Human muscle stem cells

Human muscle stem cellsRacquel N Cooper, Gillian S Butler-Browne and Vincent Mouly

Stem cells are unspecialized cells that have been defined in

many different ways but they have two important

characteristics that distinguish them from other cells in the

body. First, they can replenish their numbers for long periods

through cell division. Second, after receiving certain chemical

signals, they can produce, through asymmetric cell division, a

progeny that can differentiate or transform into specialized cells

with specific functions, such as heart, nerve or muscle. In

recent years, stem cells have received much attention owing to

their potential use in cell-based therapies for human

neurodegenerative diseases such as Parkinson’s disease,

stroke and muscular dystrophies. However, many questions

need to be resolved before stem cells with myogenic potential

are used in clinical standard protocols.

Addresses

INSERM U787, Institut de Myologie, 105 bd de l’Hopital, 75013 Paris,

France

Corresponding author: Mouly, Vincent ([email protected])

Current Opinion in Pharmacology 2006, 6:1–6

This review comes from a themed issue on

Musculoskeletal

Edited by Geoff Goldspink and Brendon Noble

1471-4892/$ – see front matter

# 2006 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.coph.2006.01.007

Introduction: skeletal muscle stem cellsSkeletal muscle needs to employ stem cells for its main-

tenance and repair. It contains a potent myogenic stem

cell population, called satellite cells, with the ability to

self-renew as well as to generate myogenic precursor cells

that can regenerate muscle in vivo. However, owing to the

gradual decrease in number of satellite cells during the

evolution of muscular dystrophies, leading to their final

exhaustion [1,2], many attempts have been undertaken to

isolate cells able to efficiently repair adult skeletal mus-

cle, following the paradigm of haematopoietic stem cells

which reconstitute blood cells.

Satellite cells

Since its identification over 40 years ago, the satellite cell

has been a popular candidate for the adult skeletal muscle

stem cell. Located beneath the basal lamina of mature

skeletal muscle fibres, they are ideally positioned for

repair of degenerating muscle fibres. These dormant cells

COPHAR 355

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are activated to proliferate upon muscle injury or when

heavily used during activities such as weight lifting or

running. This proliferation step is necessary to generate

sufficient numbers of myoblasts for muscle differentia-

tion and myotube formation. In humans and mice, these

mononuclear cells are most plentiful at birth (estimated at

32% of sublaminar nuclei) [3]. Their frequency declines

post-natally, stabilizing to between 1% and 5% of skeletal

muscle nuclei in adult mice [4]. In humans, the propor-

tion of satellite cells in skeletal muscles also decreases

with age, which could explain the decreased efficiency of

muscle regeneration in older subjects [5,6]. Satellite cells

from aged muscle also display reduced proliferative and

fusion capacity, as well as a tendency to accumulate fat, all

of which probably contribute to deteriorating regenera-

tion capacity [7].

Recent studies carried out in mice and involving single

myofibre transplantations have allowed the direct inves-

tigation of the function and behaviour of satellite cell

populations [8�]. It was found that fibres containing as

little as seven satellite cells on a transplanted myofibre

could generate over 100 new myofibres containing thou-

sands of myonuclei. These results indicate that at least

some cells in the position of satellite cells (i.e. on the edge

of the fibres) present an extended proliferative potential

and can repopulate extensively the host’s muscle with an

efficiency unknown in any other experimental situation.

Whether this population of progenitor, which also pro-

vides cells in the satellite compartment, corresponds to

bona fide stem cells or simply progenitors with extended

proliferative potential is still unknown (see review by

Collins, this issue).

The identification of multiple stem cell populations

resident in skeletal muscle has added further complexity

to our understanding of the process of muscle regenera-

tion. In adult human tissue, skeletal muscle-derived stem

cells (MDSCs) appear to be a distinct population of

immature progenitors of satellite cells, but their func-

tional properties remain unclear.

Muscle-derived stem cells

The idea that the regeneration of adult muscle is wholly

accomplished by satellite cells has been challenged by

the demonstration that muscle also contains a population

of adult stem cells termed MDSCs. These cells constitute

a unique population that appears to be distinct from

muscle satellite cells [9]. MDSCs exhibit the capacity

to reconstitute the entire haematopoietic repertoire after

intravenous injection into lethally irradiated mice [10,11],

although the cells that showed this potential were

Current Opinion in Pharmacology 2006, 6:1–6

2 Musculoskeletal

identified as having a haematopoietic origin [12]. Further-

more, muscle side population (SP) cells, which can be

isolated by their specific exclusion of the vital dye Hoechst

33342 [10,13], are a fraction of MDSCs that expresses stem

cell antigen-1, a haematopoietic stem cell marker, but no

satellite cell markers such as M-cadherin or Pax7 [14]. In

addition, transplanted SP cells isolated from bone marrow

or muscle actively participate in the formation of skeletal

myotubes during regeneration [13]. Significantly, Pax7-

deficient muscle entirely lacks myogenic satellite cells, yet

contains muscle SP cells that exhibit a high level of

haematopoietic progenitor activity [15].

This review focuses on recent advances in skeletal mus-

cle stem cell research and in particular human muscle

stem cells.

Muscle stem cell plasticitySatellite cells have long been considered monopotential

stem cells with the ability to give rise only to cells of the

myogenic lineage. However, recent experiments have

identified the existence of adult stem cells present in

most tissues that appear to exhibit the ability to differ-

entiate into many different cell types after reintroduction

in vivo [10,14,16–18]. Importantly, these cells have been

recognized as a unique population that appears to be

distinct from muscle satellite cells.

Treatment of C2C12 myoblasts, an immortalized cell line

derived from mouse limb muscle, with bone morphoge-

netic protein activates osteogenic markers, whereas treat-

ment with adipogenic inducers such as fatty acids and

thiazolidinediones activates adipocyte markers [19].

Interestingly, satellite cells derived from intact single

fibre cultures (and thought to be more representative

of true myogenic stem cells) spontaneously form adipo-

cytes and osteoblasts when cultured on Matrigel, a solu-

ble basement membrane matrix lacking strong osteogenic

or adipogenic signals [20]. The adipogenic potential of

myoblasts has also been observed in ageing mouse mus-

cles [7]. The finding that undifferentiated cells in adult

myoblast cultures co-express MyoD, Runx2 and PPARg,

key regulators for myogenesis, osteogenesis and adipo-

genesis, respectively, supports the hypothesis that satel-

lite cells have a multipotential predisposition [21] that is

revealed depending upon the environment. Interestingly,

recent data point to a possible role for wnt signalling in

maintaining the physiological balance between adipo-

genic and myogenic potential of satellite cells. Mice

deficient for Wnt 10b display an increased fat infiltration

within muscle, a phenomenon that has been described

during physiological ageing where wnt10b is downregu-

lated [22].

The plasticity of muscle stem cells has also been demon-

strated using ex vivo approaches. Mouse muscle stem cells

isolated via serial preplating contribute to regenerating

Current Opinion in Pharmacology 2006, 6:1–6

myofibres when injected directly into dystrophic muscle,

and are also detected among differentiated vascular and

nerve populations [18,23]. Furthermore, these cells,

which express the myoblast markers desmin and MyoD,

are sufficient to completely heal skull defects in vivowhen engineered to express bone morphogenetic protein

[24]. These MDSCs are also capable of reconstituting

bone marrow in lethally irradiated mice [25].

Nevertheless, these studies do have a limitation in that

they used animal models, and few studies have focused

on human stem cells. Alessandri et al. [26] have recently

shown that adult human skeletal muscle includes a

population of progenitor stem cells that can generate

stem cells of the same lineage and cells with neurogenic

properties. Consequently, human skeletal muscle could

represent an important alternative source to isolate plur-

ipotent stem cells for the development of cell-based

therapies for human myogenic and neurogenic diseases.

Likewise, neural stem cells that have been isolated from

adult human skeletal muscle offer a possible alternative

source of neural stem cells for tissue engineering of the

central nervous system [27].

Other stem cells with myogenic potentialIn recent years, many data have been published indicat-

ing the ability of multipotent adult stem cell populations

from a given tissue to differentiate into myogenic cells.

The majority of these studies were performed in rodent

models, whereas a few used human cell sources. Pioneer-

ing studies were performed by Ferrari and colleagues in

1998 [28], in which unfractionated bone marrow-derived

cells were capable of myogenic conversion. However,

repair by bone marrow-derived cells never attained

>1% of total muscle fibres during the lifespan of trans-

planted mdx mouse muscles and did not improve murine

muscular dystrophy [29]. Gussoni et al. [13] transplanted

several thousand haematopoietic stem cells from male

mice into female mdx mice and were able to show that

donor cells efficiently replenished bone marrow of reci-

pients; cells from males expressing dystrophin were found

at low levels in host muscles, indicating differentiation of

transplanted cells into muscle. Blau and colleagues have

shown more recently that bone marrow cells can partici-

pate in muscle regeneration by first being converted to

satellite cells [30].

Intra-arterial injections of wild-type mesoangioblasts —

stem cells isolated from blood vessels — into mice

suffering from limb girdle muscle dystrophy results in

complete functional recovery of all affected muscles [31].

This presents a promising solution to difficulties encoun-

tered with myoblast transplantation therapy, and makes

all muscles accessible for treatment. This is especially

important for the treatment of muscles such as the dia-

phragm, impairment of which results in severe respiratory

problems.

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Human muscle stem cells Cooper, Butler-Browne and Mouly 3

However, one would have to scale up the procedure to

repair the large muscles found in humans. This would

require large numbers of cells, and human mesoangio-

blast stem cell cultures have not yet been described.

Human-derived AC133, a newly identified glycosylated

polypeptide expressed on a population of circulating

human hematopoeitic/endothelial progenitor cells [32],

can repopulate bone marrow and differentiate into mature

endothelial cells. Torrente et al. [32] have shown that,

after injection of human AC133+ cells into skeletal muscle

of scid/mdx mice, an immunodeficient strain of mice that

lacks dystrophin, up to 10% of human dystrophin-positive

fibres could be detected, suggesting that blood-derived

human AC133+ cells participated in muscle regeneration

of dystrophic muscles. It should be noted that, although

some of these AC133+ cells were of circulating origin, no

data have been published concerning a systemic delivery

of these cells, even in animal models, which would be a

pre-requisite to successful cell treatment of major dys-

trophies such as Duchenne muscular dystrophy (DMD).

Rodriguez et al. [33] have more recently demonstrated the

isolation of adipogenic, myogenic and osteogenic lineages

Figure 1

Plasciticy of myogenic stem cells. Whereas muscle precursor cells can give

types of stem cells (e.g. from bone marrow, blood vessels [mesoangioblast

through two distinct mechanisms: by direct fusion with newly forming fibres

occupying the niche of resident stem cells (i.e. satellite cells).

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from human multipotent adipose-derived stem cells

(called hMADS). Transplantation of these hMADS into

muscles of non-immunocomprised mdx mice resulted in

long-term engraftment and higher dystrophin expression

in myofibres than previously reported for primary myo-

blasts or muscle stem cells from wild-type mice [34].

These results suggest substantial recruitment of human

multipotent adipose-derived stem cells to the muscle

lineage. However, these provocating results require

further confirmation by other groups before being trans-

ferred to the clinic.

Figure 1 provides an overview of the plasticity of skeletal

muscle and the myogenic potential of other stem cells.

Clinical approachesAttempts to reconstitute skeletal muscle in human mus-

cle disease through myoblast transfer therapy have now

been underway for more than a decade, and provide many

lessons about the obstacles that have to be surmounted in

stem cell therapy. Although early experiments in animal

models of DMD showed that transplantation of

rise to cells from the adipogenic and osteogenic lineage, several

s)] or circulating blood [AC133+]) can give rise to skeletal muscle

(whether this is an active or a passive mechanism) and/or by first

Current Opinion in Pharmacology 2006, 6:1–6

4 Musculoskeletal

committed muscle stem cells from normal muscle

resulted in partial restoration of the deficient dystrophin

protein, a high percentage of introduced cells died within

a short time after transplant [35]. Although such a high

level of cell death still needs to be estimated when human

cells are injected, clinical trials in DMD have showed no

significant clinical improvement. Roadblocks to the use-

fulness of this therapy include limited migration and

proliferative capacity of donor cells in dystrophic muscle,

problems with poor donor cell survival, immunorejection

and inefficient myogenic contribution [36]. Difficulties in

delivering myoblasts to a wide range of muscle tissue

remain to be overcome. No systemic delivery of comm-

ited myogenic cells has ever been successful, although

only systemic delivery could reply to the extended degen-

eration observed in many muscle degenerative diseases,

such as DMD, where the majority of the musculature is

targeted by the disease. Despite enormous hope and

enthusiasm for this strategy by investigators and the

muscular dystrophy community when it was introduced,

and although some progress has been made, we are still a

long way from efficient therapy.

Adult stem cells have been shown in animal models to

repair heart damage, provide therapeutic benefit for

stroke, and reverse diabetes. In human patients, these

cells have been employed successfully to relieve lupus,

multiple sclerosis and arthritis, whereas the regenerative

potential of embryonic stem cells has not yet been tested,

as efficacy and safety tests are still underway. However,

although embryonic stem cells might not be immuno-

genic per se, they may turn to be so when they engage to a

more differentiated pathway.

Stem cell research for the treatment of degenerative

diseases is in its infancy and many issues require resolu-

tion before clinical application. The problem of using

autologous or allogenic cells is crucial to the development

of therapeutic trials. Until tolerance to allogenic cells can

be safely induced, the injection of allogenic cells will

require heavy immuno-depressive treatment, which can-

not be sustained for prolonged periods of time owing to

the secondary effects of these treatments. On the other

hand, autologous cells cannot be used if they are already

exhausted by the degenerative process. There have,

however, been a few clinical trials employing autologous

adult human muscle stem cells to treat heart disease and

some muscular dystrophies.

Myocardial regeneration

The first demonstration of successful treatment for heart

disease using the patient’s own adult muscle stem cells

was performed in 2000 by Menasche et al. [37]. Auto-

logous human myoblasts were isolated from the quad-

riceps of the patient, propagated and expanded ex vivountil enough cells are obtained to be injected into the

ventricular wall. After eight months of follow-up, the

Current Opinion in Pharmacology 2006, 6:1–6

grafted cells were functionally integrated and augmented

the function of the patients’ heart. These encouraging

results underline the potential of this new approach and

further clinical trials are now underway in Europe and the

US to investigate the effectiveness of muscle stem cell

therapy in improving function of failing hearts. Autolo-

gous myoblasts are considered possible donor cells

because they possess an inherent capacity for contracti-

lity. They are resistant to ischemia and do not raise

immunologic and ethical concerns.

Muscular dystyrophies

From the results obtained with heart infarct, it was

recently proposed that autologous myoblasts could ame-

liorate the conditions of patients suffering from muscular

dystrophies in cases when the disease leaves enough

muscles spared by the pathological evolution. One such

example is fascio-scapulo-humeral muscular dystrophy,

where myoblasts isolated from muscles spared by the

disease showed normal regenerative capacity [38]. In

oculo-pharyngeal muscular dystrophy, only pharyngeal

and eyelid muscles are targeted by the disease in most

patients. A Phase I clinical trial involving autologous

transfer of myoblasts into pharyngeal muscles has

recently been launched (Perie, Lacau St Guily, Marolleau,

Butler-Browne and Mouly, unpublished).

Perspectives and conclusionsFollowing the successful use of blood stem cells to

reconstitute cells damaged in diseases such as leukaemia,

researchers have been interested in the use of other types

of stem cells to repair adult tissue damaged through injury

or degenerative disease. However, it has until now been

difficult to isolate pure populations of adult stem cells in

large numbers. Exciting results recently obtained by

Montarras et al. [39�] reveal the development of a new

purification procedure that gives direct access to muscle

stem cells that can both repair and contribute to the

progenitor cell population of damaged muscles. The

isolated cells are more efficient contributors to muscle

repair than are the cultured muscle precursor cells pre-

viously used, as 20 000 purified muscle stem cells were

sufficient to promote significant muscle repair when

grafted in dystrophic mouse muscles as compared with

one million cultured muscle precursor cells. It is sug-

gested that the higher regenerative capacity of freshly

isolated stem cells reflects the ability of these cells to

more effectively colonise grafted muscle compared with

cultured cells, which undergo modifications that make

them less efficient, partly because they tend to differ-

entiate too quickly and thereby lose their ability to

regenerate damaged cells. Collins et al. [8�] have recently

demonstrated in the mouse that the implantation of single

fibres with their resident satellite cells into regenerating

muscles of host mice results in an extremely efficient

participation of the cells originally resident to the

implanted fibre to the host’s regeneration (see review

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Human muscle stem cells Cooper, Butler-Browne and Mouly 5

by Collins, this issue). Although not yet applicable to

human therapy, the aforementioned results provide new

insight for therapeutic use of muscle precursors cells in

humans.

Stem cell therapy holds huge promise for many illnesses

that are presently incurable. This field is currently in its

early stages of development, and a huge amount of work is

a prerequisite to fulfilling these promises. Information

about scientific achievements needs to be communicated

to the public properly, in a rigorous and cautious way, so

that it does not raise excessive expectations. Despite the

numerous obstacles, the potential for stem cell-based

therapy in the treatment of muscular dystrophies and

degenerative diseases of all organ systems provides a

stimulating field for continued research.

AcknowledgementsThe laboratory has received support from Association Francaise contre lesMyopathies (AFM), the Parent Project, Universite Pierre et Marie Curie,CNRS, INSERM and EC (Program Ageing Muscle in the 5th FP andMYORES Network of Excellence, contract 511978, from the EuropeanCommission 6th Framework Programme). The authors wish to thank allthe other members of the laboratory, as well as Martin Catala and WoodyWright for fruitful discussions and Terry Partridge for sharing observationsand concepts.

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� of special interest�� of outstanding interest

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39.�

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The characteristics of Pax3+ myogenic cells were determined by FACSusing Pax3-GFP mice as a source of cells. Grafting of as few as 104 freshlyisolated cells, sorted by FACS using the same characteristics, showedthat these freshly isolated cells were 10-50 times more efficient thanmyogenic cells expanded in culture. These data can be explained eitherby the presence of a population of cells that is lost during expansion inculture, or by modifications of the cells during the expansion in vitro.

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