The existence of cells with stem cell-like abilities derived from various tissues can now be extended to include the skeletal muscle compartment. Although researchers have focused on the utilization of these cells with regard to their myogenic capacity, initially exploring more efficient cellular therapy treatments for muscular dystrophy, it is becoming increasingly apparent that such cells may one day be used in the treatment of non-myogenic disorders. Evidence regarding the existence and differentiation capacity of muscle-derived stem cells is discussed, along with current theories regarding their proposed position within the myogenic hierarchy.
The search for more effective therapies in treating diseases has many researchers revisiting basic questions regarding cellular functions and rethinking classic definitions of myogenic cellular differentiation. Indeed, skeletal muscle may represent a convenient source of stem cells for cellular and cell-mediated therapies. The literature presented examines the evidence regarding this newly discovered cell isolated from skeletal muscle. For clarity, it is important to distinguish between satellite cells and muscle-derived stem cells (MDSC). Satellite cells, referred to by many as muscle stem cells, are myogenic precursors that are capable of regenerating skeletal muscle and demonstrate self-renewal properties, however they are considered to be committed to the myogenic lineage. MDSCs, which may represent a predecessor of the satellite cell, are considered to be distinct in that they may not be restricted to the myogenic or mesenchymal tissues. This review serves to entice discussion regarding the existence of MDSCs and their potential application in regenerative medicine and gene therapy.
Stem cells: definitions, characteristics and recognition
Adult stem cells are defined by two major functions: self-renewal and multilineage differentiation. Stem cells are functionally responsible for development and regeneration of tissue and organs. Developmental signals, both biochemical and biomechanical, trigger the proliferative action of the stem cells in early development. However, as we attempt to harvest and utilize adult stem cells, it is necessary to understand the specific signals which the cell requires in order to be directed along a desired pathway. Nonetheless, at this point, we are limited to a more descriptive understanding of their behavior.
The hierarchy of multilineage differentiation leads to the terms totipotential, pluripotential, multipotential, progenitor and precursor cells. In the earliest stages of development, the totipotential zygote and early blastocyst cells give rise to a fully differentiated adult organism. Just a few divisions into development, totipotency is lost. At this stage, pluripotential cells are present that give rise to cells of all three germ layers, but are no longer capable of giving rise to an entire organism. Germ layer-specific multipotential cells emerge later in development and are present in the adult tissues to repopulate and regenerate in response to environmental cues. The organ-specific progenitor and precursor cells give rise to mature, tissue-specific, differentiated cells.1
A second defining characteristic within the classical definition of a stem cell is its self-renewal ability. In tissues in which stem cell function is essential, homeostasis is maintained by balancing any tissue loss (due to injury or apoptosis) with repopulating organ-specific cells, while at the same time preserving the ability of the tissue to undergo future rounds of re-population. In this respect, it is thought that asymmetric cellular divisions occur such that the stem cell gives rise to one cell destined for differentiation and one renewed stem cell. An alternative possibility is that individual cells of the stem cell population respond stochastically by either differentiating or self-renewing. With either mechanism a stem cell, or reserve population, is maintained.
Stem cells are also often recognized by their quiescent behavior, though it is certainly not characteristic of stem cells of early development. In general, the stem cells of adults most likely remain quiescent until activated by injury or tissue damage. Indeed, the degree of the injury may dictate the level of stem cell activation and participation in response to the insult. Precursor cells may be readily available for homeostasis, while the more potent stem cell may be kept safely quiescent until serious injury occurs.
Isolation and identification of stem cell populations from various tissues is dependent upon specific markers, usually either exclusive proteins or characteristic profiles of more common surface proteins. Currently, one of the most well-defined stem cell populations is the mouse hematopoietic stem cell, which can be readily identified by a characteristic marker profile (Sca-1-positive, c-kit-positive, and differentiated hematopoietic lineage markers-negative).234 To date, only Sca-1 has been consistently identified on the putative MDSC. It is possible that the more committed the level of the stem cell, the more unique the organ-specific marker. As the level of pluripotency increases, these organ-specific markers may diminish and sets of common markers among stem cells from various organs may become more apparent.
Muscle-derived stem cells: differentiation capability
The mature functional cell of skeletal muscle, the multinucleated myofiber, is surrounded by satellite cells that lie outside the sarcolemma, but within the basal lamina. These satellite cells, which appear to be committed precursor cells, were first described by Mauro in 1961 based on their location and morphology.5 The implantation of radiolabeled satellite cells led to the understanding of their role in muscle regeneration.6 Satellite cells in adult muscle remain quiescent until external stimuli trigger re-entry into the cell cycle. Their progeny, myoblasts, fuse to form new multinucleated myofibers.78910 Cell surface markers associated with the satellite cell phenotype, either in the quiescent or activated state, have attempted to be elucidated and include: M-cadherin, c-met, and CD34.1011121314 Figure 1 presents a proposed mechanism of skeletal muscle precursor cell differentiation within the myogenic lineage, including the MDSC.
More recent evidence supports the existence of a population of multipotential MDSCs able to differentiate into other mesodermal cell types. A population of cells, isolated from avian and mammalian skeletal tissue using a freeze–thaw procedure, was capable of differentiating into muscle, fat, bone and cartilage when stimulated in vitro with the synthetic glucocorticoid, dexamethasone.15161718 More recent characterization of human MDSCs by the same group has identified the expression of CD13 and to a lesser extent CD10 and CD56, on these cells,19 as well as the lack of hematopoietic marker expression, including CD45. These cells were named mesenchymal stem cells for their ability to differentiate into the mesodermal phenotypes. For clarity, it should be noted that a population of bone marrow stromal cells has also been termed mesenchymal stem cells, referring to their differentiation potential. Marrow-derived mesenchymal stem cells have been described as expressing the proteins SH2, SH3, CD29 CD44, CD71, CD90, CD106, CD120a and CD124.20 It is not clear at this point if these two populations are the same, are distinct, or represent different stages of maturation of the same lineage.
Muscle-derived cells have been shown to differentiate into mesenchymal tissues, functionally regenerating bone and muscle, as well as play a role in cartilage healing. Bone-morphogenic protein 2 (BMP-2) has been identified as an inducing agent that can stimulate conversion to the osteogenic pathway.21 Since this discovery, several researchers have transduced murine candidate MDSCs with an adenovirus encoding for BMP-2, and transplanted the cells into an allogeneic host to demonstrate bone matrix formation and cellular differentiation into osteoblasts and osteocytes.222324 In particular, Lee et al22 demonstrated that genetic engineering of the muscle-derived cells provided an effective cellular therapy in healing a critical-size skull bone defect. Additionally, muscle-derived cells showed improved cartilage healing in comparison to chondrocytes when seeded on to collagen gel scaffolds and placed into a full-thickness cartilage defect.25 With regard to hematopoietic differentiation, muscle-derived cells transplanted into lethally irradiated mice have been shown to reconstitute the hematopoietic system.2627 In the classic demonstration of self-renewal, Jackson et al27 used the muscle-derived reconstituted bone marrow from one animal to rescue a second animal, achieving complete multilineage engraftment of the hematopoietic compartment. In vitro experiments have demonstrated that expression of the transcription factor Pax7 is required for cellular differentiation of a specific MDSC population (SP) towards the myogenic pathway (satellite cells). Interestingly, an identical MDSC population derived from a Pax7−/− animal demonstrated a 10-fold increase in hematopoietic colony formation suggesting a potential shift in commitment.28 Such findings suggest that satellite cells and MDSCs are distinct populations, and will be discussed in further detail with regard to the potential origins of these cell populations. Current thinking regarding the multilineage differentiation capability of MDSCs is shown in Figure 2.
Putting muscle-derived stem cells to work: muscular dystrophy
The existence of MDSCs, and perhaps the most intriguing and promising application for their use, has evolved from studies investigating myoblast transplantation for the treatment of muscular dystrophies. Since 1989, when it was discovered that muscle precursor cells (satellite cells) from a normal donor could restore dystrophin expression within dystrophic host muscle, this form of cellular transplantation has revealed many facets of skeletal muscle biology and development.29 As such, it is important to discuss MDSCs, and detail their potential utility, with regard to enhancement of cellular therapies for muscular dystrophy.
Observations regarding the extremely low number of donor precursor cells that survive following transplantation into mdx mice, the widely used animal model for Duchenne's muscular dystrophy, have forced researchers to more closely examine muscle precursor cell biology.303132 It is now thought that only a few percent of donor cells survive the initial transplantation process, which includes a non-specific inflammatory reaction along with other necrotic, and yet undefined, events.333435363738 The survival of a portion of transplanted cells does not appear to be by default, rather, specific populations of muscle precursor cells may be more suited to survive this initial environment and contribute to the regeneration process.313335 Specific muscle precursor populations are now being investigated with regard to their ability to restore dystrophin expression both locally and systemically, and the results are suggestive of the existence of MDSCs.
Varying isolation techniques and the lack of a commonly accepted set of identifying proteins, or markers, has made direct comparison of the various populations difficult. This is compounded by the fact that many of the markers, particularly surface proteins, may be differentially up-regulated or down-regulated in vitro, possibly due to such factors as culture conditions and differentiation. Characterization in terms of surface markers is now commonly performed in a similar manner to that used to describe hematopoietic stem cells, leading to more definable populations. However, more work still needs to be performed to establish differences and similarities between the various populations. A summary of the defining characteristics and major findings of recently described MDSC populations is presented in Table 1. It is convenient to separate the studies based upon the isolation technique used: sorting via fluorescence-activated cell sorting versus culture techniques.
A paramount study investigating stem cells derived from both bone marrow and muscle has provided evidence of both plasticity and homing capability. By applying to skeletal muscle a sorting technique used to isolate a bone marrow ‘side population’ (SP) with hematopoietic reconstitution capacity, a population of SP cells was isolated that partially restored dystrophin expression following intravenous delivery and resided within positions consistent with muscle precursor satellite cells. Interestingly, these same muscle-derived sp cells were shown to reconstitute the hematopoietic lineage of irradiated hosts, yet displayed a different set of surface antigens than their marrow-derived counterparts (most notably lacking c-kit, CD45 and CD43).25 In addition, a similar method was also employed to demonstrate hematopoietic engraftment potential in a separate study.26 Thus, the concept of systemic delivery was demonstrated along with expanding the knowledge of muscle-derived cell plasticity, yet clinical utility for muscular dystrophies dictates more efficient delivery and/or a larger commitment to the myogenic lineage. Advances in the understanding of the molecular mechanisms and signaling pathways involved in differentiation may allow manipulation to enhance such processes.
Culture methods have also been utilized to separate distinct muscle-derived cell populations. Based upon the variable adherence of cells obtained from freshly dissociated muscle, pre-plating has been used as a purification technique to obtain MDSC populations.3438 Using such techniques to isolate cells that demonstrate slow adhesion characteristics it was shown that greater transplantation efficiency could be achieved, even without anti-inflammatory strategy.34 In a subsequent study, this purification technique was used to establish a clonal cell line that was shown to be highly efficient in regenerating dystrophin-positive myofibers upon direct injection and also demonstrated, although to a lesser extent, the ability to regenerate muscle following intravenous delivery. Further, this same population of cells was shown to differentiate within the osteogenic lineage in vivo, when appropriately stimulated with osteogenic proteins.22 Although the mechanisms behind this behavior are not yet defined, characterization of the pre-plating process has revealed the purification of subpopulations displaying cell surface proteins similar to those used to characterize hematopoietic stem cells.39
In separate studies a purified muscle-derived cell fraction, obtained by a similar pre-plating technique and enriched for two markers found on hematopoietic stem cells, displayed enhanced adhesive properties to muscle capillaries of mdx mice when delivered arterially. This population, which was also found to possess hematopoietic differentiation capability, could disseminate from the bloodstream and participate to a small extent in the restoration of dystrophin in skeletal muscle. This restoration was significantly enhanced by needle injury to the muscle following arterial delivery, but not by less damaging exercised-induced strain injury. Although this observation could be explained by mechanisms involving the physical release of cells adhered to the capillary lumen, identical cells displayed a poor commitment to myogenic differentiation when injected directly into dystrophic muscle.38 Observations such as these, further demonstrate that complex signaling, from yet unknown factors, plays a key role in directing differentiation and myogenic commitment.
The origin of muscle-derived stem cells: are they distinct from satellite cells?
Recently, models have emerged attempting to address the existence, origin, and function of MDSCs. One model is based upon the finding that satellite-like cells, sharing both myogenic and endothelial markers, can be obtained from the dorsal aorta of murine embryos.40 Such findings suggest that at least some portion of the satellite cell population may develop independently of embryonic myogenesis and may be rooted in vascular development. Such a model predicts that precursors associated with the vasculature may differentiate in a capacity directly related to the tissue in which the vasculature is associated.143540 This model is not inconsistent with many of the findings in muscular dystrophy transplantation using MDSCs, as well as other findings regarding the derivation of stem cells from other tissues of the body.
Still, some confusion exists regarding the possibility that MDSCs are a subpopulation of the satellite cells that have been described for years. Further investigation of the sp population, described earlier, is supportive of a clear distinction between conventional satellite cells and MDSCs. A difference between the sp cells and satellite cells was confirmed through the differential expression of the transcription factor Pax7. Pax7−/− mice demonstrated a complete absence of satellite cells, whereas the number of SP cells obtained from these mice was unaffected. Together the results are suggestive of a model in which the sp cells represent satellite cell progenitors, with Pax7 expression signaling commitment to the myogenic lineage.27 This is in agreement with original observations regarding the satellite cell positioning of sp cells within dystrophin-expressing myofibers following intravenous delivery in the mdx host.25 Considerable room for overlap with the previously described model is possible, considering that Pax7−/− mice display normal-appearing skeletal muscle further implies that embryonic myoblast and muscle development may occur independently of post-natal satellite cell development.1427 However, the definitive origin of the sp cells is currently unknown.
Although a certain degree of myogenic cell plasticity within defined clonal populations has been described,1321 recent studies have provided a substantial degree of evidence suggesting the presence of at least one type of primitive MDSC population. It remains unclear whether one true population of MDSCs exists, however some common themes among investigations indicate that the elucidation of these cells are more than a biologic fascination. Indeed, MDSCs seem to display abilities that make their potential clinical use for the treatment of diseases, such as muscular dystrophy, advantageous. These include, but are not limited to, their differentiation into various mesenchymal and non-mesenchymal tissues, their ability to more efficiently survive the transplantation process, and their seemingly enhanced ability to be delivered through the circulation and home to muscle. A recent demonstration of vastly improved muscle regeneration and dystrophin delivery with MDSC transplantation, in comparison to satellite cells, underscores the importance of continued investigation in this area.42 In particular, improvement in systemic delivery processes, which traditionally have been performed by direct injection into specific muscles, may circumvent one of the major clinical barriers facing the use of cellular therapies for the treatment of muscular dystrophies.
Questions that must continue to be addressed by researchers studying MDSCs are similar to those encountered by others working in the stem cell field. These include issues involving: (1) their isolation from human tissues of various ages; (2) origin and optimal isolation techniques; (3) identity of the population(s) responsible for stem cell-like capabilities; (4) expansion such that clinically relevant quantities can be obtained for use in treating diseases; (5) differentiation and signaling processes that regulate progression within a lineage; and (6) effects of genetic manipulation to correct inherent deficiencies. It remains to be seen whether researchers will be able to turn these elusive cells into vehicles for curing a host of both muscle and non-muscle diseases.
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This work was supported in part by grants to Dr Huard from the National Institutes of Health (1P60 AR44811-01, 1PO1 AR45925-01), the Pittsburgh Tissue Engineering Initiative (PTEI), the William F and Jean W Donaldson Chair at Children's Hospital of Pittsburgh, the Muscular Dystrophy Association (USA), the Parent Project (USA), and the Orris C Hirtzel and Beatrice Dewey Hirtzel Memorial Foundation.
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Jankowski, R., Deasy, B. & Huard, J. Muscle-derived stem cells. Gene Ther 9, 642–647 (2002). https://doi.org/10.1038/sj.gt.3301719
- skeletal muscle
- stem cells
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A modified preplate technique for efficient isolation and proliferation of mice muscle-derived stem cells