Download Satellite cells, myoblasts and other occasional myogenic progenitors

Seminars in Cell & Developmental Biology 16 (2005) 623–631
Review
Satellite cells, myoblasts and other occasional myogenic progenitors:
Possible origin, phenotypic features and role in muscle regeneration
Giulio Cossu a,b,∗ , Stefano Biressi a
b
a Stem Cell Research Institute, Dibit, H. San Raffaele, via Olgettina 58, 20132 Milan, Italy
Institute of Cell Biology and Tissue Engineering, San Raffaele Biomedical Science Park of Rome, II◦ Medical School,
University of Rome “La Sapienza”, via Castel Romano 100/2, 00128 Rome, Italy
Abstract
In the vertebrate embryo, skeletal muscle originates from somites and is formed in discrete steps by different classes of progenitor cells.
After myotome formation, embryonic myoblasts give rise to primary fibers in the embryo, while fetal myoblasts give rise to secondary fibers,
initially smaller and surrounding primary fibers. Satellite cells appear underneath the newly formed basal lamina that develops around each
fiber, and contribute to post-natal growth and regeneration of muscle fibers. Recently, different types of non somitic stem-progenitor cells
have been shown to contribute to muscle regeneration. The origin of these different cell types and their possible lineage relationships with
other myogenic cells as well as their possible role in muscle regeneration will be discussed. Finally, possible use of different myogenic cells
in experimental protocols of cell therapy will be briefly outlined.
© 2005 Elsevier Ltd. All rights reserved.
Keywords: Skeletal myogenesis; Muscle satellite cells; Skeletal myoblasts; Mesoangioblasts; Muscle regeneration
Contents
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2.
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9.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A brief overview of muscle development and regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Are all myogenic cells derived from somites? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The fetal stage of myogenesis is more similar to regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
At variance with myoblasts, satellite cells are already committed, quiescent cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Are myogenic cells only derived from satellite cells in regenerating muscle? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The possible lineage relationship of mesoangioblasts with other myogenic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Features of professional and occasional myogenic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Perspectives for use in cell therapy protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
Abbreviations: BMP2, bone morphogenetic protein 2; GFP, green fluorescent protein; HSC, hematopoietic stem cell; MSC, mesoderm stem cell
(referred to as non hematopoietic); PKC, protein kinase C; Shh, sonic hedgehog; SP, side population; TGF␤, transforming growth factor ␤
∗ Corresponding author. Tel.: +39 02 2643 4954; fax: +39 02 2643 4621.
E-mail address: [email protected] (G. Cossu).
1084-9521/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.semcdb.2005.07.003
A large body of experimental data is rapidly accumulating on the activation, the self-renewal and differentiation
potency of muscle satellite cells. The field has been certainly
fueled by the recent reports on the myogenic potency of
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other cell types, derived from bone marrow, vessels, brain,
adipose tissue, sinovia [1–5] and possibly still other tissues
of the mammalian body. In the last few years, several
studies have described the steps that cells originating in
the bone marrow (an to a lesser extent in other tissues)
undergo in order to acquire the ability to contribute to muscle
regeneration [6–9]. Although myogenic differentiation of
these other cells is a rare and poorly understood event, it
has nevertheless challenged the idea that all skeletal muscle
is derived and can only derive from somites. Developmental potency, revealed after experimental manipulation,
does not necessarily correspond to normal fate during
unperturbed development. Nevertheless, these data have
stimulated further work aimed to a better understanding
of activation, self-renewal and differentiation of resident
satellite cells. Consequently, the scenario of skeletal muscle
regeneration is now wider though far from complete and
several chapters in this issue will examine these aspects in
detail.
We will rather focus on the developmental origin of
satellite cells, first in comparison with embryonic and fetal
myoblasts, and then with other non-somitic myogenic progenitors. We will then outline the phenotype of these different
cells and will discuss their possible therapeutic perspectives
as a tool to treat diseases that affect primarily skeletal muscle,
such as muscular dystrophies.
2. A brief overview of muscle development and
regeneration
Muscle regeneration is currently considered as a recapitulation of muscle development. However, in order to gain
insights from pre-natal development of skeletal muscle, it
is necessary to consider that it occurs in several distinct,
though overlapping steps [10]. As schematized in Fig. 1,
skeletal myogenesis begins with myotome formation (at E
9 in the mouse) that is followed by fusion of myoblasts to
form primary fibers, at approximately day E 11–12. Some
of the myoblasts do not fuse into primary fibers but continue to proliferate and differentiate only at E 15–17, giving
rise to secondary (fetal) fibers that are originally smaller and
surround primary fibers with a donut like configuration. At
the same time, a basal lamina begins to form around each
fiber and it is only after its formation that satellite cells can
be morphologically identified as mononucleated cells laying
between the basal lamina and the fiber plasma membrane.
During peri and post-natal development, satellite cells divide
at a slow rate and part of the progeny fuse with the adjacent
fiber to contribute new nuclei and to increase to size of muscle
fibers whose nuclei cannot divide. At the end of post-natal
growth, satellite cells enter a phase of quiescence but can
be activated if the muscle tissue is damaged. They resume
mitotic activity and repair damaged fibers or fuse with other
Fig. 1. A schematic, color guided, representation of the possible origin of myogenic cells during skeletal muscle development. Different myogenic cells
originated from somites are represented in different tones of green. Dorsal aorta and different stem-progenitor cells derived from it and endowed with an
inducible myogenic potency are represented in red. Nuclear transcription factors are indicated with different colors. For simplicity, no distinction among
hematopoietic or parietal cells is made in this scheme.
G. Cossu, S. Biressi / Seminars in Cell & Developmental Biology 16 (2005) 623–631
satellite cells to form new ones. It has been recently shown
that other cell types have the potential to participate to muscle
regeneration, though their contribution appear to be minimal
under normal circumstance.
Embryonic myogenesis differs significantly from secondary or fetal myogenesis and it is this last one that shares
many common steps with regeneration. In the next section,
we will address a few unsolved issues related to myogenic
commitment and differentiation.
3. Are all myogenic cells derived from somites?
Embryonic myogenesis begins in newly formed somites
where progenitors located in the dorso-medial and in the
ventro-lateral lips of the dermamyotome respond to signals
such as Wnts and Shh, emanating from adjacent neural tube,
notochord and ectoderm, and activate basic Helix loop Helix
transcription factors (Myf5 and MyoD) that commit cells to
myogenesis. For a more detailed description of embryonic
myogenesis, several recent reviews can be consulted [10–12].
However, it is still unknown whether all myogenic progenitors are committed at once in the newly formed somite, or
rather mesoderm cells can be committed to myogenesis later
in development and even in post-natal life, as studies on non
somitic progenitors would suggest. This question is important but very difficult to be addressed experimentally. For
example, it is well established that already committed myogenic progenitors (that express Pax3 but not Myf5 or MyoD)
migrate from somites to the limb [13], but it is unknown
whether all the myoblasts that contribute to fetal myogenesis (see below) are also committed in the somite or may be
recruited locally from lateral mesoderm progenitors by signals emanating for example by newly formed primary fibers.
This hypothesis is in contrast with the widely accepted view
that all skeletal muscle (in the body) is derived from somites
[14]. Such a view stems from classic quail-chick transplantation experiments that were analyzed in almost all cases at E
6–8 in the chick, i.e. before fetal and post-natal myogenesis
take place. Therefore, the somitic origin of skeletal muscle has been demonstrated for the embryo and inferred for
later developmental stages. For example, the origin of satellite cells is presumed to be somitic, but in the only reported
study using quail-chick transplantation, [15] only about 50%
of the chick satellite cell nuclei were identified as quail in
origin, possibly due to the technical difficulty of identifying
quail heterochromatin at the ultra-structural level. An alternative explanation would be that some of those nuclei were
of chick origin and thus not derived from somites.
As a matter of fact, even assuming that all canonical
myogenic cells (embryonic and fetal myoblasts and satellite
cells) may be committed to myogenesis in the newly formed
somite, terminal differentiation occurs only in a fraction of
somitic cells; the remaining should be kept in a committed
but undifferentiated state. Very recent evidence suggest that
cells located in the central domain of the dermamyotome
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express the Pair ruled transcription factors Pax3 and Pax7
but not myogenic bHLH. These cells represent progenitors
for embryonic, fetal myoblasts and satellite cells, since depletion of both Pax3 and Pax7 abolish all myogenesis with the
exception of the myotome [16]. These novel findings suggest that different transcription factors regulate the identity
of myotomal versus later myogenic progenitors, but do not
tell us whether all Pax3/Pax7 positive cells originate from the
dermamyotome nor how the differential fate of embryonic
(that will produce primary fibers) of fetal (that will produce
secondary fibers) and of satellite cells is regulated [17]. In
Drosophila, lateral inhibition through Notch and Delta has
been shown as the probable mechanism by which adult myogenic progenitors are selected in response to Wng signaling
[18]. Recently, a similar mechanism has been shown to operate in adult muscle [19]. It thus appears likely that the same
may occur in the mammalian somite. Indeed, several Delta
and Notch isoforms are expressed in the somites [20] and
Notch inhibits myogenesis, probably through different intracellular mechanisms [21,22]. Receptors for growth factors or
other signaling molecules such as BMP or TGF beta may be
targets for Notch signaling. However, direct evidence for a
role of Notch in diversifying cell fate in mammalian somites
is still missing.
One reason for the existence of these multiple phases and
progenitors for vertebrate myogenesis can be found in the
need of maintaining a precursor pool of dividing myogenic
cells (to cope with the growing size of the embryo) and at
the same time to generate contracting and post-mitotic skeletal muscle fibers (to allow early movements of the embryo).
A possible mechanism to ensure that certain myoblasts will
differentiate in an environment that is permissive for proliferation, may be based on the inability of these myoblasts
to respond to growth factors and/or to molecules which
inhibit differentiation. Some years ago, we proposed a possible mechanism by which TGF␤ might influence the process
of primary fiber formation in vivo, by inhibiting differentiation of fetal but not of embryonic myoblasts [23]. Thanks to
a micro-array analysis of Myf5-GFP purified embryonic and
fetal myoblasts, we are currently testing the role that decorin,
byglican, PKC␪ [24], SMAD-6 and cited1 (all differentially
expressed in the two populations) play in regulating this phenomenon.
4. The fetal stage of myogenesis is more similar to
regeneration
Embryonic myogenesis occurs in somites and later in the
limbs in the absence of already formed muscle fibers. In
the case of the somite, myogenic progenitor cells differentiate synchronously (for each somite) into the differentiated,
mononucleated muscle cells of the myotome [12]. Pattern is
simple, with all muscle cells aligned along the whole craniocaudal length of the somite, likely as a remnant of muscle
differentiation in fish. Embryonic myogenesis in the limb
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also occurs in the absence of pre-existing muscle fibers, but
here the pattern is more complex with groups of progenitor cells that begin to differentiate and fuse into discrete
clusters, corresponding to the major muscle masses of the
dorsal and ventral aspects of the developing limb. Based
on transplantation experiments, it was concluded that nonmuscle cells from the somatopleura create a pattern that is
filled by embryonic myoblasts that migrate into the limb
from co-axial somites. In fact, when limb buds were transplanted onto a chorio-allantoid membrane before myoblast
migration, an apparently normal but muscle-less limb forms,
with patterned connective scaffolds that should have been
filled by myoblasts and rather remained empty [25]. At
around day E 11 (in the mouse), embryonic myoblasts
that have invaded the limb begin to fuse into multinucleated myotubes and do not form mononucleated myocytes as
their somitic counterparts. Roughly at the same day (E 11)
embryonic myoblasts (Pax3/Pax7+) that invade the myotome
[26], fuse into myotubes, likely incorporating the initially
mononuleated myocytes of the myotome, even though this
has not been formally demonstrated. Moreover, in the Myf5
null mice, that fail to form a myotome, myogenesis proceeds in a relatively normal sequence [27,28], suggesting
that formation of a myotome is not a necessary step for
later muscle development. The point that we would like
to stress here is that the earliest myogenesis occurs without pre-existing myofibers, a unique situation that could be
reproduced only if one day we would like to create in vitro
muscles de novo. In all other cases, i.e. fetal myogenesis,
post-natal muscle growth and muscle regeneration, myogenic
progenitors will form new muscle cells within a pre-existing
micro-environment composed of growing, damaged or regenerating muscle fibers. In all these cases, myogenic progenitors
must face a choice at the time of differentiation: they must
either fuse with a pre-existing fiber or fuse with similar cells
into a new myotube that will eventually mature into a new
fiber. For example, during fetal myogenesis, fetal myoblasts
mainly fuse with each other, giving rise to secondary (fetal)
fibers that are originally smaller and surround primary fibers
with a donut like configuration. However, it has been shown
that a minority of fetal myoblasts fuse with primary fibers
[29]. We have currently no idea if this is a random choice or is
dictated by some signaling molecule, even though it has been
proposed that VLA-4-VCAM-1 interactions influence alignment of secondary myoblasts along primary myotubes and/or
the fusion of secondary myoblasts [30]. A similar situation
may occur during regeneration and we do not know which
signals promote fusion of satellite cells to a pre-existing fiber
or rather induce formation of a new fiber.
5. At variance with myoblasts, satellite cells are
already committed, quiescent cells
There is a second important difference between embryonic myogenesis and regeneration. In somites, commitment
to a myogenic fate is induced by signals emanating from
neighboring tissues in “naı̈ve” proliferating cells [10–12]. In
contrast, during post-natal muscle regeneration, a quiescent
but already myogenically committed cell is activated by signals emanating from infiltrating cells and damaged muscle
fibers [31].
As detailed elsewhere in this issue, satellite cells are classically defined as quiescent mononucleated cells, located
between the sarcolemma and the basal lamina of adult skeletal muscle [32]. When activated, following muscle damage,
they undergo a number of cells divisions producing fusion
competent cells and other cells that return to quiescence, thus
maintaining a progenitor pool. This fact has led to the suggestion that they represent a type of stem cells [33,34].
Old work from our and other laboratories identified specific features of satellite cells (morphology, resistance to
phorbol esters but susceptibility to TGF␤ induced block
of differentiation, early expression of acetylcholine receptors and acetyl-cholinesterase) that characterize them as a
class of myogenic cell different from embryonic and fetal
myoblasts [35]. Satellite cells are first detectable when the
basal lamina forms in vivo. In this period, intensive myoblast
fusion occurs, leading to a drastic reduction of myogenic
mononucleated cells. Since satellite cells do not undergo
differentiation at this time, the control of proliferation and
differentiation in these cells must be different, so as to allow
the persistence of mononucleated undifferentiated myogenic
cells in the post-natal and in the adult muscle.
6. Are myogenic cells only derived from satellite cells
in regenerating muscle?
Satellite cells are the only relatively well-defined myogenic cell in post-natal life. It is currently assumed but not
experimentally proved that they represent a single cell type,
with a common embryological origin, although they may be
kept in different stages of the differentiation pathway.
During the last 10 years, a number of observations had
pointed to the unorthodox appearance of muscle cells in a
variety of tissues or cell culture systems that were neither
myogenic nor derived from somites. For example, spontaneous myogenic differentiation of cells from the brain has
been repeatedly documented, but it was only through the
insertion of the reporter gene LacZ, into the Myf5 locus that it
was possible to unequivocally identify Myf5 expressing cells
in the nervous system and to show that these cells co-express
neural and muscle markers [36]. Hence, even though there
is still no clue as to the physiological significance of these
findings, they provided an indication for potential myogenic
precursors in sites other than muscle.
Similarly, several laboratories had shown that primary
fibroblasts from different organs are able to undergo muscle differentiation at low but significant frequency when
co-cultured with myogenic cells [37]. Now, we can propose
that possibly multi-potent mesoderm stem cells (see below)
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present in preparation of primary cells were responsible for
myogenic differentiation.
A search for donor tissues that may contribute myogenic cells for muscle regeneration identified bone marrow
as a possible source. By transplanting genetically-marked
bone marrow into immune-deficient mice, it was shown that
marrow-derived cells may undergo myogenic differentiation
and, moreover that they can circulate [1].
These results raised questions on the origin of these circulating myogenic progenitors and their possible relationship
with resident satellite cells. Indeed, we found that the large
majority of clones with the typical morphology of mouse
adult satellite cells were derived from dorsal aorta and not
from somites, the presumed source of all skeletal myogenic
cells of the body. In vitro, these aorta-derived myogenic cells
express a number of myogenic and endothelial markers that
are also expressed by satellite cells. In vivo, aorta-derived
myogenic progenitors participate in muscle regeneration and
fuse with resident satellite cells [38]. These data suggest that
a subset of post-natal satellite cells may be rooted in a vascular lineage. Whether these myogenic vascular cells arise
from a primordial pericyte or from endothelial cells proper,
as suggested by transient expression of endothelial markers, is not currently known. However, the circle in the end
may close up in the paraxial mesoderm. Through a genetic
labeling approach that allows the identification of clones of
differentiated cells originating from a single founder cell in
the unperturbed mouse embryo, it was very recently shown
that clones expressing alpha cardiac actin can be found both
in the myotome and in the dorsal aorta (Esner and Buckingham, ms in preparation). These clones occur with high
frequency and are often composed of few cells, suggesting
a founder cell that had undergone few divisions before differentiation. Moreover, embryos in which the GFP reporter
gene had been targeted into the Pax3 locus, allowed to follow
migration of Pax3 expressing cells from the paraxial mesoderm to the dorsal aorta, suggesting that progenitors for these
vessel associated multi-potent stem cells (that indeed express
high level of Pax3, but not Pax7) may indeed originate from
the paraxial mesoderm, that anyway forms the roof of the
aorta, and then use angiogenesis as a route to be distributed
to all mesoderm tissues during fetal histogenesis. Whatever
their origin may be, these cells may be multi-potent, since
clones of dorsal aorta can give rise to osteoblast-like cells in
the presence of BMP-2; indeed, multi-potency is preserved
even in adult muscle satellite cells, as proven by the fact that
BMP2 can switch them to an osteogenetic fate [39]. Based
on these data, we postulated that when a blood vessel penetrates into a muscle anlagen, these progenitors should find
themselves into a muscle field and thus adopt a satellite cell
fate (mimicked by appropriate culture conditions); when the
vessel grows inside a different tissue, these cells may adopt
the specific fate of that tissue and contribute to local histogenesis [40]. This would explain the wide distribution of rare
cells with a residual myogenic potency in the many if not all
the tissues of the post-natal body. Upon proper culture condi-
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tions, clones from embryonic but also post-natal vessels can
be established in culture and maintained for many passages
in vitro, while retaining their multi-potency [41]. We termed
these vessel-derived cells “mesoangioblasts”. Multi-potent
mesenchymal cells, capable of producing osteoblasts, chondroblasts, adipocytes and even skeletal muscle, have long
been known to be present in the bone marrow and are a
subject of intense investigation [42]. Whether the cells originating from embryonic vessels represent the progenitors of
multi-potent mesenchymal cells, or a separate lineage with
at least part of the same developmental potential, remains to
be investigated.
7. The possible lineage relationship of
mesoangioblasts with other myogenic cells
Mesoangioblasts are derived from the vessel wall and so
are mesenchymal stem cells and multi-potent adult progenitors; thus, the vascular niche in the bone marrow and possibly
in all mesoderm is a site where different types of multi-potent
(and potentially myogenic cells) are found. Furthermore,
hematopoietic stem cells (HSC), which also show some level
of myogenic potency, are present in the same anatomical site,
within the bone marrow and other hematopoietic tissues.
Until now the best differentially expressed marker that
distinguish HSC from the other mesoderm stem cells (MSC)
is CD45. Both CD45 positive HSC and at least a subset of
CD45 negative mesoderm stem cells may ultimately derive
from a ill defined “hemangioblast” population, present in the
embryo but possibly persisting as a rare cell in post-natal life
(for a detailed discussion see [40]).
A question relevant to muscle regeneration is whether
there is any lineage relationship between one or more types
of HSC or MSC and muscle satellite cells. In other words, it
is possible that either HSC or MSC or both may home to a
satellite cell position and become satellite cells. Evidence for
this event has been claimed of the basis of co-expression of a
satellite cell markers (M-Cadherin, CD34, Pax7) and a donor
cell marker (GFP, LacZ, etc.) in a cell located underneath
the basal lamina but outside the sarcolemma, after injection
or bone marrow transplantation. Even though this event has
been found to be rare when analyzed in vivo, a real possibility exists that it may occur constantly during late fetal
and post-natal muscle growth, so that it may feed a significant proportion of cells into the satellite cell compartment or
contribute directly to regenerating fibers (Fig. 2). Obviously,
experiments carried out in a short period of time would miss
the alternative origin of satellite cells that may have been
derived from HCS or other MSC, long before the time of
analysis and thus be scored as resident cells. Furthermore, in
all these experiments, a damage to skeletal muscle and often
a depletion of the resident pool of myogenic cells are required
to provide a selective advantage to donor cells. This means
that it will be very difficult to know what is the turn-over of
satellite cells and what part of this turn-over may be carried
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Fig. 2. A schematic representation of the possible origin of myogenic cells in regenerating skeletal muscle. Regenerated fibers (characterized by centrally
located nuclei) are formed by fusion of resident satellite cells (red) that may derive, although for a very minor part, from cells derived from the micro-vasculature,
either as circulating cells (violet) or cells of the vessel wall (green). No distinction is made between endothelial cells and pericytes.
out by non resident progenitors cells in the healthy muscle of
a normal mammal or in the course of a primary myopathy.
8. Features of professional and occasional myogenic
cells
From what argued above, it is possible to draw the following conclusions: (i) all embryonic myoblasts are derived
from somites; (ii) fetal myoblasts are most likely derived
from somites but it cannot be formally excluded that some
may also come from lateral mesoderm; (iii) satellite cells
are thought to be derived from somites but the evidence is
circumstantial and more direct experiments are difficult to
envision; (iv) mesoderm stem cells of various kind appear to
originate and reside within the vascular niche (even though
they may derive at least in part from paraxial mesoderm) and
thus can be found in many if not all the mesoderm. They are
normally not fated to give rise to skeletal muscle, but can do
so if exposed to a “myogenic field” as it could happen during
muscle development or regeneration.
Until recently, it was almost impossible to isolate mouse
embryonic and fetal myoblasts from other mesoderm cells
(fibroblasts) and thus there was very little data on genes
expressed before differentiation. The recent availability of
a Myf5-GFP mouse [43] has allowed to isolate committed
(Myf5+) but undifferentiated (MyHC−) embryonic and fetal
myoblasts and their gene expression profile has bees studied by micro-array analysis. The results will be reported
elsewhere. For what concerns the issue treated here, it is sufficient to say that the profile of gene expression of embryonic
myoblasts is similar to that of fetal myoblasts but more distant from that of satellite cells and mesoangioblasts [44] that
indeed share some similarity.
When explanted in culture, these different types of
cells differ dramatically in their behavior (Table 1) even
though their morphology (Fig. 3) and expression of different
markers (Table 2) is not dramatically different. Embryonic
Table 1
Features of different myogenic progenitor cells under various experimental conditions
Cell type
Origin
Proliferation
Self-renewal
Commitment
Differentiation
Embryonic myoblasts
Fetal myoblasts
Satellite cells
MSC
HSC
Somite
Somite
Somite, vasculature?
Vessel wall
Bone marrow microvasculature
Very low
Low
High
High
Low (in vitro)
No
No
Yes
Yes
Yes
Wnt–Shh
Wnt–Shh?
?
?
Wnt?
Spontaneous, muscle absent
Spontaneous, muscle present
Spontaneous, muscle present
Induced by muscle cells
Induced by muscle cells
For simplicity, MSC refer to mesoangioblasts, mesenchymal stem cells and multi-potent adult progenitors. HSC cells refer to hematopoietic stem cells,
independently from the selection method (lineage negative, expression of markers such as c-Kit, CD34, Sca-1, dye exclusion—SP population).
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Fig. 3. Different morphology of embryonic (A) and fetal (B) myoblasts, of satellite cells (C) and mesoangioblasts (D) in culture. Phase contrast.
myoblasts are elongated cells that immediately differentiate into mononucleated or oligonucleated myotubes and do
not respond to growth factors in the medium. Related to
this, their differentiation is also not inhibited by molecules
such as TGF␤ and phorbol esters [35]. Fetal myoblasts
show a triangular shape and proliferate to a limited extent
in response to growth factors, differentiate into large multinucleated myotubes and this differentiation is inhibited by
TGF␤ and phorbol esters. Satellite cells are the only clonogenic cell in the myogenic lineage of the mouse; they also
respond to growth factors but undergo senescence after a lim-
ited number of passages in vitro. They show a round shape
morphology and also form large myotubes whose differentiation is sensitive to TGF␤ but not to phorbol esters.
Mesoangioblasts (as an example of MSC) show a similar morphology to satellite cells and fetal myoblasts but
proliferate continuously in vitro and fail to undergo myogenesis in vitro unless co-cultured with differentiating myogenic
cells [41]. In terms of expression of regulatory genes, only
embryonic and fetal myoblasts and satellite cells express
myogenic bHLH and MEF2. Fetal myoblasts like satellite
cells express high levels of VCAM1 and ␣7-integrin, as
Table 2
Expression of different surface molecules and transcription factors in different myogenic progenitor cells
Cell type
CD45 Tal-1
CD34
Flk-1
SMA
MyoD Myf5
Pax3
Pax7
␣7-integrin
Vcam1
PKC␪
Myotome
Emb mb
Fetal mb
Satellite
MSC
HSC
−
−
−
−
−
+
±
−
±
±
+/−
+
±
±
±
±
+/−
+
+
+
+
+
+
−
+
−
−
−
−
−
−
+
+
+
+/?
?
−
+
+
+
?
?
−
±
+
+
±
−
−
±
+
+
+
−
−
−
+
−
−
−
Expression: (+); weak expression: (±); no expression: (−); expression in one but not in other types of mesoderm stem cells: (+/−). For example, CD34 is
expressed by mesoangioblasts but not by mesenchymal stem cells or multi-potent adult progenitors; Pax3 and Pax7 are expressed in all myogenic cells (but the
myotome) before progressing into the myogenic differentiation pathway at which time they also express Myf5 and MyoD. Pax3 but not Pax7 is expressed in
mesoangioblasts: expression unknown in other mesoderm stem cells.
630
G. Cossu, S. Biressi / Seminars in Cell & Developmental Biology 16 (2005) 623–631
previously reported [30], whereas embryonic myoblasts do
not. Mesoangioblasts express low level of VCAM1 but not
␣7-integrin. Some of the endothelial markers expressed by
mesoangioblasts such as CD34 are also expressed on satellite cells but not in myoblasts [38]. Beside some variation
in marker expression (such as CD34), what has been stated
for mesoangioblasts applies also to mesenchymal stem cells,
multi-potent adult progenitors and, to a lesser extent, to
endothelial progenitor cells, some of which may be resident in
muscle endothelium and have myogenic potency [4]. Indeed,
we still do not know how many different types of mesoderm
stem cells exist in the mammals. A clearly different situation
is represented by HSC or their SP (side population) fraction.
These cells are CD45, Tal1 positive, like none of the others,
are still difficult to be expanded in vitro and the extent of their
muscle differentiation in vivo is very limited [45], although
controversy exists as to whether it is due to myogenic commitment or fusion [7,8].
sively demonstrated, even though significant morphological,
biochemical and functional rescue has been achieved by intraarterial delivery of mesoangioblasts in a mouse model of
muscular dystrophy [47].
However, functional correction of a large muscle may represent a completely different problem, probably calling for
experimentation in a large animal model such as the dystrophic dog [48]. Despite the elevated cost, the need to produce a number of specie-specific reagents, the high variability
among different dystrophic pups and the ethical concerns
related to the use of dogs for research, it is likely that cell
therapy protocols in this animal will bring further information as to the feasibility of similar protocols in dystrophic
patients. Hopefully, in a few years time, phase I clinical trials with stem cells may start and set the stage for one more,
and at least in part successful attack to defeat these genetic
diseases.
Acknowledgements
9. Perspectives for use in cell therapy protocols
The scenario described above is complex and likely will
be expanded, refined and possibly modified by the rapidly
accumulating data from the many laboratories involved in
this area of research.
Nevertheless, a quest for a therapy for muscular dystrophy
and other primary muscle diseases, raises the additional need
to choose among these myogenic progenitors those which
may best fit the requirements for a successful restoration of
muscle morphology and function [46].
To this aim, selection of the appropriate cell type should
meet the following criteria: (a) accessible source (e.g. blood,
bone marrow, fat aspirate, muscle or skin biopsy); (b) ability to grow as a relatively homogeneous population in vitro
for extended periods without loss of differentiation potency
(since it appears currently unlikely that cells may be acutely
isolated in numbers sufficient for therapeutic purposes); (c)
susceptibility to in vitro transduction with vectors encoding therapeutic genes (these vectors should themselves meet
criteria of efficiency, safety and long term expression); (d)
ability to reach the sites of muscle degeneration/regeneration
through a systemic route and in response to cytokines released
by dystrophic muscle; (e) ability to differentiate in situ into
new muscle fibers with high efficiency and to give rise to
physiologically normal muscle cells.
Embryonic and fetal myoblasts that may be derived from
aborted material do not grow extensively in vitro and thus
will not be considered. Satellite cells on the other hand fulfill
all the requested criteria but one, unfortunately a crucial one.
They cannot reach the site of regeneration through a systemic
route and thus need to be delivered locally through intramuscular injection.
On the other hand, mesoderm stem cells can reach skeletal
muscle through the circulatory route, but their ability to repair
skeletal muscle in primary myopathies is still to be conclu-
Our work is supported by Telethon, the European Community, the Duchenne Parent Project, the Association Francoise
contra les Myopathies, the Muscular Dystrophy Association
and the Italian Ministries or Health and of Research.
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