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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 1. 2. 3. 4. 5. 6. 7. 8. 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623 624 625 625 626 626 627 628 630 630 630 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 624 G. Cossu, S. Biressi / Seminars in Cell & Developmental Biology 16 (2005) 623–631 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 625 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 626 G. Cossu, S. Biressi / Seminars in Cell & Developmental Biology 16 (2005) 623–631 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) G. Cossu, S. Biressi / Seminars in Cell & Developmental Biology 16 (2005) 623–631 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- 627 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 G. Cossu, S. Biressi / Seminars in Cell & Developmental Biology 16 (2005) 623–631 628 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). G. Cossu, S. Biressi / Seminars in Cell & Developmental Biology 16 (2005) 623–631 629 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. References [1] Ferrari G, Cusella-De Angelis MG, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, et al. Muscle regeneration by bone marrowderived myogenic progenitors. Science 1998;279:1528–30. 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