMR 1Diastolic function; 1mechanoenergetics;1EF; 2scar size

Makkar et al87 Swine Chronic MI Allogeneic Surgical Echocardiography;LV angiography

Preserved EF; no change in LVchamber dimensions

Perin et al107 Canine Subacute MI Allogeneic IC/TESI Echocardiography 1EF; 2EDV; 2ESV

Price et al108 Swine Acute MI Allogeneic IV Echocardiography 1EF; 2hypertrophy

Halkos et al109 Swine Chronic MI Allogeneic IV LV angiography No change in EF

Hashemi et al110 Swine Acute MI Allogeneic TESI CMR 2Scar size

Schuleri et al111 Swine Acute MI Allogeneic TESI CMR 1Myocardial perfusion

Gyongyosi et al112 Swine Subacute MI Allogeneic TESI CMR 2Scar size

Qi et al113 Swine Acute MI Autologous IC CMR 1EF; 2scar size

TESI indicates transendocardial stem cell injection; CMR, cardiac magnetic resonance imaging; IC, intracoronary infusion; ESV, end-systolic volume; IV, intravenousinfusion; 1, increase; and 2, decrease.




delivery. However, intracoronary infusion may be able toeffectively deliver cells to ischemic tissue after full reperfu-sion therapy after an acute MI. Advantages of this techniqueare the familiarity of angioplasty techniques to interventionalcardiologists and the ability to deliver cells during percuta-neous intervention for acute MI.118

Most preclinical studies of intracoronary infusion of MSCshave focused on models of acute MI.107,113,119,120 Cardiacmagnetic resonance imaging (MRI) was used to investigatethe effects of intracoronary infusion of iron oxide–labeledMSCs or placebo in swine at approximately 5 days after LADMI.113 Delayed-enhancement (DE) cardiac MRI showed an8% reduction in scar size in MSC-treated swine comparedwith increased scar size in placebo-infused animals. EF wasunchanged in placebo-infused animals, whereas a 15-pointimprovement in EF was observed in MSC-treated swine. At 8weeks after infusion, Prussian blue staining identified en-graftment of iron oxide–labeled MSCs in the peri-infarctzone.

In an ischemia-reperfusion canine model of acute MI,intracoronary infusion was compared with electroanatomicguided transendocardial injection of allogeneic MSCs (100million cells) at 7 days after MI with a no-infusion placebocontrol arm.107 Two animals died in the intracoronary infu-sion arm, 1 because of microvascular plugging associatedwith MSC infusion and another of intestinal ischemia, withno deaths in the other groups. Echocardiography at 21 daysafter transplantation showed no difference in EF or LVchamber sizes in the MSC intracoronary infusion groupcompared with placebo, whereas EF, end-diastolic volume(EDV), and end-systolic volume improved in the transendo-cardial MSC injection group. Histological analysis showedthat intracoronary infusion produces a more uniformly dis-tributed pattern of MSCs, concentrated in the border zone andnormal myocardium. Transendocardial injection was alsoused to deliver MSCs to the border zone and normal myo-cardium and achieved higher MSC concentration per squaremicron than intracoronary delivery.

Catheter-Based Intramyocardial Injectionof MSCsTransendocardial stem cell injection delivers MSCs directlyinto the myocardium with a catheter navigated in the LV byfluoroscopic guidance or electroanatomic mapping.118 Tran-sendocardial stem cell injection uses a needle deployed fromthe tip of a catheter that engages and infuses cells into themyocardium. Perforation of myocardium, with the potentialfor cardiac tamponade, and induction of arrhythmias must bemonitored with transendocardial stem cell injection, but theminimally invasive ability to directly inject cells into scar andborder zones makes it an attractive delivery technique.112,115

In a series of acute MI studies in swine, our groupdemonstrated the ability of transendocardial MSC injection toimprove cardiac function and reduce scar size (Figure5).40,86,121 Allogeneic MSCs were administered to the borderand infarct zones at 3 days after MI, and DE cardiac MRIshowed approximately 50% reduction in scar size at 8 weeksafter transplantation, whereas control animals had no changein scar size.86 On gross pathological examination, MSC-treated animals had infarct confined to the midmyocardium,with visible myocardium present at both the subendocardialand subepicardial zones. The subendocardial tissue, alsopresent on DE-MRI, was consistent with new tissue growthand was not evident on control animals. Cardiac MRIdemonstrated an increase in EF from 25% to 42% at 8 weeksafter injection, whereas the nontreated animals had minimalchange in EF. Pressure-volume loops showed improved LVrelaxation (LV end-diastolic pressure and relaxation time)and systolic compliance (end-systolic pressure-volume rela-tionship) in MSC-treated animals compared with placebo.Additional work by our group has also shown that transen-docardial MSC injection improves resting myocardial bloodflow by first-pass gadolinium perfusion MRI at 8 weeks afterinjection compared with placebo.121 Vessel density assessedby von Willebrand factor expression was similar in MSC- andplacebo-treated swine, but MSC-injected animals had signif-icantly larger vessels that likely contributed to improvedtissue perfusion.

Figure 5. Impact of allogeneic MSC therapy on infarct size. DE-MRI images of (A) swine with chronic ischemic cardiomyopathyinjected with 200 million allogeneic MSCs before and 3 months after therapy, showing a 17% reduction in scar size, and (B) a controlanimal showing no change in scar size (red arrows indicate gadolinium-enhanced scar).




In another study, 3 allogeneic MSC doses (2.4�107,2.4�108, or 4.4�108) versus placebo delivered by electro-anatomic mapping–guided transendocardial stem cell injec-tion were compared at 3 days after MI.110 DE-MRI at 12weeks after injection showed reduced infarct size in all MSCdosage groups, whereas placebo-injected animals had in-creased infarct size. Interestingly, there was no dose-dependent effect on scar size, and EF at 12 weeks was similarin all groups. In another study, transendocardial injection ofautologous MSCs also produced a significant reduction inDE-MRI–derived scar size at 10 days after injection com-pared with placebo injection.112

To investigate the impact of MSCs on healed scars, ourgroup studied transendocardial allogeneic MSC injection inswine with 3-month-old anterior wall infarcts.21 At 3 monthsafter MI, remodeling in swine is nearly complete and resultsin depressed LV function.111 DE-MRI at 12 weeks afterinjection showed that MSC therapy resulted in a 30%reduction in scar size, whereas the placebo-injected animalshad unchanged scars. Furthermore, the circumferential extentof scar decreased approximately 14% in MSC-treated swineand increased slightly in placebo-injected animals. Whentagged MRI was used to assess regional LV function, peakEulerian circumferential shortening demonstrated improvedcontractility of the infarct and border zones in MSC-treatedanimals, whereas further declines in function were evident inthe placebo group. Scar size reduction and increased regionalfunction led to improved global LV function in MSC-treatedanimals, whereas EF remained depressed in placebo-injectedswine. These findings are consistent with the ability ofintramyocardial injection of MSCs to lead to reverse remod-eling in the chronically injured heart.

Direct Surgical Intramyocardial Injection of MSCsThe largest preclinical experience with allogeneic and autol-ogous MSC therapy for cardiac disease used direct surgicalintramyocardial injection.20,22,42,85,87,88,122 Surgical injection isthe most invasive delivery technique because it requireseither a thoracotomy or sternotomy for cell delivery, but itallows direct visualization of scarred myocardium and needleengagement. If perforation occurs, it can be controlled at thetime of injection with sutures. Direct surgical injection ofMSCs has been studied in large animals with acute MI,subacute MI, and chronic ischemic cardiomyopathy.

In a sheep model of acute LAD coronary artery occlusionwithout reperfusion, BM-derived STRO-3–positive mesen-chymal precursor cells were injected directly into the infarctborder zone 1 hour after MI.42,88 As discussed above, STRO-3–positive mesenchymal precursor cells are a subset ofBM-MSCs with extensive capacity for proliferation anddifferentiation.123 Animals were allocated to 1 of 4 differentcell dosages (25, 75, 225, or 450 million cells) or placebo.Echocardiography at 8 weeks after injection demonstratedimproved EF in all mesenchymal precursor cell–treatedanimals compared with placebo. Compared with placebo-treated animals, EDV was significantly smaller in the 2low-dose groups (25 and 75 million cells), but it wasunchanged in the 2 high-dose groups (225 and 450 millioncells). EF improved significantly in all cell-treated animals

compared with placebo. Echocardiography-derived MI lengthwas significantly smaller in the 3 low-dose groups (25, 75,and 225 million) compared with placebo but was not signif-icantly different in the high-dose (450 million)–treated groupcompared with placebo. These data suggest that there may bea threshold effect with this particular subpopulation of MSCs.

In an ischemia-reperfusion swine model of subacute MI,autologous MSCs (60 million cells) were injected directlyinto 2-week-old infarcts via a midline sternotomy.20 Sonomi-crometry crystals were placed in the infarct region duringexposure of the heart to assess wall thickness and regionalcontractility. At 4 weeks after injection, a trend towardimproved end-diastolic wall thickness and improved systolicwall thickening was seen in MSC-treated animals comparedwith placebo.

Our group reported the results of direct surgical injectionsof autologous MSCs in a swine model of ischemic cardio-myopathy.22 Animals underwent LAD coronary artery occlu-sion followed by reperfusion, and at 12 weeks after MI, theyreceived either 20 million or 200 million autologous MSCs orplacebo injections to the infarct and border zone. Serialcardiac enzymes (troponin I, creatine kinase, and creatinekinase-MB) and white blood cells obtained after injectionsshowed no difference between groups. Cardiac MRI was usedto assess LV function, scar size, and myocardial blood flow.DE-MRI conducted at 12 weeks after injection demonstrateda trend toward decreased infarct size in low-dose MSC–treated animals and a significant reduction in infarct size inhigh-dose MSC-treated animals. Furthermore, decreased cir-cumferential extent of scar was evident in MSC-treatedanimals, whereas there was a trend in scar expansion inplacebo-treated animals. Regional contractility as assessed bytagged MRI–derived peak Eulerian circumferential shorten-ing showed improved contractility of the infarct border zonesof low- and high-dose MSC–treated animals. The high-doseMSC–treated animals also had significant improvements inregional contractility of the infarct zone. First-pass myocar-dial perfusion MRI showed improved myocardial blood flowin both high- and low-dose MSC–treated animals comparedwith no change in placebo. The reduction in scar size,improved regional contractility, and increased myocardialblood flow resulted in increased EF of MSC-treated animalscompared with unchanged EF in placebo-treated animals.

In a swine model of LAD coronary artery occlusionwithout reperfusion, allogeneic BM MSCs (200 million cells)or placebo was surgically injected during thoracotomy at 30days after MI.87 Sixty days after injection, global cardiacfunction assessed by contrast LV angiography showed dete-rioration in EF of placebo-treated animals, whereas MSC-treated animals had preserved EF. Echocardiography-derivedLV chamber dimensions were not different between MSC orplacebo-treated animals. Infarct size determined by planim-etry imaging showed no difference between treated andplacebo groups. This is a particularly important study becauseit stands in contrast to the totality of large-animal experiencewith reperfusion models, in which MI size is reduced byallogeneic and autologous MSC injection.

MSC therapy has also been studied in dogs with chronicischemic hibernating myocardium.85 Dogs underwent amer-




oid constrictor placement around the LAD coronary artery toinduce chronic ischemia, and 30 days later, direct surgicalinjections of allogeneic BM-MSCs (100 million cells) orplacebo were administered to the ischemic territory duringthoracotomy. Serial cardiac enzymes (creatine kinase-MBand troponin I), C-reactive protein, and white blood cellsobtained after injections showed no comparable levels be-tween groups. Cardiac function assessed by echocardiogra-phy showed significant attenuation in EF in MSC-treatedanimals compared with placebo at 30 days after injection.Quantitative morphometry assessment of scar size showed atrend toward reduced fibrosis in the anterolateral wall ofMSC-treated animals compared with controls.

In summary, MSC therapy improves LV function, reducesscar size, and increases myocardial tissue perfusion inpost-MI large-animal models regardless of delivery methodor species. Intramyocardial injection appears to have thehighest retention rate, and accordingly, compared with intra-coronary infusion, it has a greater impact on LV func-tion.107,115 The optimal time to deliver MSCs in the post-MIsetting, whether acutely during the remodeling phase or in thechronic setting after a healed scar is developed, needs betterand more rigorous definition in future studies. Other impor-tant lessons from cell injection trials include the importanceof reperfusion.

Genetically and PharmacologicallyModified MSCs

Several preclinical studies have investigated the effects ofgenetically modified MSCs and concomitant pharmacologi-cal therapy on cardiac repair.84,124,125 In an acute MI swinemodel of ischemia-reperfusion, direct surgical injections ofautologous MSCs (30 million cells) alone or in combinationwith oral simvastatin were compared with placebo-injectedswine with and without oral simvastatin therapy. At 6 weeksafter injection, myocardial single-photon emission tomogra-phy (SPECT) assessment of myocardial perfusion showed areduction in the perfusion defect in both the simvastatin aloneand combination (simvastatin�MSC) groups compared withcontrol; however, there was no added benefit to improvingmyocardial perfusion by adding MSC therapy to simvastatinalone. Cardiac MRI showed improvement in EF in thecombination (simvastatin�MSC) group compared with con-trol. There was no difference in EDV between groups, andend-systolic volume was significantly less in the combination(simvastatin�MSC) group than in controls. DE-MRI showedreduced scar size in both simvastatin alone and combination(simvastatin and MSC) groups compared with placebo, butnot between groups. Systolic wall thickening improved onlyin the combination (simvastatin�MSC) group compared withcontrol. Histology showed increased engraftment of MSCswith concomitant treatment with simvastatin compared withMSC therapy alone, which suggests that a statin may improveretention and survival of MSCs in post-MI hearts.

In a swine model of chronic ischemic cardiomyopathy, theeffect of adding HGF to BM-MSC therapy was investi-gated.124 Four weeks after permanent LAD occlusion, swinewere administered autologous MSCs (5 million cells) alone,combination HGF-MSCs (5 million cells), or placebo via

intracoronary infusion to the non–infarct-related artery. Gatedmyocardial SPECT showed no change in myocardial perfu-sion of placebo-treated swine; however, both the MSC-aloneand MSC-HGF groups had significant improvements at 4weeks, with no difference in myocardial perfusion betweenMSC alone and MSC-HGF groups. SPECT-derived EFshowed significant improvement in MSC-only and MSC-HGF–treated animals compared with no change in controls.Histological analysis showed increased vessel density inMSC-only and MSC-HGF–treated animals compared withplacebo but no difference between treated groups. Takentogether, the results of this study suggest that the addition ofHGF to MSC therapy does not provide additional benefit overMSC therapy alone.

Heme oxygenase-1 (HO-1) is an inducible enzyme thatrapidly degrades heme, which results in antiapoptotic andantiinflammatory activity, and in vitro studies have shownHO-1 to enhance the tolerance of MSCs to hypoxia-reoxy-genation injury.126 BM-MSCs transfected to overexpressHO-1 were administered to cells or placebo via intracoronaryinfusion after acute MI in a swine model of ischemia-reperfusion.125 Cardiac MRI at 3 months after injectiondemonstrated improved EF in HO-1–transfected MSCs com-pared with placebo- and plasmid-transfected MSCs. EDVwas similar among groups, whereas end-systolic volume wassignificantly lower in the HO-1–transfected MSC group.Western blot demonstrated significantly enhanced myocar-dial expression of vascular endothelial growth factor andreduced expression of tumor necrosis factor-� and IL-6 inHo-1–transfected MSC-treated hearts compared with control.Histological analysis demonstrated increased capillary andarteriolar density in HO-1 MSC-treated hearts. Taken to-gether, HO-1 overexpression in MSCs appears to be associ-ated with decreased inflammatory cytokine levels and im-proved angiogenesis when transplanted to post-MI swinehearts.

In another genetically modified MSC study, BM-MSCswere transfected with Akt, an enzyme that has been shown toprotect cardiomyocytes against apoptosis after ischemia-reperfusion injury.127,128 Transfected Akt-MSCs, MSCsalone, or placebo was administered via intracoronary infusion3 days after MI. Coronary angiograms after intracoronaryMSC infusion showed several cases of slow coronary flowthat required administration of intracoronary adenosine, ni-troglycerin, or nicorandil. At 4 weeks after transplantation,SPECT showed significantly improved EF in Akt-MSC–treated animals compared with MSC alone, whereas bothshowed improved LV function compared with placebo. In-farct size by SPECT was significantly less in both MSC-onlyand Akt-MSC–treated animals compared with placebo. Fur-thermore, the infarct size was significantly smaller in Akt-MSC–treated animals than with MSC alone, which suggeststhat Akt-MSCs may provide additional scar size reduction.

Safety of MSC TherapyAlthough evidence continues to accrue that MSCs have greatpotential as a new therapy to treat damaged myocardium, it iscrucial to keep safety concerns in mind. Two key concerns thatwarrant mention include proarrhythmia and tumor formation.129




Intravenous infusion of MSCs in swine was shown to alter theelectrophysiological properties of the myocardium108; contraryto these findings in swine, intravenous infusion of allogeneicMSCs in humans with acute MI demonstrated fewer ventric-ular arrhythmias than with placebo infusion.23 Furthermore,other clinical trials have not shown increased arrhythmiasafter MSC therapy.130,131 In the setting of ischemic cardio-myopathy, programmed electric stimulation did not detect ahigher level of inducibility in MSC-treated pigs.

Several reports have raised concerns about tumor forma-tion by use of BM-cultured MSCs.129,132 Murine-derivedBM-MSCs were used in these studies and were shown toundergo chromosomal abnormalities that resulted in tumorformation in numerous organs. As discussed previously, ina series of large-animal preclinical studies by our groupand others, MSCs were shown to be safe in large animals,with no evidence of tumor formation or ectopic tissuegrowth.22,40,42,85–88,107–110,119–121,133 Furthermore, there havebeen no reports of ectopic tissue growth in the early-phasehuman studies using MSCs.23,130,131 Nonetheless, these re-ports of tumorigenesis in murine models highlight the impor-tance of continued long-term surveillance of patients treatedwith MSCs.

Clinical Trials of MSC Therapy forCardiac Repair

Acute MIOn the basis of rigorous preclinical testing highlighted abovethat demonstrated the safety of MSC delivery to patients withcardiac disease, clinical trials have been initiated for bothacute MI and ischemic cardiomyopathy. Phase I/II clinicaldata have been reported with intravenous therapy, intracoro-nary infusion, and intramyocardial injection.23,130,131 Chenand colleagues130 investigated the effects of intracoronaryinfusion of autologous BM-MSCs (8–10�109, n�34) orsaline (n�35) in patients with subacute MI. Positron emissiontomographic imaging showed improvement in perfusion de-fects at 3 months after BM-MSC therapy, and left ventricu-lography demonstrated improved EF and LV chamber sizesin MSC-treated patients compared with placebo. Importantly,this study showed that MSC infusion was safe, with no deathsreported during follow-up, and electrocardiographic monitor-ing showed no arrhythmias.

Our group was part of a randomized study investigatingallogeneic MSCs (n�39) versus placebo (n�21) adminis-tered intravenously after acute MI.23 This dose-ranging studyshowed that intravenous allogeneic MSCs are safe in post-MIpatients, with no difference in adverse events betweengroups. Interestingly, follow-up electrocardiograms demon-strated a reduction in ventricular arrhythmias and improvedpulmonary function in MSC-treated patients. Additionally,echocardiography showed MSC-treated patients experienceda 6% increase in EF at 3 months.

Ischemic CardiomyopathyOn the basis of the large amount of mechanistic data availableand the extensive translational findings that MSCs canstimulate reverse remodeling in porcine models of ischemic

cardiomyopathy, clinical trial programs are under way to testthe safety and efficacy of MSCs and MSC precursor cells forpatients with cardiac injury due to previous MI. Our groupdirectly tested the hypothesis that transendocardial, intramyo-cardial injection of MSCs (and/or whole BM) produced acardiac phenotype of reverse remodeling by use of cardiacMRI imaging. In this study, 8 patients with chronic ischemiccardiomyopathy were administered BM-MSCs (n�4) ormononuclear cells (n�4) to the scar and border zone.131 Wedemonstrated reverse remodeling and improved regionalcontractility of the treated scar, which was evident as early as3 months after injection and persisted at 12 months (Figure6). The improved regional contractility strongly correlatedwith the reduction in both EDV (r2�0.69, P�0.04) andend-systolic volume (r2�0.83, P�0.01). Importantly, weused serial whole-body computed tomography scans thatshowed no evidence of ectopic tissue growth at 1 year aftertransplantation, and serial Holter electrocardiographic record-ings showed no sustained arrhythmias. Taken together, theearly-phase clinical trial data demonstrate that MSC therapyfor post-MI is safe and has favorable effects on cardiacstructure and function.

To follow up on this finding, 2 larger studies are underway: the Transendocardial Autologous Cells in IschemicHeart Failure Trial (TAC-HFT) and Percutaneous Stem CellInjection Delivery Effects on Neomyogenesis (POSEIDON).TAC-HFT is a randomized, double-blinded, placebo-controlled trial comparing BM-MSCs versus mononuclearcells, and POSEIDON is comparing the effects of allogeneicversus autologous MSC therapy in patients with ischemiccardiomyopathy.24 Additionally, we have completed enroll-ment in the Prospective Randomized Study of MesenchymalStem Cell Therapy in Patients Undergoing Cardiac Surgery(PROMETHEUS), a trial investigating MSC therapy deliv-ered during coronary artery bypass surgery; and recently weinitiated POSEIDON-DCM to study MSC therapy for thetreatment of idiopathic dilated cardiomyopathy. The next fewyears will witness the results of these early clinical trialefforts.

Several other clinical trial efforts are also under way,including the use of mesenchymal precursor cells and MSCstreated ex vivo with cytokines to enhance cardiopoiesis. Theresults of the C-CURE clinical trial (Safety, Feasibility andEfficacy of Guided Bone Marrow–Derived MesenchymalCardiopoietic Cells for the Treatment of Heart Failure Sec-ondary to Ischemic Cardiomyopathy) were recently provi-sionally reported, which included 45 patients with ischemicheart failure randomized to standard medical care (n�24) orguided cardiopoietic-MSC therapy (n�21) delivered to via-ble but dysfunctional myocardium by electromechanicalguidance.134 At 6-month follow-up, there was no evidence ofcardiopoietic-MSC–induced arrhythmias or toxicity. In thecardiopoietic-MSC arm, there was a significant improvementin clinical performance as assessed by the 6-minute walk test.In addition, EDV and end-systolic volume were decreased bycardiopoietic-MSC therapy compared with controls, and asignificant improvement in EF of approximately 5% wasdetected in the cardiopoietic-MSC group.




Strategies and Future DirectionsRetention of stem cells in the heart is low by any method ofdelivery,115 and strategies to enhance engraftment and differ-entiation are needed. Guided cardiopoiesis has been proposedas a technique to enhance the cardiac phenotype of MSCs andhas been shown to increase engraftment in murine hearts.98

The exact mechanism of action of MSCs (whether viaparacrine signaling, cell fusion, cell-cell interaction, or dif-ferentiation to cardiomyocytes and vascular cells) is stillhighly debated and unresolved.21,40,89 The mechanisms ofaction of MSCs in cardiac repair are likely multifaceted, andthe data accumulated to date in large-animal models andhumans have shown that MSC therapy for cardiac disease issafe and provides substantial improvements in cardiac struc-ture and function.

In summary, understanding of the endogenous roles ofMSCs and their therapeutic potential has evolved substan-tially over the past few decades. Initially considered bystand-ers of uncertain significance, MSCs are now appreciated asessential cells that govern tissue homeostasis by regulatingniches. This property, along with a capacity for multipotentialdifferentiation, has greatly facilitated a role for these cells asa cell-based therapeutic agent. Enhanced by ease of prepara-tion and immunoprivilege, MSCs have been tested in preclin-ical models and proof-of-concept clinical trials for theirability to both prevent and reverse ventricular remodeling.The early demonstrations of these effects, coupled with aremarkable safety profile, have set the stage for pivotaltesting of these cells as a therapeutic agent. Should these trialsbe positive and start to reveal clinical benefits for MSC-based

therapy, a truly transformative approach to cardiac therapeu-tics will have been achieved.

Sources of FundingDr Hare is funded by National Institutes Health grants U54-HL081028 (Specialized Center for Cell Based Therapy), P20-HL101443, and R01 grants HL084275, HL110737-01, HL107110,and HL094849.

DisclosuresDr Hare has consulted for Osiris Therapeutics, has a research grantfrom Biocardia, and is listed as an inventor on a patent for celltherapy filed by the University of Miami.

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Figure 6. Intramyocardial injection ofautologous MSCs improves regionalcontractility. Tagged cardiac MRI strainmaps (A) before MSC injection showing alateral infarct (white arrows) withdecreased contractility (red) and (B)after injection showing improvedcontractility (green/blue). CorrespondingDE-MRI (DE-CMR) 16-segment model ofinfarct size (A) before MSC injection and (D)1 year after injection (less transmularitydepicted by decreased orange/yellow).Ecc indicates Eulerian circumferential short-ening. Panels A and B are reproduced fromWilliams et al,131 courtesy of the AmericanHeart Association.




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Adam R. Williams and Joshua M. HareTherapeutic Implications for Cardiac Disease

Mesenchymal Stem Cells: Biology, Pathophysiology, Translational Findings, and

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