Tissue-Specific Progenitor and Stem Cells

Concise Review: Skeletal Muscle Stem Cells andCardiac Lineage: Potential for Heart Repair

NARMEEN HASSAN,a,* JASON TCHAO,b,* KIMIMASA TOBITAa,b,c

Key Words. Adult stem cells x Cell transplantation x Tissue-specific stem cells xProgenitor cells x Cardiac

ABSTRACT

Valuable and ample resources have been spent over the last two decades in pursuit of interventionalstrategies to treat the unmet demand of heart failure patients to restore myocardial structure andfunction. At present, it is clear that full restoration of myocardial structure and function is outsideour reach from both clinical and basic research studies, but it may be achievable with a combinationof ongoing research, creativity, and perseverance. Since the 1990s, skeletal myoblasts have been ex-tensively investigated for cardiac cell therapy of congestive heart failure. Whereas the Myoblast Au-tologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial revealed that transplanted skeletalmyoblasts did not integrate into the host myocardium and also did not transdifferentiate into cardi-omyocytes despite some beneficial effects on recipient myocardial function, recent studies suggestthat skeletal muscle-derived stem cells have the ability to adopt a cardiomyocyte phenotype in vitroand in vivo. This brief review endeavors to summarize the importance of skeletal muscle stem cellsand how they can play a key role to surpass current results in the future and enhance the efficaciousimplementationof regenerative cell therapy forheart failure. STEMCELLSTRANSLATIONALMEDICINE

2014;3:1–12

INTRODUCTION

In the United States, an estimated 15.4 millionpeople have coronary heart disease, of whom7.6 million are affected by myocardial infarctionand 2.1 million by congestive heart failure [1].In the last two decades, the classic concept ofthe heart as an organ with extremely limited re-generative capacity has been analyzed. The celltherapy approaches were limited at the turn ofthe newmillennium to the transfer of noncardiaccell types such as smoothmuscle cells, embryonicstem cells, mesenchymal bone marrow stromalcells, hematopoietic stem cells [2], and skeletalmyoblasts [3] into an injured heart. The idea ofthese engraftments was to stimulate repair andregeneration of the injured myocardium [4–6].The field of cardiac regenerative medicine wasbrought into greater focus with the recognitionof cardiac stem cells in 2001 [7] and the abilityto antigenically select cardiac progenitor cells ex-perimentally [8]. The capacity to generate cardio-myocytes in the adult human heart is 1% at theage of 25, and it decreases to 0.45% at the ageof 75 [9]. This rationale, together with publishedresearch reports and experimental studies [10],indicated that theheart is in fact capableof regen-eration, but therapeutic strategies are needed tostimulate this process in cardiac pathologies. Theuse of fetal cardiac cells poses ethical, logistical,and technical issues, and there still is no means

for effectively mobilizing a hypothetical poolof resident stem cells. Long-term functional im-provement requires stem cells with true cardio-myogenic properties and angiogenic potential.Presently, it is not clear whether such a perfectstem cell exists. However, a number of stem celltypes show promise for autologous cardiomyo-plasty, including multipotent stem cells isolatedfrom skeletal muscle, which we highlight in thisreview.

STEM CELLS ARE A PROMISING SOURCEFOR CARDIOMYOPLASTY

Unspecialized cells that are capable of continuousself-renewal, while maintaining the ability to dif-ferentiate into multiple different cell types, aredefined as stem cells. There are, broadly, threemajor categories of stem cells: embryonic stemcells, adult stem cells, and induced pluripotentstem (iPS) cells. Embryonic stem cells are undif-ferentiated cells, which are present during em-bryonic development and possess pluripotentdifferentiation capacity. Adult stem cells can beisolated from postnatal tissues and are multipo-tent. Sim et al. in 2002 [3] and Murry et al. in2004 [11] concluded that none of the adult stemcell sources convincingly demonstrated a poten-tial for significant long-term engraftment and dif-ferentiation into functional cardiomyocytes. The

aDepartment ofDevelopmental Biology,bDepartment ofBioengineering, andcMcGowan Institute ofRegenerative Medicine,University of Pittsburgh,Pittsburgh, Pennsylvania,USA

*Contributed equally as firstauthors.

Correspondence: KimimasaTobita, M.D., Rangos ResearchCenter, Room 8121, 4401 PennAvenue, Pittsburgh, Pennsylvania15224, USA. Telephone: 412-692-9902; E-Mail: kit3@pitt.edu

Received July 5, 2013; acceptedfor publication September 13,2013.

©AlphaMed Press1066-5099/2013/$20.00/0

http://dx.doi.org/10.5966/sctm.2013-0122

STEM CELLS TRANSLATIONAL MEDICINE 2014;3:1–12 www.StemCellsTM.com ©AlphaMed Press 2014

TISSUE-SPECIFIC PROGENITOR AND STEM CELLS

technique developed by Takahashi and Yamanaka in 2006 [12]demonstrated that the introduction of a quartet of transcriptionfactors, Oct4, Sox2, Klf4, and c-Myc, into terminally differentiatedcells (e.g., skin fibroblasts) changed these cells into an embryonicstem cell-like state known as iPS cells. Human iPS cells are similarto human embryonic cells in morphology, proliferation, surfaceantigens, gene expression, telomerase activity, epigenetic statusof pluripotent cell-specific genes, and cardiac potential [13]. How-ever, the maturation of iPS cell-derived cardiomyocytes remainsnonuniform [14, 15]. Human implantation of embryonic and iPScell-derived cardiomyocytes is limited by significant safety andethical issues. Studies have revealed significant genetic and epi-genetic abnormalities in iPS cells, higher than embryonic stemcells or fibroblasts. The mutation rates are estimated to be 10times as high as those of fibroblasts [16]. Chromosome p12 hasbeen found to be over-represented in iPS cell cultures, a charac-teristic associated with testicular germ cell tumors [16]. Othermutations are also associatedwith cell cycle regulation and onco-genesis, which raises the risk of teratogenesis in vivo. High-throughput functional genomics approaches areneeded tobetterunderstand the nature and consequences of thesemutations. iPStechnology avoids the ethical dilemma of embryo destruction re-lated to embryonic stem cells, but other ethical issues, includinginformed consent and genetic anonymity of donors, must still becarefully considered. Although iPS technology holds great prom-ise for the regeneration of the heart and other organs, clinical useof iPS technology in ischemic heart disease remains untested.From this point of view, adult stem cells remain a more practicaloption at the present time.

Significant efforts in the field of cardiac cell therapy have fo-cused on bone marrow-derived cells, especially bone marrowmononuclear cells such as hematopoietic and mesenchymal stemcells.Mesenchymal stemcells havenumerousdesirable character-istics, including positive paracrine effects, angiogenic potential,and an immunoprivileged profile. Studies have shown thatmesen-chymal stem cells, but not hematopoietic stem cells, can differen-tiate into cardiomyocytes in vivo [17, 18], with slightly tosignificantlypositive results inclinical trials [19,20].However, bonemarrow mesenchymal stem cells do not differentiate into cardio-myocytes in significant numbers invitro, suggesting that their ther-apeutic benefit is predominantly paracrine rather than throughtransdifferentiation [21]. Challenges includeexpanding these stemcells in clinically relevant numbers and developing safe methodsfor cardiomyocyte differentiation. (5-Azacytidine used for in vitrodifferentiation of mesenchymal stem cells into cardiomyocytes istoxic [17].) More recent work has focused on cardiac stem cells,which are found in niches within the heart and can be isolatedby myocardial biopsies. Studies using immunosuppressed ratshave shown that human cardiac stem cells can give rise tomyocar-diumwith improved function [22]. In light of these results, clinicaltrials havebeen initiated [23, 24].Whereas initial results of the SCI-PIO trial are promising, the CADUCEUS trial showed no differencein ventricular function compared with control despite a reductionof infarct size. Isolationof cardiac stemcells is also invasiveandcar-ries higher risk comparedwith isolation fromother tissues. Furtherinvestigation isneededtosafelyandeffectively isolate,expand,dif-ferentiate, and deliver these stem cells.

Cellular cardiomyoplasty of autologous skeletal muscle cellsinto the myocardium to reinforce its structure and function canbe used as an alternative to heart transplantation [25]. Transdif-ferentiation directly converts a specialized cell type to another

specialized cell type, bypassing a pluripotent state. In the 1980s,itwas shown that fibroblasts can be converted into skeletalmusclecells with the use of the MyoD transcription factor [26]. In recentyears, direct reprogramming of cardiac fibroblasts into functionalcardiomyocytes by transcription factors GATA4, Tbx5, and Mef2cboth in vitro and in vivo has been reported [15, 27]. However,the low reprogramming efficiency of this approach and the useof viral vectors are undesirable characteristics. It is interesting tospeculate that direct reprogramming to cardiomyocytes mightbe more effective in skeletal muscle stem cells, which are pre-endowed with myogenic potential. Lymphocytes were convertedinto macrophages by using a transcription factor called C/EBPa[28]. But once they are in one committed lineage, it is a challengetomake themconvert to another unrelated cell type, and transdif-ferentiation efficiency is often low. Szabo et al. [29] also provedthat blood cells can be generated from fibroblasts, making it likelythat transcription factors can induce large jumpsbetweendistantlyrelated cell types. This opened up the prospect that any desiredspecialized cell could be generated from essentially any other celltype. Because skeletal and cardiac muscle both arise from myo-genic, mesodermal lineages and share some characteristics, trans-differentiation from skeletal muscle progenitors to cardiac musclewould require less comprehensive alteration compared with dis-tantly relatedcell types,at least intheory.Cellsareconsidered idealif they are derived from relatively easy to obtain tissue, readilygrown in large numbers, available off the shelf for acute indica-tions, nonimmunogenic, nonarrhythmogenic, and able to regener-ate healthy myocardium [30]. Although the skeletal muscle stemcell isolation procedure is invasive comparedwith isolation of cellsfrombloodorurine, skeletalmuscle comprises the largest percent-age of total bodymass [31]. These desirable characteristics andde-velopmental similarity to cardiac muscle, described below, makeskeletal muscle-derived cells a strong candidate to repopulatedamaged myocardium.

SKELETAL MUSCLE AND CARDIAC MUSCLE: DIFFERENCE AND

SIMILARITY DURING DEVELOPMENT

It is awidely acceptednotion that terminally differentiatedmaturecardiac muscle does not express proteins that are specific to skel-etalmuscle. However, developing skeletalmuscle hasbeenprovento be similar to cardiac muscle by various studies, suggesting thatthey have a similar cell lineage. Different isoforms ofmyosin heavychain (MHC) are present in different muscle fiber types to meettheir functional needs. There are four major MHC isoforms in ratskeletal muscle. I/b is a slow type, and IIa, IIx/IId, and IIb are fasttypes. These are equivalent to skeletal muscle-specific fast MHC(sk-fMHC) [32–34]. In the adult myocardium, a-MHC and b-MHCare present, with the latter being identical to I/b-MHC found inskeletal muscle. Noncardiac MHCs play a major role during theearly myofibrillogenesis of nascent cardiomyocytes in assemblingsarcomere structure [35]. Recently, it was found that sk-fMHCwasexpressed in developing rat myocardium, and its expression wasattenuated in postnatal life [36].

The thin filament system in skeletal and cardiacmuscle is reg-ulated by a family of proteins known as troponins, which formpart of the troponin complex. Troponin I is encoded by three dif-ferent genes. Cardiac troponin I was previously shown to be ex-clusively expressed in the myocardium in adults [37], butrecently, it was revealed that it is also expressed in developing

2 Muscle Stem Cells for Heart Repair

©AlphaMed Press 2014 STEM CELLS TRANSLATIONAL MEDICINE

skeletal muscle [36]. It is distinct from the fast and slow forms inskeletalmuscle [37]. Cardiac troponin T is a component of the tro-ponin complex. It is encoded by the gene TNNT2 and allows acto-myosin interaction and contraction to occur in response to Ca2+.TNNT2 is a common mutation in familial hypertrophic cardiomy-opathy, but surprisingly, it has been found that distinct TNNT2mutations also lead to dilated cardiomyopathy [38]. It is alsoexpressed in skeletal muscle during injury. Apart from this, skel-etal muscle-specific troponins are transiently present in the im-mature heart [39]. In the early phases of myogenesis in skeletalmuscle, cardiac-like excitation-contraction coupling mechanismsdominate, whereas skeletal muscle-like excitation-contractioncoupling dominates in more mature muscle [40, 41]. Thus, be-tween cardiac and skeletal muscle, there is a strong overlap inthe genes encoding key proteins responsible for contractility,which is a hallmark of striated muscle.

Cardiac andskeletalmusclesalsosharecommonmetabolic reg-ulatoryproteins.Fattyacid-bindingprotein3 isamemberofa familyof binding proteins and is mainly expressed in cardiac and skeletalmuscle cells, and it has been linked to fatty acid metabolism, traf-ficking, and signaling [42]. UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamime kinase (GNE) is involved in the early devel-opment of both cardiac and smoothmuscle. GNE-deficient cardiaccells degraded very soon, and their beating capacity decayed rap-idly. Skeletal muscle-committed cells were identified in the GNE-deficient embryonic stem cell cultures by the expression of Pax7,MyoD, andMHCmarkers. GNE is strongly involved in cardiac tissueand skeletal muscle early survival and organization, as shown bythese results [43].

A common finding in certain types of muscular dystrophy, in-cluding Emery-Dreifuss, Duchenne, and Becker type dystrophies,is chronic cardiac diseases [44]. Some types are caused by muta-tions in the b-sarcoglycan-associated proteins [45], which areexpressed both in cardiac and skeletal muscle. Limb-girdle mus-cular dystrophy type 2E is caused by mutations in Sgcb gene,and Sgcb-deficient mice develop severe cardiomyopathy and se-vere muscular dystrophy, suggesting that cardiac and skeletalmuscle pathologies arise from a common origin [46]. All of thesestudies indicate how closely cardiac and skeletalmuscle are inter-linked, especially in their immature phase. Recently, our groupfound that skeletal and cardiac muscle share similar transcriptionfactors and structural proteins during development [36]. How-ever, the molecular mechanisms that regulate the fate determi-nation between these two lineages of striated muscle remainpoorly understood.

SKELETAL MYOBLASTS: AN EARLY CANDIDATE FOR

CELLULAR CARDIOMYOPLASTY

Given their phenotypic similarity to cardiac muscle, ease ofisolation/expansion, and relative resilience to hypoxia, skeletalmyoblasts were considered an appealing cell source for cellularcardiomyoplasty [47–54]. Skeletal myoblasts have shown prom-ise for ischemic heart regeneration, making them potentialcandidates for early cardiomyoplasty studies [55]. Skeletal myo-blasts were the first cell type to enter the clinical arena of heartregeneration. Lavine and Upcott in 1937 [56] performed the firstof its kind surgery in which his team treated myocardial ischemiaby using a skeletal muscle graft to the heart. The patient was typ-ically acromegalic. Before the procedure, he could not walk 5

yards without any rest, but after the surgery, he was able to walk140 yards without any shortness of breath. Early animal studiesconducted in porcine animal models showed that myoblast-derived muscle grafts remained functionally isolated from thesurrounding myocardium. The outcome did not reveal an in-creased frequency in ventricular tachycardia or fibrillation [57].Menasche et al. [49] in 2001 transplanted autologous skeletalmyoblasts into the postinfarction scar during coronary artery by-pass grafting of remotemyocardial areas. However, in thedistinctcase of skeletal myoblasts, the results of several preclinical andclinical studies have shown improved functional outcomes fol-lowing transplantation of these myogenic progenitors into post-infarction scar.

Table 1 summarizes the potential advantages and disadvan-tages of using various stem cells types for cardiac repair. Arrhyth-mogenic complications have raised concern over the safety ofadoptive transfer of skeletal muscle cell therapy [3, 11, 46–48,58]. There is a risk of ventricular arrhythmias [49, 59–62]. The factremains that myoblast transplantation has its limitations, asimplanted cells have limited survival and do not spread uniformlyat the siteof injury [48]. Skeletalmyoblastsdonotdifferentiate intocardiomyocytes but rather into multinucleated myotubes after in-jection into the heart. These myotubes form islands of conductionblock in the heart and lack gap junctions. This results in electricalinhomogeneities that slow conduction velocity and predisposepatients to re-entrant ventricular arrhythmias [63]. Whereas skel-etal myoblasts (and other skeletal muscle-derived cells) showpromise of improved myocardial contractile function, risk of ar-rhythmia is an issue that needs to be addressed to advance thistherapy.

CARDIOMYOCYTE DIFFERENTIATION POTENTIAL FROM SKELETALMUSCLE-DERIVED CELLS

Since skeletal myoblasts do not differentiate into functioning car-diomyocytes in vivo, efforts have been made to isolate cells fromskeletal muscle that can differentiate into cardiomyocytes in vitroor in vivo.Theskeletalmuscle-derived stemcell (MDSC) is a somaticstem cell that is different from skeletal myoblasts (satellite cell)in its multipotent properties [64–66]. Table 2 summarizes the dif-ferences between skeletal myoblasts and MDSCs. MDSCs areobtained fromskeletalmuscle via apreplatemethodor fluorescentactivated cell sorting of surface markers and are easily expandedunder in vitro culture environment as an autologous cell source[54, 65, 66]. MDSCs differentiate into skeletal muscle, bone, ten-don, nerve, endothelial, hematopoietic, and smooth muscle cells[54, 64–67]. They have long-term proliferation capacity and en-hanced resistance to hypoxia. Several studies have shown thatMDSCs can differentiate into functioning cardiomyocytes usingvarious strategies. Clause et al. [68] demonstrated that rat MDSCscoulddifferentiate intocardiomyocyte-likecellsexpressingcardiac-specific genes and proteins by combining cell aggregation with 3Dculture in a collagen bioreactor.Winitsky et al. [69] isolated nonad-herent cells, skeletal-based precursors of cardiomyocytes (SPOC)cells (skeletal-based precursors of cardiomyocytes), from adultmurine skeletal muscle that become beating floating cells. TheyareCD342/CD452/c-kit2. Themuscle-derivedsidepopulationcellsexpress the surface marker Sca-1 (65%), and the beating cellsdevelop out of the Sca-12 pool, which makes up 20%–40% ofthe total isolated cells. Cells expressing the cardiac-specific

Hassan, Tchao, Tobita 3

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marker Nkx2.5 and spontaneously contracting in culture weredetected in reverse transcription-polymerase chain reactionanalysis. The presence of specificmRNA codingmarkers for skel-etal (myogenin, Myf5) and cardiac muscle (GATA4) and smoothmuscle actin was seen in differentiated cells. Interestingly, allthe above-mentionedmarkerswere alsodetected in floating pro-liferating progenitor cells. This supports the idea that stem cellscan express markers of multiple lineages in their differentiatingstate [70]. Skeletal muscle interstitium-derivedmultipotent stemcells (Sk-34 and Sk-DN cells) differentiated into cardiomyocytes ina cardiac environment [50, 71]. After 4 weeks, the transplantedcells became cardiomyocytes with desmosomes and intercalateddiscs associated with gap junctions [50]. Myotube formation was

not observed. Sk-34 and Sk-DN cell differentiation is based ona cardiac environment. Table 3 lists the studies done over theyears showing cardiomyocyte differentiation and cardiac repairpotential from skeletal muscle-derived stem/progenitor cells.Taken together, these studies emphasize the importance of iso-lation techniques and culture conditions in obtaining the desiredtarget cell. For example, Parker et al. [72] showed that activationof Notch signaling during ex vivo expansion of muscle-derivedcells can significantly improve their engraftment.

There are a number of problems faced during isolation ofthese cells. Cell surface markers define muscle-derived stemand progenitor cell populations. Some of these markers are re-stricted to these populations, whereas other markers are shared

Table 1. Advantages and disadvantages of various stem cell sources

Cell type Advantages Disadvantages

Skeletal myoblast (satellite cell) Contractile phenotypeProduction of proangiogenic factorsCan be rapidly harvested and easily expandedin vitro prior to autologous transplantation.

Relatively resistant to hypoxic conditionsImproved functional outcomes followingtransplantation

Arrythmogenic complicationsLow long-term survival rateNonuniform spreading at the site of injury with

conventional delivery methodsHigh rate of rejectionLack of differentiation into cardiomyocytes in vivoLimited expansion compared with pluripotent

stem cellsInvasive isolation procedure

Bone marrow mesenchymalstem cells

Has been tested in clinical settings with positiveoutcomes

Immunoprivileged profilePotent paracrine and proangiogenic effectsleading to improved cardiac function

Differentiate into cardiomyocytes and endothelialcells in vivo

No widely used protocols for in vitro cardiomyocytedifferentiation with nontoxic agents

Lack of well-defined markers for cell isolationLimited expansion ability in vitroInvasive isolation procedure

Adipose stem cells Phenotype similar to that of bone marrowmesenchymal stem cells

Can differentiate into cardiomyocytes in vitroEasy to obtain large numbers of multipotent stemcells by liposuction

Augment cardiac function similar to bone marrowmesenchymal stem cells

Therapeutic benefit mainly from paracrine effectsand angiogenesis rather than differentiationinto cardiomyocytes

No widely used protocols for in vitro cardiomyocytedifferentiation with nontoxic agents or withoutcoculture with cardiomyocytes

Somewhat invasive isolation procedureLimited expansion ability in vitro

Induced pluripotent stem cells Can theoretically be expanded indefinitely in vitro,providing an unlimited source of stem cells

Similar in morphology, proliferation, surfaceantigens, gene expression, telomerase activity,epigenetic status of pluripotent cell specificgenes, and cardiac potential to humanembryonic cells

AutologousPluripotent

Risk of teratogenesisMaturation stages are not uniformChromatin modificationRisk of unwanted cellular transdifferentiationEpigenetic reprogrammingPotential immunogenicitySomatic cell isolation can be invasive from some

tissuesUntested in clinical setting

Table 2. Comparison of MDSCs and skeletal myoblasts

Characteristic MDSCs Satellite cells

Differentiation potential Osteogenic, chondrogenic, neural, endothelial,hematopoietic, cardiac, myogenic [31]

Myogenic [108]

Marker profile Sca-1+/2, Flk-1+, Pax72, M-cadherin2 [31] Sca-12, Flk-12, Pax7+, M-cadherin+ [31]

Regeneration potential Better [81] Good [81]

Engraftment Over 3 months [54] Several weeks [54]

Ischemia tolerance Better [54] Good [54]

Clinical outcomes Preclinical; MDSC sheets can improve heartfunction and prevent arrhythmias in animalmodels [90]

Studies suggest improved ventricularfunction but variable incidence of arrhythmias[62, 109]

Abbreviation: MDSC, muscle-derived stem cell.

4 Muscle Stem Cells for Heart Repair

©AlphaMed Press 2014 STEM CELLS TRANSLATIONAL MEDICINE

Table3.

Studiesofcardiacdifferentiation/rep

airfrom

skeletalmusclestem

cells

Study

Species

Isolationmethod

Culture

conditions

Timein

culture

Cardiomyocyte

induction/

differentiation

Key

findings

Winitskyet

al.[69

],20

05Mouse

Platingandpassaging

nonadheren

tSca-12

/+cells

50:50DMEM

/F12

,5%FBS,

EGF,bFG

F,insulin,

transferrin,selen

ium,

ethanolamine

Upto

4weeks

Spontaneo

uslybeating

phen

otype

Theinjected

cells

inamouse

modelofacute

infarctionarestillvisibleafter3monthsas

striated

heartmusclerestricted

tothe

regionofthecardiacinfarct.

Roelletal.[98

],20

07Transgen

icmice

Cardiac

andskeletal

musclecells

expressing

EGFP

under

controlofthe

human

a-actinpromoter

wereharvested

from

transgen

icmice

Invivo

transplantation

Upto

2weeks

Transplantedinto

syngeneic

wild-typemice

Skeletalmyoblastsgenetically

engineered

toexpressCx43

conferredaprotection

similar

tothatofem

bryoniccardiomyocytesagainst

indu

cedventriculartachycardia.

Invernicietal.[110],200

8Human

Isolatedfrom

the

brachioradialis

and

paravertebralm

uscleand

culturedinflasks

coated

withtype

Icollagen

TypeIcollagen/SCM,

20–50

mg/lplatelet-derived

growth

factorBB,102

7

to10

29M9cis-RA

Upto

10days

Retinoicacidtreatm

ent

Expression

ofcardiomyogeniclineage-specific

antibo

dies

withimmun

ostaining;CD133,

CD105,desm

in,and

vimentin.

Spon

taneou

scontraction.

Cardiac-like

respon

setoincreasedextracellular

calcium.

Retinoicacid-treated

skeletalmusclestem

cells

expressedconnexin43

whencocultured

with

mou

secardiomyocytes.

Cells

weredetectableeven

5weeks

after

injectionwhentransplanted

into

ischem

icheart.

Tamakietal.[50

],20

08Mouse

CD34

+/CD45

2(Sk-34

)stem

cells

wereen

zymatically

isolatedfrom

green

fluorescentprotein

transgen

icmice

DMEM

,5%–10

%FCS;IM

DM/

10%FCS;20

%FCS/IM

DM

6days

Invitroclonalculture

and

coculture

withem

bryonic

cardiomyocytes,invivo

transplantation

into

anude

ratmyocardialinfarction

model

Highexpression

ofGATA

-4,N

kx2-5,Isl-1,M

ef2,

andHand2

was

seen

oncoculturetogether

withform

ationof

gap-junction

sand

synchronou

scontractionfollowing

sphere-like

colony

form

ation.

Donor

cells

exhibitedtypicalcardiom

yocyte

structurewith

form

ationof

gap-junctio

ns,as

wellasintercalated

discsanddesm

osom

esbetweendonorandrecipientand/or

donor

anddonorcells

at4weeks

after

transplantationoffreshlyisolated

Sk-34cells.

Arsicet

al.[70],2008

Mouse

Serialpreplatingtechnique;

nonadheren

tcell

population

isolated

50:50DMEM

/F12

Upto

2months

Plated

onCCL14

6fibroblast

MDSC

exhibitedSca-1(65%

),Bcrp-1(80%

),MyoD(15%

),Neuronalb

IIItubulin(25%

),GFAP(30%

),andNkx2.5(1%).

Tamakietal.[71

],20

10Mouse

CD34

2/452

(Sk-DN)stemcells

wereen

zymaticallyisolated

from

greenfluorescent

proteintransgen

icmice

DMEM

,5%–10

%FCS;IM

DM/

10%FCS,10

ng/mlbFG

F,20

ng/mlEGF

Upto

2weeks

Singlecell-derived

single-sphereim

plantation

into

myocardium

Donorcells

exhibited

typicalcardiomyocyte

structure

withtheform

ationof

gap-junctions

betweendonorandrecipient

cells

after4weeks

ofimplantation.

Expression

ofspecificmRN

Asfor

cardiomyocytes,such

ascardiacactin

and

GATA-4,N

kx2-5,Isl-1,M

ef2,andHand2.

Cellfusion-independentdifferentiation.

Hassan, Tchao, Tobita 5

www.StemCellsTM.com ©AlphaMed Press 2014

Table3.

(Con

t’d)

Study

Species

Isolationmethod

Culture

conditions

Timein

culture

Cardiomyocyte

induction/

differentiation

Key

findings

Clauseet

al.[68],2010

Rat

Modifiedpreplating

technique

DMEM

,10%

FBS,10

%horse

serum,collagentype

I/Matrigeltissueconstruct,

DMEM

,5%FBSfor

differentiation

Upto

9days

Three-dim

ensionalgel

bioreactor

MDSCsexpressed

cardiacgenes/proteins

afteraggregateform

ationand3D

bioreactorculture.

MDSC-CMsexpressed

Cx43gapjunctionsand

generated

cardiac-likecalcium

transien

ts.

MDSC-CMsgenerated

strain-dep

enden

tactive

contractileforce.

Neefet

al.[111],2012

Neo

natalmice

Prep

latingtechnique

40%Ham

’sF10med

ium,20%

FBS,2.5ng/mlbFG

F,40

%DMEM

,2%horseserum,

pen

icillin/strep

tomycin

(1:100),fungizone(1:1,000)

Upto

14days

Collagenconstructs

Coup

lingofcells

from

pre-engineeredtissue

constructsto

cellsinem

bryonicheartslices

showed

slower

conductionvelocities

than

myocardialcellswiththeem

bryonicheart

slices,lower

maxim

um

stim

ulation

freq

uen

cies,higher

sensitivities

tothegap

junctioninhibitor,skeletalmyoblast-based

transplantablegraftsthat

electrically

coupledto

myocardium.

PerumalSrinivasan

etal.[11

2],201

2Neo

natalmice

Prep

latingtechnique

40%Ham

’sF10med

ium,20%

FBS,2.5ng/mlbFG

F,40

%DMEM

,2%horseserum,

pen

icillin/strep

tomycin

(1:100),fungizone(1:1,000)

Upto

14days

Collagenconstructs,serum

starvation

Skeletalmyoblastswithintissueconstructs

dem

onstratedlong-term

survival,o

rdered

alignmen

t,preserved

ability

todifferentiateinto

contractilemyotubes.

Theexpressionofgapjunctionandcell

adheren

ceproteinswas

maintained

or

increasedthroughoutd

ifferentiationwhen

thetissueconstructsweresubjected

topassive

longitudinaltensilestress.

Oshim

aet

al.[54],2005

Mouse

Modifiedpreplating

technique

DMEM

,10%

FBS,10

%horse

serum,1%pen

icillin/

streptomycin,0.5%chick

embryoextract

12weeks

Invivo

transplantation

(C57

BL/6mice)

Compared

withmyoblasts,MDSCs

dem

onstratedmore

persisten

ten

graftm

ent,expressionofconnexin43,

increasedangiogenesis,b

ettertolerance

foroxidativestress,greater

functional

improvemen

t,andadoptionofacardiac

phen

otypeat

lowfreq

uen

cy.

Okadaet

al.[75],20

08Human

Fluorescen

ceactivatedcell

sortingbyCD34,C

D56,and

CD14

4

DMEM

,10%

FBS,10

%horse

serum,1%pen

icillin/

streptomycin,1%chick

embryoextract

6weeks

Invivo

transplantation

(NOD/SCID

mice)

Human

myogenic-endothelialcells(CD34

2/

CD45

+/CD14

4+)aresimilarto

murine

MDSCsintheircapacityforheartrepair.

They

improved

cardiacfunctio

n,reduced

scarring,stimulated

neovascularization

,and

demon

stratedlong-term

engraftm

ent

better

than

myoblastsor

endo

thelialcells.

Xianget

al.[113],201

1Mouse,h

uman

Modifiedpreplating

technique

DMEM

,10%

FBS,10

%horse

serum,1%pen

icillin/

streptomycin,0.5%chick

embryoextract

3weeks

Invivo

transplantation

(NOD/SCID

mice)

Wnt11-transducedMDSCsshow

edgreater

engraftm

entandcardiomyogenic

differentiationcomparedwith

controlM

DSCs.

SomeWnt11-transducedMDSCsbeat

spontaneouslyinvitro.

6 Muscle Stem Cells for Heart Repair

©AlphaMed Press 2014 STEM CELLS TRANSLATIONAL MEDICINE

with other cell populations. The marker profile-based isolationmethods exclusively depend on the expression of cell surfacemarkers that are variable and may change under in vitro cell cul-ture conditions. Thus, thesemethods have their limitations. Pres-ently, the most prevalent method for isolating MDSCs is themodified preplate technique. It is a marker profile-independenttechnique that is based on variable adherence of freshly dissoci-atedmuscle cells to collagen-coated flasks. However, the isolatedpopulations are heterogeneous [73]. Multiple nonsatellite stemcell fractions reside in skeletalmuscle, although it remainsunclearhow these different populations are related. Okada et al. [74]demonstrated that slowly adhering cells showed greater regener-ative potential and oxidative/inflammatory stress tolerance com-pared with rapidly adhering cells. Myogenic-endothelial cells,cells that express both myogenic and endothelial markers (CD56,CD34, and CD144), can be isolated from skeletal muscle and havebeen shown to have superior regenerative properties and stresstolerance compared with myoblasts [75, 76]. The blood vesselsof skeletal muscle also contain stem cells, perivascular stem cells,whichdemonstratemyogenic andmultipotent potential [77]. Theyare isolated on the basis of CD146 expression and lack of CD34,CD45, CD144, and CD56 expression.

In vitro generated MDSC-derived cardiomyocytes are moresimilar functionally and biochemically to fetal cardiomyocytes[78]. However, the rate of cardiomyocyte differentiation alone islikely not sufficient to explain the functional improvements gainedfromMDSC transplantation. Another possiblemechanismbywhichMDSCs exert therapeutic benefit is by acting as a reservoir to se-crete paracrine factors that promote angiogenesis and cell survivalwithin the injured microenvironment [54]. Transplanted undiffer-entiated human skeletal myoblasts (CD56+) in an immunodeficientnude ratmodel demonstrated expression of human-specificmatrixmetalloproteinase 2 (MMP-2) TNNI3, CNN3, proangiogenic factors(PGF), TNNT2, PAX7, transforming growth factor-b (TGF-b), andinsulin-like growth factor-1 (IGF-1) 1 month after transplant. Geneexpression showed upregulation of PGF, antiapoptotics (BAG-1,BCL-2), heart development (TNNT2, TNNC1), and extracellular ma-trix remodeling (MMP-2, MMP-7) in skeletal myoblasts. The datasuggested that myoblast-secreted factors may contribute to thebeneficial effects ofmyogenic cell transplantation in infarctedmyo-cardium [79].

The paracrineeffectexertedbyMDSCsandother stemcells hasa positive effect within the injured myocardium. Bioactive mole-cules such as basic fibroblast factor (bFGF), vascular endothelialgrowth factor (VEGF), platelet-derived growth factor, SDF-1, hepa-tocyte growth factor (HGF), and IGF-1 confer cardioprotectiveeffects. Inaddition, secretionofmolecules suchasVEGF, angiopoie-tin, and bFGF increase blood vessel formation in the ischemicregion, improving perfusion and function. HGF and IGF-1 also acti-vate resident stem cells and promote endogenous repair mecha-nisms. Additional factors such as TGF-b, MMPs, and tissueinhibitorofmetalloproteinasereduce fibroblastproliferation, fibro-sis, and left ventricular dilation [80, 81]. Okada et al. [82] showedthat a subpopulation of slowly adhering MDSCs showed enhancedparacrine factor secretion, increased stress tolerance, engraftment,and angiogenesis compared with rapidly adhering MDSCs. Theyalso found increased cardiomyocyte proliferationwithin the infarctregion, suggesting the enhancement of endogeneous repairmech-anisms. The selective enrichment of certain types of MDSCs mayimprove their regenerative effects through enhancement of para-crineeffects. StudiesofMDSCtransplantationhaveshownthatonlyTa

ble3.

(Con

t’d)

Study

Species

Isolationmethod

Culture

conditions

Timein

culture

Cardiomyocyte

induction/

differentiation

Key

findings

Sekiya

etal.[90],2013

Mouse

Modifiedpreplating

technique

DMEM

,10%

FBS,10

%horse

serum,1%pen

icillin/

streptomycin,0.5%chick

embryoextract

12weeks

Invivo

transplantation

(NOD/SCID

mice)

MDSCstransplantedas

cellsheetsshowed

areductionofleftventriculardilationand

sustained

contraction.

MDSC

sheetsform

edaligned

myotubes

with

increasedcapillaryden

sity

compared

with

directinjectionofMDSCs.

MDSC

sheetsallowed

betterfunctional

recovery

witharrhythmias.

Abbreviations:3D

,three-dim

ensional;bFG

F,basicfibroblastgrowth

factor;CM,cardiomyocyte;D

MEM

,Dulbecco’smodifiedEagle’smed

ium;EGF,ep

idermalgrowth

factor;EG

FP,enhancedgreenfluorescent

protein;FBS,fetalbovineserum;FCS,fetalcalfserum;IMDM,Iscove’smodifiedDulbecco’smed

ium;M

DSC,m

uscle-derived

stem

cell;RA,retinoicacid;SCM,stem

cellmed

ium.

Hassan, Tchao, Tobita 7

www.StemCellsTM.com ©AlphaMed Press 2014

a small fraction of donor cells demonstrates long-term engraft-ment, and themajority of cells within the regenerating tissue comefrom the host via the chemoattraction of postnatal stem cells fromthe vasculature [83]. Thus, the paracrine or bystander effect ofMDSCs may be as important as, if not more important than, myo-genic differentiation in contributing to cardiac repair.

TISSUE-ENGINEERING APPROACHES TO CARDIAC REPAIR

Transvascular methods of cell delivery are commonly used to treatrecently infarcted myocardium. Intracoronary artery infusion candeliver a high concentration of cells homogeneously to the siteof injury. Direct needle injection into the ventricular wall is pre-ferred for patients late in the disease process when occlusion ofthe coronary artery prevents transvascular delivery [84]. However,it often results inpoor engraftment, poor survival, and further dam-age by needle injury. Tissue-engineering approaches may providea more effective means of cell transplantation. Two main engi-neered tissue transplantation approaches for cardiac tissue arethree-dimensional scaffolds and cell sheets. One of the most chal-lenging issues in tissueengineering is the reconstructionofmyocar-dium in three dimensions. Three-dimensional (3D) scaffolds arenow available [85, 86]. Cell sheets are used for treating advancedheart failure. Myocardial cell sheets decrease fibrosis and increasevascularization in the injured heart [87–90]. In 2006, Zimmermanet al. [91] produced convincing evidence that a heart tissue canbe produced with enough contractile force to support a failingheart.Neonatal ratheart cellswereseededwith liquidcollagentypeI and Matrigel (BD Biosciences, San Diego, CA, http://www.bdbiosciences.com) in molds. The rings were grafted onto the in-farcted ratheart andafter1monthshowedpositive results. Furtherdilation of the heart was prevented, and systolic wall thicknessincreased. In 2011, Fujimoto et al. also proved that engineeredcardiac tissue from immature cardiomyocytes can functionally inte-grate intodamagedmyocardium. In this study,engineered fetal car-diac tissue proved to be more successful than neonatal tissue.Engineered fetal cardiac tissue implantation increased left ventriclecontraction and showed higher cardiomyocyte proliferation thanengineered neonatal cardiac tissue [92].

The major issues concerning the therapeutic application ofstem cells remain unresolved because of three primary factors:poor survival, marginal proliferation, and limited functionalengraftment/commitment within the host tissue. Furthermore,the cells should be prepared to combat against apoptotic, ne-crotic, and hypoxic conditions prevalent within the damaged tis-sue. Finally, if cellsmanage to proliferate, they can be functionallyincapable of appropriate lineage commitment and can end in anoncogenic transformation [93].

The in vivo studies with MDSCs show promising results formeeting some of these challenges. Repair of the infarcted heartwith MDSCs is more effective than conventional myoblasts [54].MDSCs injected into the myocardium of infarcted heart showedoutstanding survival and engraftment, promoted angiogenesis,and increased left ventricular function in amore effectivemannerin comparisonwith the transplantation of skeletalmyoblasts [94].In a postinfarctedmyocardium, transplantedMDSCs sustained itscontractile function, preventing left ventricular chamber remod-eling. Some transplanted donor MDSCs differentiated into bothsk-fMHC- and/or cardiac troponin I-positive muscle cells [54,75]. Clause et al. [68] demonstrated that MDSCs cultured withina three-dimensional engineered tissue construct differentiated

into an immature functioning cardiomyocyte phenotype. Trans-plantation of tissue-engineered cell sheets has negated someof the limitations by eliminating use of trypsin, direct needle in-jury to the heart, and by covering larger infarcted area [95, 96].Reduction in left ventricle (LV) dilation and sustained LV contrac-tion were displayed with the implanted MDSC sheet. It yieldedbetter functional recovery of chronic infarctedmyocardium com-paredwith directMDSC injectionwithout any significant arrhyth-mic events, indicating that this cell sheet delivery system couldmarkedly improve the myocardial regenerative potential of theMDSCs [90]. These studies suggest that tissue-engineering ap-proachesmay provide ameans to further improve upon the func-tional benefits derived from MDSC transplantation.

Tissue-engineering approaches help to address one of themajor limitations of current delivery modalities—the immediateandmassive attrition of naked cells that occurs after cell delivery,which leads to low retention of implanted cells even 24 hoursafter the procedure. However, current tissue-engineering ap-proaches focus on building complex 3D structures, which pre-cludes catheter-based delivery and necessitates surgery. Usingthe cell patch approach, transplanted cells are initially localizedto the epicardial region. A recent development, which improvescell retention, is the delivery of hydrogel-cell liquid suspensionsvia catheter [97]. The hydrogel polymerizes en route and trapscells in the target tissue. If successful, this method can combinethe demonstrated safety of catheter-based delivery with the abil-ity of a tissue-engineered scaffold to improve engraftment.

FUTURE CHALLENGES AND OPPORTUNITIES

Although skeletal muscle stem/progenitor cells have benefits,a large number of injected cells undergo early death. The first pro-spective randomized placebo-controlled phase II skeletal myoblasttrial (MAGIC trial) exhibited lack of efficacy and was discontinuedprematurely [48]. The trial’s disappointing results were due to sev-eral reasons: a low rate of initial cell retention, a high rate of sub-sequent cell death, and the inability of engrafted myoblasts toestablish functional electromechanical connections with the hostcardiomyocytes [3]. In addition, a trend toward excess arrhythmiaswas observed in myoblast-treated patients despite the use of theprophylactic antiarrhythmic drug amiodarone. This raised safetyconcerns that had already been raised by earlier phase 1 trials. Incontrast, SEISMIC trial [63] argued that injectionof autologous skel-etal myoblasts in heart failure patients is safe and may providesymptomatic relief, as a trend toward increased exercise tolerancewasobserved in the cell-treated group.Nevertheless, no significanteffect on global left ventricular ejection fraction was detected. Allthis taken together indicates that more work needs to be doneto bridge the gap between preclinical animal models and clinicaltrials.

In light of the mixed results from clinical trials, further workhas gone into eliminating the heterogeneity of transplanted cellsand improving electromechanical coupling between transplantedand host myocytes. Transplantation of myogenic-endothelialcells that express CD56, CD146, and Ulex, purified from humanskeletal muscle into the ischemic heart, drastically improved leftventricular function, reduced scar tissue, and promoted angio-genesis [54]. Connexin-43 is the predominant gap junction ofthe ventricular myocardium. Skeletal myoblasts lack connexin-43 after fusion into elongated contractile myotubes. In cellularmonolayers, conduction velocity was slowed and re-entry-induced

8 Muscle Stem Cells for Heart Repair

©AlphaMed Press 2014 STEM CELLS TRANSLATIONAL MEDICINE

arrhythmias were promoted when skeletal myoblasts werecocultured with neonatal cardiomyocytes in vitro and studiedwith high-resolution optical mapping. The proarrhythmic effectwas reducedwhen engineered cells overexpressed connexin-43[98]. The findings were later tested in an animal model [11].Methods to improve electromechanical compatibility betweenengrafted muscle and host myocardium are currently underinvestigation.

Issues related to electromechanical compatibility betweencardiac and skeletal muscle tissue could be ameliorated by gen-erating cells from MDSCs that have a cardiomyocyte-like pheno-type. The heart also contains resident stem cells. Oh et al. [99]identified in 2003 an independent population of Sca-1+ cardiacstem cells as a subgroup of cells (constituting ∼14%) isolated inthe noncardiomyocyte cell fraction of the adult mouse heart ina whole heart digestion. Sca-1+ cells coexpressed CD31 andCD38 and lacked c-Kit, CD34, and CD45 when freshly isolated.Ninety-three percent of the side population was Sca-1+. Freshlyisolated Sca-1+ cells did express the early cardiac-specific tran-scription factors GATA4, Mef2C, and Tef-1 but not Nkx2.5 orgenes encoding cardiac sarcomeric proteins. Sca-1+ cells en-graftedat amuchhigher rate than Sca-12 cells in amousemodelof ischemia-reperfusion after 2weeks and could be found form-ing new cardiomyocytes. Cardiac stem cells in bulk cultureupregulated GATA-4 expression resulting in enhanced cardio-myocyte differentiation, suggesting that the GATA-4 high c-kit+

cardiac stem cells have potent cardiac regenerative potential.The study also demonstrated spontaneous differentiation intoskeletalmyocytes [100].Hasanet al. [101] establishedcardiacplu-ripotent stem cell-like cells from the left atriumof adult rat heartsthat could differentiate into beating cardiomyocytes in the

methylcellulose-based medium containing interleukin-3 andstem cell factor, which contributed to the differentiation into car-diac troponin I-positive cells. Distinctly small populations of plu-ripotent stem cell-like cells from the left atrium coexpressedGATA4 and myogenin, which are markers specific to cardiomyo-cytes and skeletal myocytes, respectively. These could differenti-ate intoboth cardiac and skeletalmyocytes. These studies suggestthe possibility that cardiac and skeletal muscle can arise froma common myogenic progenitor, and stem cells purified fromskeletal muscle may have similar differentiation potential, asdemonstrated by studies of cardiomyocyte differentiation fromMDSCs.However, thepathways thatdeterminewhether a cell dif-ferentiates into a cardiomyocyte or skeletal muscle cell are onlybeginning to be unraveled.

A hypothesis is presented in Figure 1 showing how skeletalmuscle stem/progenitor cells can be induced to become cardiacmuscle with post-transcriptional modification. Microribonucleicacids (micro-RNAs, miRs) are post-transcriptional regulators ofcardiac and skeletal myogenesis, including miR206, which specif-ically promotes skeletal myogenesis [102–104] as part of anintrinsic cell-regulatory program. Crippa et al. [105] isolated car-diac progenitors from neonatal sarcoglycan-null mouse heartsaffected by dilated cardiomyopathy, and they spontaneously dif-ferentiated into skeletal muscle fibers both in vitro and whentransplanted into regenerating muscles or infarcted hearts. Theabsence of expression of miR669q and with downregulation ofmiR669a was associated with differentiation potential. Skeletalmyogenesis was prevented by miR669a and miR669q acting up-stream of myogenic regulatory factors by directly targeting theMyoD untranslated region. Successful conversion of cardiac cellsinto skeletal muscle fibers opened a huge area of discussion if the

Figure 1. MicroRNAs post-transcriptionally regulatemyogenesis. miR-206 specifically promotes skeletal muscle differentiation, whereasmiR-669 promotes cardiac differentiation by inhibiting MyoD, a skeletal muscle transcription factor. By modulating expression of these and othermuscle-specific microRNAs, stem cells can be induced to differentiate into cardiac or skeletal muscle. The local microenvironment and solublefactors also play important roles in stem cell differentiation. Abbreviations: CM, cardiomyocyte; ECM, extracellular matrix; MHC, myosin heavychain; MIR, microRNA.

Hassan, Tchao, Tobita 9

www.StemCellsTM.com ©AlphaMed Press 2014

reverse is possible. MDSCs have genes similar to those of cardiacand skeletal muscle, and cardiac and skeletal muscles are inter-changeable because of miR669. The physical environment mayalso present important environmental cues [106]. Three-dimensional cultures and cardiac-specific extracellular matrixhave been shown to promote cardiomyocyte induction [36, 68,107].

CONCLUSION

In any endeavor as complex and perplexing as stem cell research,controversies and disagreements are to be expected. Whereasearly clinical trials of skeletal myoblast transplantation yieldedmixed results and did not meet initial expectations, more recentworks have shown that improved isolationmethods, novel tissue-engineering approaches, and differentiation strategies may im-prove the efficacy of myogenic stem cell transplantation. Skeletalmuscle-derived stem cells remain the only stem cell that can

differentiate into a muscle phenotype and can be isolated easilyin abundant numbers. Cardiovascular regenerative medicine isstill in its early stages, but recent studies show renewed promise.The field of regenerative medicine has been quite progressive, asa search on PubMed for the keywords “cardiac, stem cell, heart,”yields more than 9,000 references as of today with almost 2,000review articles to summarize all current understanding. This fieldwas nonexistent a little over a decade ago.

AUTHOR CONTRIBUTIONS

N.H. and J.T.: conception and design, manuscript writing; K.T.:conception and design, manuscript writing, final approval ofmanuscript.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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