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Myogenic Specification of Adult Stem Cells in Skeletal Muscle
... The presence of cells with the potential of mesenchymal stem cells was revealed in muscles of birds, rabbits and human beings [19]. Depending on isolation method or described features they are called either muscle-derived stem cells (MDSCs) [20] , [21] , or musclederived hematopoietic stem cells (MD-HSCs) [22]. It is not clear whether these cells are differently called but are in fact the same population or if they are rather distinct groups of cells. ...
... To describe muscle stem cell phenotype the presence of stem cell antigen-1 (sca-1), CD34 (marker of hematopoietic and satellite cells), CD45, c-kit (markers of hematopoietic cells) and desmin (marker of myogenic cells) were determined (Table 1 ). Conflicting data were reported [21][22][23][24][25][26][27][28], what can suggest that several distinct populations of multipotential musclederived stem cells exist. On the other hand, obtaining various expression profiles could be caused by using different methods of protein detection – western blot, immunocytochemistry or flow cytometry. ...
... Initially, the term "side population" was associated mainly with a subpopulation of hematopoietic stem cells [38]. However, later studies revealed that high level of ABCG2 expression and effluxing the Hoechst 33342 dye characterize some types of stem cells in other tissues and organs (for example skeletal muscle tissue, mammary gland or lungs [21,39,40] and also some cancer cells [41] ). Therefore, side population phenotype is rather associated with low level of differentiation then certain features or origin of cells. ...
Both skeletal muscle and bone marrow tissue contain myogenic stem cells. The population residing in muscles is heterogenic. Predominant in number are "typical" satellite cells - muscle progenitors migrating from somites during embryonic life. Another population is group of multipotent muscle stem cells which, at least in part, are derived from bone marrow. These cells are tracked by gradient of growth factors releasing from muscle during injury or exercise. Recruited bone marrow-derived cells gradually change their phenotype becoming muscle stem cells and eventually can attain satellite cell position and express Pax7 protein. Mesenchymal stem cells (MSC) isolated directly from bone marrow also display myogenic potential, although methods of induction of myogenic differentiation in vitro have not been optimized yet. Concerning efforts of exploiting myogenic stem cells in cell-mediated therapies it is important to understand the cause of impaired regenerative potential of aged muscle. Up to now, most of research data suggest that majority of age related changes in skeletal muscles are reversible, thus depending on extrinsic factors. However, irreversible intrinsic features of muscle stem cells are also taken into consideration.
... Post-natal muscle regeneration is mediated by satellite cells (SC), a population of "professional" muscle stem cells. Other cell populations with myogenic potential have been isolated from skeletal muscle, such as side population cells, pericytes, and myogenic endothelial cells (Asakura, Seale et al. 2002;Zheng, Cao et al. 2007;Crisan, Yap et al. 2008); however, the importance of these for muscle ...
... During proliferation, cyclin D1 functions to activate cyclin dependent kinases (CDK) 4 and 6, promoting the activity of the transcription factor E2F and allowing entry into S-phase. Several NF-κB binding sites have been found within the cyclin D1 promoter (Asakura, Seale et al. 2002). Although preferential binding is reported to be by a nonclassical p52/B-cell lymphoma 3-encoded protein (Bcl3) heterodimer, there appears to be some redundancy, as cyclin D1 transcription was not found to change in p52, Bcl3, p50, c-Rel, or p65 null (-/-) mouse embryonic fibroblasts (MEFs). ...
... Declining NF-κB activity in turn lowers YY1 and increases MyoD at the same time. MyoD can then replace YY1 at myogenic gene promoters and recruit other activators, including CREB-binding protein (CBP)/p300 (Asakura, Seale et al. 2002;Wang, Garzon et al. 2008). ...
Duchenne muscular dystrophy (DMD) is a fatal disease characterized by progressive skeletal muscle degeneration. Inhibition of the transcription factor nuclear factor-κ B (NF-κB), and more specifically the p65 subunit, significantly improves the phenotype of mdx mice, a murine DMD model. However, the ubiquity of NF-κB stands as an obstacle to clinical translation. In this dissertation, we explore the roles of NF-κB/p65 in the regenerative capacity of muscle-derived stem cells (MDSCs) with the goal of identifying alternative approaches to DMD treatment. We found that both cell proliferation and myogenic potential were increased in MDSCs lacking one allele of p65 (p65+/-). In wild type MDSCs, in vitro pharmacologic inhibition of the upstream activating kinase, IKKβ, increased myotube formation in a dose-dependent manner. When transplanted into mdx hind limb muscle, p65+/- MDSCs resulted in significantly larger engraftments. Furthermore, engraftments in cardiotoxin (CTX) injured muscle were associated with reduced local host necrosis and inflammation. Not only were p65+/- MDSCs found to be more resistant to oxidative stress, but we found that p65 depletion improved the anti-inflammatory capacity of MDSCs in vitro and in vivo via upregulation of hepatocyte growth factor (HGF). Moreover, accelerated regeneration in p65 haploinsufficient mdx mice (mdx;p65+/-) coincided with HGF upregulation. Intraperitoneal injection of a musculotropic adeno-associated virus carrying shRNA targeting HGF reversed the phenotypic improvements of mdx;p65+/- mice, increasing both muscle inflammation and necrosis. These data implicate NF-κB/p65 in muscle stem cell proliferation, differentiation, survival, and growth factor gene expression, further underlining the danger of broadly targeting such an important pathway. Finally, this research has also identified HGF as a downstream effector of NF-κB/p65 inhibition in mdx mice. Thus, delivery of HGF or activation of its receptor, MET, may represent a new approach to reduce chronic inflammation and preserve muscle fiber integrity in DMD.
... In addition to SCs, many other cell populations in skeletal muscle have myogenic potential. These cells include pericytes, pericyte-like mesoangioblasts, muscle-derived stem cells, side population cells, and CD133 + stem cells (Ten Broek et al. 2010;Asakura et al. 2002;Torrente et al. 2004;Galvez et al. 2006;Peault et al. 2007;Negroni et al. 2009;Dellavalle et al. 2007). Among them, we mainly focus on pericytes and pericyte-like mesoangioblasts owing to their unique features and promising therapeutic potential. ...
This review chapter describes the current knowledge about the nature of pericytes in the gut, their interaction with endothelial cells in blood vessels, and their pathophysiological functions in the setting of chronic liver disease. In particular, it focuses on the role of these vascular cell types and related molecular signaling pathways in pathological angiogenesis associated with liver disease and in the establishment of the gut-vascular barrier and the potential implications in liver disease through the gut-liver axis.
... Whilst the cells constituting the side population do not typically enter myogenic differentiation, when cultured in isolation, subjected intramuscular transplantation, co--cultured with myoblasts or induced to express MyoD, they do form myocytes (Asakura et al. 2002). Interestingly, satellite cells also appear to be pluripotent and can differentiate to osteocytes or adipocytes following exposure to bone morphogenic proteins or adipogenic factor, even where MyoD, ...
Sporadic inclusion body myositis (IBM) is the commonest myopathy acquired after 50 years of age and causes significant disability. No effective treatment exists despite several clinical trials of immunotherapies. This reflects a lack of pre-clinical disease models. The work described in this thesis began with development of such a system, in which disease relevant pathological outcome measures were characterised in vitro using primary satellite cell cultures. Through over-expression of β-Amyloid Precursor Protein or exposure to inflammatory mediators IL1β and TNFα, IBM-like pathology was induced. Myotubes demonstrated ubiquinated intracellular aggregates, increased expression of MHC Class I, cytoplasmic translocation of TDP-43, ER stress, calcium dyshomeostasis and activation of NFκB. As some of these are proposed to be central pathogenic mechanisms in IBM, they provide a panel of tools on which to assess new IBM treatment strategies. The effects of heat shock response augmentation were examined using Arimoclomol, a co-inducer of the transcription factor HSF-1 that drives expression of key endogenous Heat Shock Proteins. Arimoclomol treatment ameliorated several IBM-relevant features, represented by improved cell viability, reduced ER stress, inhibition of NFκB and reduced cytoplasmic translocation of TDP-43. These data supported further evaluation of HSR manipulation as a potential therapy for IBM. Therefore, Arimoclomol was advanced into a randomised, placebo-controlled clinical trial involving 24 patients with IBM over one year. The primary outcome measure was safety and tolerability. Muscle biopsy was performed before and after the treatment phase to evaluate HSP expression and histopathological changes. Together, this in vitro model and clinical trial represent a novel translational research pathway in IBM, the lack of which has hampered the development of effective treatments for this disease.
... Likewise, Sherwood et al. demonstrated similar FACS isolation of myogenic cells using positive markers CXCR4 and β1-integrin and negative markers CD45, Sca-1, and Mac-1, and cell purity was confirmed by growing the sorted cells into myotubes in cell culture [54]. Because of the heterogeneity of the satellite cell population, with certain sub-populations displaying unique surface marker profiles, various additional marker combinations specific to satellite cells have been identified [55,56]. Consistent with the work described above, Bosnakovski et al identified CD34 as a positive marker and CD45 as a negative marker for satellite cells sorted from a Pax7- ZsGreen + mouse [57]. ...
Engineered skeletal muscle holds promise as a source of graft tissue for the repair of traumatic injuries such as volumetric muscle loss. The resident skeletal muscle stem cell, the satellite cell, has been identified as an ideal progenitor for tissue engineering due to its role as an essential player in the potent skeletal muscle regeneration mechanism. A significant challenge facing tissue engineers, however, is the isolation of sufficiently large satellite cell populations with high purity. The two common isolation techniques, single fiber explant culture and enzymatic dissociation, can yield either a highly pure satellite cell population or a suitably large number or cells but fail to do both simultaneously. As a result, it is often necessary to use a purification technique such as pre-plating or cell sorting to enrich the satellite cell population post-isolation. Furthermore, the absence of complex chemical and biophysical cues influencing the in vivo satellite cell "niche" complicates the culture of isolated satellite cells. Techniques under investigation to maximize myogenic proliferation and differentiation in vitro are described in this article, along with current methods for isolating and purifying satellite cells.
... Many of these cell types have been demonstrated to proliferate following muscle injury and either have the capacity to become myogenic and contribute to skeletal muscle or remain non-myogenic but nevertheless support the process of muscle regeneration (reviewed in [33] [34] [35]). Cells such as satellite cells, pericytes and mesoangioblasts, side population cells, PW1+ cells, and CD133+ progenitor cells can become myogenic and fuse into injured skeletal muscle [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47]. Additionally, fibro-adipogenic (FAP) cells support muscle repair by promoting the differentiation of other muscle precursor cells [48] [49]. ...
While changes in muscle protein synthesis and degradation have long been known to contribute to muscle wasting, a body of literature has arisen which suggests that regulation of the satellite cell and its ensuing regenerative program are impaired in atrophied muscle. Lessons learned from cancer cachexia suggest that this regulation is simply not a consequence, but a contributing factor to the wasting process. In addition to satellite cells, evidence from mouse models of cancer cachexia also suggests that non-satellite progenitor cells from the muscle microenvironment are also involved. This chapter in the series reviews the evidence of dysfunctional muscle repair in multiple wasting conditions. Potential mechanisms for this dysfunctional regeneration are discussed, particularly in the context of cancer cachexia.
... In mouse, Pax7+ cells are crucial for muscle to sustain its regenerative ability (Relaix and Zammit, 2012 ). Many muscleresident cell populations, which do not express Pax7, have previously been shown to be able to contribute to muscle regeneration, such as side-population cells (Gussoni et al., 1999; Asakura et al., 2002 ), muscle-derived stem cells (Petersen et al., 2002) and CD133+ progenitor cells (Torrente et al., 2004). Even though these particular cell populations have not been described in zebrafish, it is possible that such a non-pax7- expressing cell population acts to repair small injuries to the muscle tissue, whereas pax7a-expressing cells only regenerate muscle following larger insult. ...
Skeletal muscle regeneration in vertebrates occurs by the activation of quiescent progenitor cells that express pax7 to repair and replace damaged myofibers. We have developed a mechanical injury paradigm in zebrafish to determine whether developmental stage and injury size affect the regeneration dynamics of skeletal muscle. We found that both small focal injuries, and large injuries affecting the entire myotome, lead to expression of myf5 and myogenin, which was prolonged in older larvae, indicating a slower process of regeneration. We characterized the endogenous behavior of a population of muscle-resident Pax7-expressing cells using a pax7a:eGFP transgenic line and found that GFP+ cell migration in the myotome dramatically declined between 5 and 7 days post-fertilization (dpf). Following a small single myotome injury, GFP+ cells responded by extending processes, before migrating to the injured myofibers. Furthermore, these cells responded more rapidly to injury in 4 dpf larvae compared to 7 dpf. Interestingly, we did not see GFP+ myofibers after repair of small injuries, indicating that pax7a-expressing cells did not contribute to myofiber formation in this injury context. On the contrary, numerous GFP+ myofibers could be observed after an extensive single myotome injury. Both injury models were accompanied by an increased number of proliferating GFP+ cells, which was more pronounced in larvae injured at 4 dpf than 7 dpf. This indicates intriguing developmental differences, at these early ages. Our data also suggests an interesting disparity in the role that pax7a-expressing muscle progenitor cells play during skeletal muscle regeneration, which may reflect the extent of muscle damage.
... However, the engraftment efficiency needs to be improved to provide a significant therapeutic outcome. cells which have been tested for systemic cell therapy in mice [23]. These cells can be purified by cell sorter based on their efficient efflux of vital DNA dye Hoescht 33342. ...
... SPs have been induced to undergo myogenic commitment by co-culturing them together with myoblasts or forcing Pax7 or MyoD expression [132]. A subpopulation of SPs characterized by CD45 -/ Sca-1 + / ABCG2 + / Pax7 + / Syndecan- 4 + marker profile has been found to differentiate into myofibers and undergo self-renewal in the quiescent compartment after in vivo injections [45] . ...
Satellite cells are unipotent stem cells involved in muscle regeneration. However, the skeletal muscle microenvironment exerts a dominant influence over stem cell function. The cell intrinsic complexity of the skeletal muscle niche located within the connective tissue between fibres includes motor neurons, tendons, blood vessels, immune response mediators and interstitial cells. All these cell types modulate the trafficking of stimuli responsible of muscle fibre regeneration. In addition, several types of stem cell have been discovered in skeletal muscle tissue, mainly located in the interstitium. The majority of these stem cells appear to directly contribute to myogenic differentiation, although some are mainly implicated in paracrine effects. This review focuses on one of these classes of stem cells known as adult stem cells, which following their identification in the last decade have been used for therapeutic purposes, mainly in animal models of chronic muscle degeneration. Emerging literature identifies other myogenic progenitors generated from pluripotent stem cells as potential candidates for the treatment of skeletal muscle degeneration. However, adult stem cells still represent the gold standard for future comparative studies.
... Skeletal muscles serve as a niche not only for satellite cells but also for a few other populations of stem cells. These include muscle side population cells that were identified based on their ability to exclude Hoechst 33342 dye from their cytoplasm as well as the presence of stem cell antigen Sca1 and CD45 proteins [6]. In 2002 Asakura and Rudnicki demonstrated that these cells could fuse with myoblasts in vitro and also contribute to the formation of 1% of new myofibres when transplanted into the damaged anterior tibialis muscle of SCID mice [7]. ...
... Multipotent cells have also been isolated from primary cultured skeletal muscle cells [69, 70], but these are distinct from SP cells [71]. Primary cultured myoblasts differentiate into osteoblasts and adipocytes, as well as myotubes, under appropriate culture conditions [72][73][74][75]. The existence of mesenchymal stem cell pools in muscle and other tissues, combined with evidence that ectopic BMP expression can induce these cells to differentiate along the osteogenic pathway, provides an alternative, and perhaps more abundant, source of MSC progenitors for skeletal gene therapy applications. ...
Skeletal gene therapy is an attractive new approach to the treatment of bone disorders. Impressive advances in our knowledge of the molecular genetic basis of skeletal disorders and fracture healing have led to the development of novel therapeutics based on ectopic expression of one or more genes in patient cells that can influence repair or regenerative processes in bone. Although still a relatively immature field, proof-of-principle for enhanced bone formation through skeletal gene therapy has already been established. The challenge now is to more precisely define optimal cellular targets and therapeutic genes, and to develop safe and efficient ways to deliver therapeutic genes to target cells. In this review, we will highlight some of the exciting advances that have been made in skeletal gene therapy in recent years, with a focus on treatment of localized skeletal lesions. Strengths and weaknesses of current approaches will be discussed, as will strategies for improved safety and therapeutic outcome in the future. Skeletal gene therapy can have an enormous impact on patient care. The next 5 years will present us with unparalleled opportunities to develop more effective therapeutic strategies and overcome obstacles presented by current gene transfer technologies.
... In this perspective, the availability of an animal model that mimics the characteristics of human muscle injury is crucial to study the pathophysiological mechanisms of regeneration and possible therapies. Many animal models of muscle injury have been proposed such as injection of cardiotoxin [23,24], mdx transgenic mice, crioinjury, manual clamping, laceration, strain and induced by exercise but none reproduces re-injuries which are the most common type of damage in the orthopedic field. Thus, the main purpose of this study was to investigate whether BMMC injected directly into damaged muscles could improve muscle function in a rat model with repetitive muscle injuries. ...
Skeletal muscle injury is the most common problem in orthopedic and sports medicine, and severe injury leads to fibrosis and muscle dysfunction. Conventional treatment for successive muscle injury is currently controversial, although new therapies, like cell therapy, seem to be promise. We developed a model of successive injuries in rat to evaluate the therapeutic potential of bone marrow mesenchymal cells (BMMC) injected directly into the injured muscle. Functional and histological assays were performed 14 and 28 days after the injury protocol by isometric tension recording and picrosirius/Hematoxilin & Eosin staining, respectively. We also evaluated the presence and the fate of BMMC on treated muscles; and muscle fiber regeneration. BMMC treatment increased maximal skeletal muscle contraction 14 and 28 days after muscle injury compared to non-treated group (4.5 ± 1.7 vs 2.5 ± 0.98 N/cm2, p<0.05 and 8.4 ± 2.3 vs. 5.7 ± 1.3 N/cm2, p<0.05 respectively). Furthermore, BMMC treatment increased muscle fiber cross-sectional area and the presence of mature muscle fiber 28 days after muscle injury. However, there was no difference in collagen deposition between groups. Immunoassays for cytoskeleton markers of skeletal and smooth muscle cells revealed an apparent integration of the BMMC within the muscle. These data suggest that BMMC transplantation accelerates and improves muscle function recovery in our extensive muscle re-injury model.
... Skeletal muscles serve as a niche not only for satellite cells but also for a few other populations of stem cells. These include muscle side population cells that were identified based on their ability to exclude Hoechst 33342 dye from their cytoplasm as well as the presence of stem cell antigen Sca1 and CD45 proteins [6]. In 2002 Asakura and Rudnicki demonstrated that these cells could fuse with myoblasts in vitro and also contribute to the formation of 1% of new myofibres when transplanted into the damaged anterior tibialis muscle of SCID mice [7]. ...
Understanding the mechanism of stem cell mobilization into injured skeletal muscles is a prerequisite step for the development of muscle disease therapies. Many of the currently studied stem cell types present myogenic potential; however, when introduced either into the blood stream or directly into the tissue, they are not able to efficiently engraft injured muscle. For this reason their use in therapy is still limited. Previously, we have shown that stromal-derived factor-1 (Sdf-1) caused the mobilization of endogenous (not transplanted) stem cells into injured skeletal muscle improving regeneration. Here, we demonstrate that the beneficial effect of Sdf-1 relies on the upregulation of the tetraspanin CD9 expression in stem cells.
The expression pattern of adhesion proteins, including CD9, was analysed after Sdf-1 treatment during regeneration of rat skeletal muscles and mouse Pax7-/- skeletal muscles, that are characterized by the decreased number of satellite cells. Next, we examined the changes in CD9 level in satellite cells-derived myoblasts, bone marrow-derived mesenchymal stem cells, and embryonic stem cells after Sdf-1 treatment or silencing expression of CXCR4 and CXCR7. Finally, we examined the potential of stem cells to fuse with myoblasts after Sdf-1 treatment.
In vivo analyses of Pax7-/- mice strongly suggest that Sdf-1-mediates increase in CD9 levels also in mobilized stem cells. In the absence of CXCR4 receptor the effect of Sdf-1 on CD9 expression is blocked. Next, in vitro studies show that Sdf-1 increases the level of CD9 not only in satellite cell-derived myoblasts but also in bone marrow derived mesenchymal stem cells, as well as embryonic stem cells. Importantly, the Sdf-1 treated cells migrate and fuse with myoblasts more effectively.
We suggest that Sdf-1 binding CXCR4 receptor improves skeletal muscle regeneration by upregulating expression of CD9 and thus, impacting at stem cells mobilization to the injured muscles.
... Once they have been activated and are proliferating, these cells are referred to as myoblasts and express muscle regulatory transcription factors (MRF) that regulate cell cycle exit and differentiation . Other populations of resident [77] and circulating stem cells with myogenic potential may be involved in muscle regeneration in adulthood787980. Following proliferation and migration from neighboring fibers, the myoblasts fuse with each other to form strips of multinucleated myotubes, a phenomenon observed during myogenesis in vitro [81] and during embryonic development in vivo [82,83]. ...
In tissues characterized by a high turnover or following acute injury, regeneration replaces damaged cells and is
involved in adaptation to external cues, leading to homeostasis of many tissues during adult life. An understanding
of the mechanics underlying tissue regeneration is highly relevant to regenerative medicine-based interventions. In
order to investigate the existence a leitmotif of tissue regeneration, we compared the cellular aspects of
regeneration of skin, nerve and skeletal muscle, three organs characterized by different types of anatomical and
functional organization. Epidermis is a stratified squamous epithelium that migrates from the edge of the wound
on the underlying dermis to rebuild lost tissue. Peripheral neurons are elongated cells whose neurites are organized
in bundles, within an endoneurium of connective tissue; they either die upon injury or undergo remodeling and
axon regrowth. Skeletal muscle is characterized by elongated syncytial cells, i.e. muscle fibers, that can temporarily
survive in broken pieces; satellite cells residing along the fibers form new fibers, which ultimately fuse with the old
ones as well as with each other to restore the previous organization. Satellite cell asymmetrical division grants a
reservoir of undifferentiated cells, while other stem cell populations of muscle and non-muscle origin participate in
muscle renewal. Following damage, all the tissues analyzed here go through three phases: inflammation,
regeneration and maturation. Another common feature is the occurrence of cellular de-differentiation and/or
differentiation events, including gene transcription, which are typical of embryonic development. Nonetheless,
various strategies are used by different tissues to replace their lost parts. The epidermis regenerates ex novo,
whereas neurons restore their missing parts; muscle fibers use a mixed strategy, based on the regrowth of missing
parts through reconstruction by means of newborn fibers. The choice of either strategy is influenced by the
anatomical, physical and chemical features of the cells as well as by the extracellular matrix typical of a given tissue,
which points to the existence of differential, evolutionary-based mechanisms for specific tissue regeneration. The
shared, ordered sequence of steps that characterize the regeneration processes examined suggests it may be
possible to model this extremely important phenomenon to reproduce multicellular organisms.
... SP cells express Sca-1 and c-Kit and may be either CD45 negative or CD45 positive : despite the absence of Pax7 and desmin expression, they can differentiate into myoblasts within muscle or in coculture with SC. They become hematopoietic cells after intravenous injection [51,115116117118119. All these results indicate that SP cells, though possessing a constitutive haematopoietic potential, yield myoblasts upon appropriate conditions. ...
The skeletal muscle has the capacity to repair damage by the activation and differentiation of fiber sub-laminar satellite cells. Regeneration impairment due to reduced satellite cells number and/or functional capacity leads to fiber substitution with ectopic tissues including fat and fibrous tissue and to the loss of muscle functions. Muscle mesenchymal cells that in physiological conditions sustain or directly contribute to regeneration differentiate in adipocytes in patients with persistent damage and inflammation of the skeletal muscle. These cells comprise the fibro-adipogenic precursors, the PW1-expressing cells and some interstitial cells associated with vessels (pericytes, mesoangioblasts and myoendothelial cells). Resident fibroblasts that are responsible for collagen deposition and extracellular matrix remodeling during regeneration yield fibrotic tissue and can differentiate into adipose cells. Some authors have also proposed that satellite cells themselves could transdifferentiate into adipocytes, although recent results by lineage tracing techniques seem to put this theory to discussion. This review summarizes findings about muscle resident mesenchymal cell differentiation in adipocytes and recapitulates the molecular mediators involved in intramuscular adipose tissue deposition.
... Sca-1 (stem cell antigen-1), a member of the Ly-6 family, was first described as one of the cell surface antigens expressed on hematopoietic stem cells [57]. Multipotent stem cells derived from bone marrow and skeletal muscles express Sca-1 [58][59][60]. Thereafter, several groups identified various heterogeneous subpopulations of Sca- 1+ cells based on different subsets of markers co-expressed with Sca-1 [61]. ...
Cardiovascular disease remains the number one cause of morbidity and mortality in the world. With great advances in medical and interventional therapies, patients who suffer from acute myocardial infarction have a longer life expectancy than before, but gradually develop chronic heart failure in their later life due to irreversible loss of cardiomyocytes. So far, heart transplantation is the only therapeutic option for advanced heart failure. However, the shortage of donor organs largely limits its role as the gold standard therapy. In the past decades, stem cell-based regenerative medicine has been proposed as a promising approach for the treatment of heart failure based on numerous animal studies. A variety of potential stem cell types, including skeletal myoblasts and bone marrowderived stem cells, have been investigated in clinical trials for cardiac repair and regeneration, but have shown mixed results in heart functional improvement or life-threatening disadvantages such as ventricular arrhythmia. On the other hand, due to the advantages of autologous origin and cardiac-committed lineage, cardiac stem cell therapy has emerged as a promising cell-based strategy for treatment of HF. Thus, this review discusses the current therapies for heart failure and further focuses on stem cell therapy using different endogenous cardiac stem cells, purified by stem cell surface markers (e.g., c-kit or Sca-1) or derived from explants via the formation of cardiospheres. In addition, the potential effect of patient age on cell-based therapy for heart disease is discussed.
... Recent reports have provided clear evidence that multipotent adult stem cells exist in many more organs and tissues than previously expected. Mesenchymal cells capable of differentiation into a variety of specific cell phenotypes have been isolated from tissues such as bone marrow, muscle, fat, periosteum and synovial membrane both from rodents and humans [8, 12, 13, 24, 25]. Although bone marrow provides a universal source of MSCs, adipose tissue also possesses abundant and easily accessible MSCs. ...
A b s t r a c t I In nt tr ro od du uc ct ti io on n: : Adipose-derived stem cells (ADSCs) have been shown to differentiate into osteoblasts, adipocytes or myoblasts. However, it is not certain that ADSCs are equal to bone marrow stem cells (BMSCs) in their osteogenic differentiation potential. The purpose of this study was to answer the question. M Ma at te er ri ia al l a an nd d m me et th ho od ds s: : Mesenchymal stem cells (MSCs) were isolated from bone marrow and fat of adult rats. After cell expansion in culture media and three passages, osteogenesis was induced on a monolayer culture with osteogenic medium containing dexamethasone, b-glycerophosphate and ascorbate. After 4 weeks, expression of the osteocalcin gene was analyzed by RT-PCR, alkaline phosphatase (ALP) activity assayed, and Alizarin Red S and Von Kossa staining were done. Cell viability and apoptosis were also assayed by MTT and flow cytometry, respectively. R Re es su ul lt ts s: : In the test of osteogenesis, the osteoblastic differentiation of ADSCs as demonstrated by ALP activity was less than that of the BMSCs. The amount of matrix mineralization shown by Alizarin Red S and Von Kossa staining also showed statistical differences between the two MSCs. The incidence of apoptotic cells among ADSCs was higher than BMSCs. The flow cytometry proves that cell growth reduction is due to a decrease of the cells entering the S phase of the cell cycle. The data demonstrated by MTT assay indicated that viable cells among ADSCs were lower than BMSCs in control groups. C Co on nc cl lu us si io on ns s: : The results of our study suggest that ADSCs may have an inferior potential for osteogenesis compared with BMSCs. K Ke ey y w wo or rd ds s: : osteogenesis, bone marrow stem cells, adipose-derived stem cells, rat.
... Although it is generally accepted that satellite cells represent the primary cell population responsible for post-natal growth and repair, there exists other populations of cells that demonstrate the ability to aid in the repair of skeletal muscle (Wagers and Conboy 2005). Such populations include the muscle-resident side population (muSP) cells (Asakura, Seale et al. 2002) and interstitial PW1 + /Pax7cells (PICs) (Mitchell, Pannerec et al. 2010). There has also been evidence that bone marrow cells can contribute to myofiber repair (Fukada, Miyagoe-Suzuki et al. 2002;LaBarge and Blau 2002), although at a much lower frequency. ...
... Muscle satellite cells mainly contribute to muscle regeneration while several studies have revealed that non-satellite cells, such as bone marrow derived stem cells (Dreyfus et al., 2004; Dezawa et al., 2005), side population (SP) cells (Gussoni et al., 1999; Asakura et al., 2002), mesoangioblasts/pericytes (Dellavalle et al., 2007) and CD133+ cells (Torrente et al., 2004), are able to contribute in myotube formation as well as satellite cell reconstitution during muscle regeneration. These cells do not initially express Pax7. ...
Adult skeletal muscle possesses extraordinary regeneration capacities. After muscle injury or exercise, large numbers of newly formed muscle fibers are generated within a week as a result of expansion and differentiation of a self-renewing pool of muscle stem cells termed muscle satellite cells. Normally, satellite cells are mitotically quiescent and reside beneath the basal lamina of muscle fibers. Upon regeneration, satellite cells are activated, and give rise to daughter myogenic precursor cells. After several rounds of proliferation, these myogenic precursor cells contribute to the formation of new muscle fibers. During cell division, a minor population of myogenic precursor cells returns to quiescent satellite cells as a self-renewal process. Currently, accumulating evidence has revealed the essential roles of satellite cells in muscle regeneration and the regulatory mechanisms, while it still remains to be elucidated how satellite cell self-renewal is molecularly regulated and how satellite cells are important in aging and diseased muscle. The number of satellite cells is decreased due to the changing niche during ageing, resulting in attenuation of muscle regeneration capacity. Additionally, in Duchenne muscular dystrophy (DMD) patients, the loss of satellite cell regenerative capacity and decreased satellite cell number due to continuous needs for satellite cells lead to progressive muscle weakness with chronic degeneration. Thus, it is necessary to replenish muscle satellite cells continuously. This review outlines recent findings regarding satellite cell heterogeneity, asymmetric division and molecular mechanisms in satellite cell self-renewal which is crucial for maintenance of satellite cells as a muscle stem cell pool throughout life. In addition, we discuss roles in the stem cell niche for satellite cell maintenance, as well as related cell therapies for approaching treatment of DMD.
... Sca-1 was first reported as a cell surface marker of HSCs and, in combination with c-Kit, allows the definition of a population that contains the vast majority of HSCs, although they are still present at low frequency (around 10%) [44,95]. It has also been claimed that Sca-1 is expressed by the progenitor compartment of a number of other tissues, such as the skeletal muscle [95,96], mammary gland [95,97], kidney [95,98], liver [99], and prostate [95,100]. While some of these reports are not consensually accepted, others remain unchallenged. ...
The identification, in the adult, of cardiomyocyte (CM) turnover events and of cardiac progenitor cells (CPCs) has revolutionized the field of cardiovascular medicine. However, the low rate of CPCs differentiation events reported both in vitro and in vivo, even after injury, raised concerns on the biological significance of these subsets. In this Comprehensive Review, we discuss the current understanding of cardiac Lin-Sca-1+ cells in light of what is also known for similar phenotype cell-compartments in other organs.
... Mesenchymal progenitor cells have been identified in skeletal muscle that directly or indirectly contributes to repair in response to injury [7]. These multi-potent stem cells have been isolated by using unique cell surface markers and thus classified as side population cells [8,9], pericytes [10], muscle-derived stem cells [11] , musclederived MSCs (mMSCs) [12,13] , fibro/adipogenic pro- genitors [14], and PW1 + interstitial cells [15], and some degree of overlap likely exists between these cell populations . Whereas some of these cells can become myogenic, the majority have limited capacity for myogenic differentiation and are primed to secrete factors essential for indirect repair of skeletal muscle. ...
Introduction
Mesenchymal stem cells (MSCs) reside in a variety of tissues and provide a stromal role in regulating progenitor cell function. Current studies focus on identifying the specific factors in the niche that can alter the MSC secretome, ultimately determining the effectiveness and timing of tissue repair. The purpose of the present study was to evaluate the extent to which substrate and mechanical strain simultaneously regulate MSC quantity, gene expression, and secretome.
Methods
MSCs (Sca-1+CD45-) isolated from murine skeletal muscle (muscle-derived MSCs, or mMSCs) via fluorescence-activated cell sorting were seeded onto laminin (LAM)- or collagen type 1 (COL)-coated membranes and exposed to a single bout of mechanical strain (10%, 1 Hz, 5 hours).
Results
mMSC proliferation was not directly affected by substrate or strain; however, gene expression of growth and inflammatory factors and extracellular matrix (ECM) proteins was downregulated in mMSCs grown on COL in a manner independent of strain. Focal adhesion kinase (FAK) may be involved in substrate regulation of mMSC secretome as FAK phosphorylation was significantly elevated 24 hours post-strain in mMSCs plated on LAM but not COL (P <0.05). Conditioned media (CM) from mMSCs exposed to both LAM and strain increased myoblast quantity 5.6-fold 24 hours post-treatment compared with myoblasts treated with serum-free media (P <0.05). This response was delayed in myoblasts treated with CM from mMSCs grown on COL.
Conclusions
Here, we demonstrate that exposure to COL, the primary ECM component associated with tissue fibrosis, downregulates genes associated with growth and inflammation in mMSCs and delays the ability for mMSCs to stimulate myoblast proliferation.
... For these reasons, these cells became feasible to therapeutic application for Duchenne muscular dystrophy (DMD). Different groups demonstrated that transplantation of MSCs into murine model of DMD replenished the host satellite cell compartment—allowing the expression of dystrophin and ameliorating the dystrophic phenotype—and also remained as a pool of quiescent satellite cells [53, 54]. Similarly, de Bari and coworkers isolated MSCs from human synovial membrane and injected them into mdx mice, showing MSCs persistence into host muscles up to six months [55]. ...
Extraordinary progress in understanding several key features of stem cells has been made in the last ten years, including definition of the niche, and identification of signals regulating mobilization and homing as well as partial understanding of the mechanisms controlling self-renewal, commitment, and differentiation. This progress produced invaluable tools for the development of rational cell therapy protocols that have yielded positive results in preclinical models of genetic and acquired diseases and, in several cases, have entered clinical experimentation with positive outcome. Adult mesenchymal stem cells (MSCs) are nonhematopoietic cells with multilineage potential to differentiate into various tissues of mesodermal origin. They can be isolated from bone marrow and other tissues and have the capacity to extensively proliferate in vitro. Moreover, MSCs have also been shown to produce anti-inflammatory molecules which can modulate humoral and cellular immune responses. Considering their regenerative potential and immunoregulatory effect, MSC therapy is a promising tool in the treatment of degenerative, inflammatory, and autoimmune diseases. It is obvious that much work remains to be done to increase our knowledge of the mechanisms regulating development, homeostasis, and tissue repair and thus to provide new tools to implement the efficacy of cell therapy trials.
... Other types of stem cells capable of assuming a myogenic cell fate and of regenerating muscles have been described. Those include bone marrow stem cells (Ferrari et al., 1998; Bittner et al., 1999; Gussoni et al., 1999 ), muscle side population (SP) cells (Gussoni et al., 1999; Asakura et al., 2002; Rivier et al., 2004; Doyle et al., 2011), muscle-derived stem cells (Lee et al., 2000), mesangioblasts (Minasi et al., 2002; Sampaolesi et al., 2003; Galli et al., 2005; Morosetti et al., 2006), pericytes (Dellavalle et al., 2007; Peault et al., 2007), embryonic stem cells (ESCs) (Bhagavati and Xu, 2004; Barberi et al., 2007; Darabi et al., 2009; Filareto et al., 2012) and induced pluripotent stem cells (iPSCs) (Chang et al., 2009; Kazuki et al., 2010; Darabi et al., 2012; Tedesco et al., 2012). Therapeutic approaches to muscle disorders and targeting stem cells have focused primarily on demonstrating the feasibility of restoring dystrophin expression following transplantation of cells isolated from healthy donors. ...
The progressive loss of muscle mass characteristic of many muscular dystrophies impairs the efficacy of most of the gene and molecular therapies currently being pursued for the treatment of those disorders. It is becoming increasingly evident that a therapeutic application, to be effective, needs to target not only mature myofibers, but also muscle progenitors cells or muscle stem cells able to form new muscle tissue and to restore myofibers lost as the result of the diseases or during normal homeostasis so as to guarantee effective and lost lasting effects. Correction of the genetic defect using oligodeoxynucleotides (ODNs) or engineered nucleases holds great potential for the treatment of many of the musculoskeletal disorders. The encouraging results obtained by studying in vitro systems and model organisms have set the groundwork for what is likely to become an emerging field in the area of molecular and regenerative medicine. Furthermore, the ability to isolate and expand from patients various types of muscle progenitor cells capable of committing to the myogenic lineage provides the opportunity to establish cell lines that can be used for transplantation following ex vivo manipulation and expansion. The purpose of this article is to provide a perspective on approaches aimed at correcting the genetic defect using gene editing strategies and currently under development for the treatment of Duchenne muscular dystrophy (DMD), the most sever of the neuromuscular disorders. Emphasis will be placed on describing the potential of using the patient own stem cell as source of transplantation and the challenges that gene editing technologies face in the field of regenerative biology.
Skeletal muscle regeneration
is mainly enabled by a population of adult stem cells known as satellite cells. Satellite cells have been shown to be indispensable for adult skeletal muscle repair and regeneration. In the last two decades, other stem/progenitor cell populations resident in the skeletal muscle interstitium have been identified as “collaborators” of satellite cells during regeneration. They also appear to have a key role in replacing skeletal muscle with adipose, fibrous, or bone tissue in pathological conditions. Here, we review the role and known functions of these different interstitial skeletal muscle cell types and discuss their role in skeletal muscle tissue homeostasis, regeneration, and disease, including their therapeutic potential for cell transplantation protocols.
Background
Fluorescent-activated cell sorting (FACS) has enabled the direct isolation of highly enriched skeletal muscle stem cell, or satellite cell, populations from postnatal tissue. Several distinct surface marker panels containing different positively selecting surface antigens have been used to distinguish muscle satellite cells from other non-myogenic cell types. Because functional and transcriptional heterogeneity is known to exist within the satellite cell population, a direct comparison of results obtained in different laboratories has been complicated by a lack of clarity as to whether commonly utilized surface marker combinations select for distinct or overlapping subsets of the satellite cell pool. This study therefore sought to evaluate phenotypic and functional overlap among popular satellite cell sorting paradigms.
Methods
Utilizing a transgenic Pax7-zsGreen reporter mouse, we compared the overlap between the fluorescent signal of canonical paired homeobox protein 7 (Pax7) expressing satellite cells to cells identified by combinations of surface markers previously published for satellite cells isolation. We designed two panels for mouse skeletal muscle analysis, each composed of markers that exclude hematopoietic and stromal cells (CD45, CD11b, Ter119, CD31, and Sca1), combined with previously published antibody clones recognizing surface markers present on satellite cells (β1-integrin/CXCR4, α7-integrin/CD34, and Vcam1). Cell populations were comparatively analyzed by flow cytometry and FACS sorted for functional assessment of myogenic activity.
Results
Consistent with prior reports, each of the commonly used surface marker schemes evaluated here identified a highly enriched satellite cell population, with 89–90 % positivity for Pax7 expression based on zsGreen fluorescence. Distinct surface marker panels were also equivalent in their ability to identify the majority of the satellite cell pool, with 90–93 % of all Pax7-zsGreen positive cells marked by each of the surface marker schemes. The direct comparison among surface marker schemes validated their selection for highly overlapping subsets of cells. Functional analysis in vitro showed no differences in the abilities of cells sorted by these different methods to grow in culture and differentiate.
Conclusions
This study demonstrates the equivalency of several previously published and widely utilized surface marker schemes for isolating a highly purified and myogenically active population of satellite cells from the mouse skeletal muscle, which should facilitate cross-comparison of data across laboratories.
Electronic supplementary material
The online version of this article (doi:10.1186/s13395-016-0106-6) contains supplementary material, which is available to authorized users.
The heart has a limited ability to regenerate. It is important to identify therapeutic strategies that enhance cardiac regeneration in order to replace cardiomyocytes lost during the progression of heart failure. Cardiac progenitor cells are interesting targets for new regenerative therapies because they are self-renewing, multipotent cells located in the heart. Cardiac side population cells (cSPCs), the first cardiac progenitor cells identified in the adult heart, have the ability to differentiate into cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts. They become activated in response to cardiac injury and transplantation of cSPCs into the injured heart improves cardiac function. In this review, we will discuss the current literature on the progenitor cell properties and therapeutic potential of cSPCs. This body of work demonstrates the great promise cSPCs hold as targets for new regenerative strategies.
Skeletal muscle can repair muscle tissue damage, but significant loss of muscle tissue or its long-lasting chronic degeneration makes injured skeletal muscle tissue difficult to restore. It has been demonstrated that non-thermal atmospheric pressure plasma (NTP) can be used in many biological areas including regenerative medicine. Therefore, we determined whether NTP, as a non-contact biological external stimulator that generates biological catalyzers, can induce regeneration of injured muscle without biomaterials. Treatment with NTP in the defected muscle of a Sprague Dawley (SD) rat increased the number of proliferating muscle cells 7 days after plasma treatment (dapt) and rapidly induced formation of muscle tissue and muscle cell differentiation at 14 dapt. In addition, in vitro experiments also showed that NTP could induce muscle cell proliferation and differentiation of human muscle cells. Taken together, our results demonstrated that NTP promotes restoration of muscle defects through control of cell proliferation and differentiation without biological or structural supporters, suggesting that NTP has the potential for use in muscle tissue engineering and regenerative therapies.
Regenerative medicine is an area of intense excitement and potential. Despite the increasing rate of end-stage renal disease, dialysis and transplantation remain the only treatment options to date. However, there is hope that stem cells and regenerative medicine may procure additional therapeutic options for renal disease. Such new treatment options may include induction of repair using endogenous or exogenous stem cells or the reprogramming of the kidney to reinitiate development. This chapter reviews the current state of understanding with respect to stem cell functions in the kidney, regenerative principles in kidney diseases, as well as clinical implications and implementation of regenerative medicine in renal disease.
Promoting the paracrine effects of human mesenchymal stem cell (hMSC) therapy may contribute to improvements in patient outcomes. Here we develop an innovative strategy to enhance the paracrine effects of hMSCs. In a mouse hindlimb ischaemia model, we examine the effects of hMSCs in which a novel triple-catalytic enzyme is introduced to stably produce prostacyclin (PGI2-hMSCs). We show that PGI2-hMSCs facilitate perfusion recovery and enhance running capability as compared with control hMSCs or iloprost (a stable PGI2 analogue). Transplanted PGI2-hMSCs do not incorporate long term into host tissue, but rather they mediate host regeneration and muscle mass gain in a paracrine manner. Mechanistically, this involves long noncoding RNA H19 in promoting PGI2-hMSC-associated survival and proliferation of host progenitor cells under hypoxic conditions. Together, our data reveal the novel ability of PGI2-hMSCs to stimulate host regenerative processes and improve physical function by regulating long noncoding RNA in resident progenitor cells.
Although not numerous, satellite cells in skeletal muscle are capable to generate a large number of myoblasts upon injury. A further process of regenerative myogenesis leads to the muscle regeneration. Satellite cells are stem cells specific to skeletal muscle tissue. Their activation initiates the sequence of expression in myogenic regulatory factors and thus myogenic program. Besides the ability to provide new myoblasts, these cells also have selfrenewing capacity. Satellite cells are a heterogeneous group of cells considering their function and distinction markers. Several other types of stem cells with myogenic ability (in experimental conditions) have been isolated from the skeletal muscle. Therapeutic approaches to treat some forms of muscular diseases have been based on myogenic potential of both groups of stem cells. © 2015, Croatian Medical Association and School of Medicine. All rights reserved.
Understanding the regulation of the stem cell fate is fundamental for designing novel regenerative medicine strategies. Previous studies have suggested that pharmacological treatments with small molecules provide a robust and reversible regulation of the stem cell program. Previously, we showed that treatment with a vanadium compound influences muscle cell fate in vitro. In this study, we demonstrate that treatment with the phosphotyrosine phosphatase inhibitor bisperoxovanadium (BpV) drives primary muscle cells to a poised stem cell stage, with enhanced function in muscle regeneration in vivo following transplantation into injured muscles. Importantly, BpV-treated cells displayed increased self-renewal potential in vivo and replenished the niche in both satellite and interstitial cell compartments. Moreover, we found that BpV treatment induces specific activating chromatin modifications at the promoter regions of genes associated with stem cell fate, including Sca-1 and Pw1. Thus, our findings indicate that BpV resets the cell fate program by specific epigenetic regulations, such that the committed myogenic cell fate is redirected to an earlier progenitor cell fate stage, which leads to an enhanced regenerative stem cell potential.-Smeriglio, P., Alonso-Martin, S., Masciarelli, S., Madaro, L., Iosue, I., Marrocco, V., Relaix, F., Fazi, F., Marazzi, G., Sassoon, D. A., Bouché, M. Phosphotyrosine phosphatase inhibitor bisperoxovanadium endows myogenic cells with enhanced muscle stem cell functions via epigenetic modulation of Sca-1 and Pw1 promoters.
Stem cells are traditionally studied in the context of embryonic development, yet studies confirm that a fraction remains in the adult organism for the purpose of daily remodeling and rejuvenation of multiple tissues following injury. Adult stem cells (ASCs) are found in close proximity to vessels and respond to tissue-specific cues in the microenvironment that dictate their fate and function. Exercise can dramatically alter strain sensing, extracellular matrix composition, and inflammation, and such changes in the niche likely alter ASC quantity and function postexercise. The field of stem cell biology is still in its infancy and identification and terminology of ASCs continues to evolve; thus, current information regarding exercise and stem cells is lacking. This chapter summarizes the literature that reports on the ASC response to acute exercise and exercise training, with particular emphasis on hematopoietic stem cells, endothelial progenitor cells, and mesenchymal stem cells.
Low intensity laser irradiation (LILI) was found to promote the regeneration of skeletal muscle in vivo but the cellular mechanisms are not fully understood. Myoblasts, normally quiescent and inactivated in adult skeletal muscle, are a type of myogenic progenitor cells and considered as the major candidates responsible for muscle regeneration. The aim of the present study was to study the effect of LILI on the growth potential and cell-cycle progression of the cultured myoblasts. Primary myoblasts isolated from rat hind legs were cultured in nutrient-deficient medium for 36 hours and then irradiated by helium-neon laser at a certain energy density. Immunohistochemical and flow cytometric analysis revealed that laser irradiation could increase the expression of cellular proliferation marker and the amount of cell subpopulations in the proliferative phase as compared with the nonirradiated control group. Meanwhile, the expressions of cell-cycle regulatory proteins in the laser-treated myoblasts were markedly upregulated as compared to the unirradiated cells, indicating that LILI could promote the reentry of quiescent myoblasts into the cell division cycle. These results suggest that LILI at certain fluences could promote their proliferation, thus contributing to the skeletal muscle regeneration following trauma and myopathic diseases.
Background:
Although mechanisms underlying heterotopic ossification remain unknown, certain risk factors can influence heterotopic bone formation. The purpose of this study was to determine whether flaps used in periarticular reconstruction had any effect on heterotopic ossification formation.
Methods:
A retrospective review of periarticular injuries requiring flap coverage from 2003 through 2014 was performed. Flap types, Injury Severity Scores, functional outcomes, and complications were reviewed. Radiology findings were assessed to determine heterotopic ossification rates and grades.
Results:
Three hundred eighty-nine flaps were performed for traumatic extremity coverage over the 13-year study period. Sixty-nine of these flaps were used for periarticular coverage. The rate of periarticular heterotopic ossification was 47 percent for fasciocutaneous versus 54 percent for muscle-based flap coverage (p = 0.88). There were no significant differences in Injury Severity Score (p = 0.44) or overall heterotopic ossification formation (p = 0.97) between groups; however, the grade of heterotopic ossification within muscle-based flap cohort was significantly higher (1.70 for muscle versus 1.06 for the fasciocutaneous cohort; p = 0.002).
Conclusions:
Combat-related trauma is associated with high rates of heterotopic ossification, with an overall formation rate exceeding 85 percent for our patients requiring periarticular flap coverage. Although no difference in the rate of heterotopic ossification formation was found between fasciocutaneous and muscle flap coverages, a significantly increased severity of heterotopic ossification was seen in periarticular muscle-based flaps. These findings suggest that flap composition might not affect the rate of heterotopic ossification formation but may have an effect on ectopic bone formation severity.
Adult skeletal muscle maintains a homeostatic state with modest levels of cellular turnover, unlike the skin or blood. However, the muscle is highly sensitive to tissue injury, which unleashes a cascade of regenerative and inflammatory processes. Muscle regeneration involves crosstalk between numerous cytokine signaling axes, and the coordinated activity of multiple muscle-resident and circulating progenitor populations. Satellite cells, closely associated with myofibers, are established as the canonical muscle stem cell, with self-renewal and myofiber-regenerating capacity. However, a heterogeneous group of mesenchymal progenitor cells residing within the muscle interstitium are also highly responsive to muscle injury and exhibit varying degrees of regenerative potential. These cells interact with satellite cells via direct and indirect mechanisms to regulate regeneration or repair. We describe the known phylogenetic and functional relationships of the multiple progenitor populations residing within skeletal muscle, their putative roles in the coordination of injury repair, and their possible contributions to health and disease.
Muscular dystrophy is a devastating disease in which no treatment or cure exists. A promising therapy for muscular dystrophy is the transplantation of cells that are able to contribute to existing muscle fibers or generate new muscle fibers. While several cell populations have been shown to demonstrate this phenomenon in mouse models, the mechanism by which these cells are able to differentiate into myogenic cells is largely unknown. The goal of this research was to (i) create HSV vectors that are useful for expressing muscle development genes in developing embryos in culture (ii) design strategies to produce and characterize large and diverse HSV vector libraries of expressed genes from different cells or tissues and (iii) design methods by which this expression vector library can be screened for expressed gene functions that participate in skeletal muscle lineage determination. The outcome of these studies were intended to provide a method for exploiting the high level infectivity and growth of HSV in embryonic stem (ES) cells and germ layer derivatives, the virus ability to accommodate large DNA inserts, and the ability to create vector libraries potentially useful in selection of lineage determining genes based on complementation of virus growth. The engineering and characterization of a replication defective HSV vector that is useful for the delivery of genes to embryonic stem cells and their derivatives was described. The deletion of ICP4, ICP22 and the conversion of the immediate early genes ICP0 and ICP27 to the early gene class by promoter exchange generated a vector, JDββHE. JDββHE was found to be non-toxic to ES cells and vector infection of ES cells did not interfere with germ layers formation. This vector was engineered to be dependent on a single IE gene ICP4 for replication. Gene expression from the JDββHE HSV vector, including the expression of eGFP and the early myogenic gene Pax3, was demonstrated in infected ES cells and cells of the embryoid body in a robust and transient manner. In order to discover genes which contribute to muscle differentiation, a cDNA library was constructed within HSV viral vectors in order to be used in the identification of novel gene functions which play a role in the activation early myogenic promoters. For this purpose, the HSV genome was modified to contain BAC elements as well as the Gateway recombination system, which facilitated efficient incorporation of cDNAs into the vector genome. HSV cDNA vectors expressed gene products at the transcriptional and protein level. Functional analysis of the cDNA inserts demonstrated that the library consisted of at least 15,000 unique genes. Some of these genes have been shown to participate in biological functions related to muscle differentiation, such as Id1 and Cand2.Finally, a conditional replication strategy was developed by which the recently produced HSV cDNA library could be used to identify genes that activate early myogenic promoters. This study exploited the characteristic of the JDββHE vector in which viral replication can be restored upon introduction of the essential viral gene ICP4. By placing this essential viral gene under the control of the early myogenic promoter Pax3, viral replication was found to be dependent on activation of this cellular promoter in a Pax3-expressing rhabdomyosarcoma cell line. In a similar manner, HSV cDNA library vectors that express a Pax3 activator will be able to replicate in the presence of the conditional ICP4 construct, thereby capturing the relevant gene product by the release of viral progeny into the supernatant. The future use of this strategy to identify gene products that specifically activate an early myogenic promoter would lead to the testing of these gene products for their ability to induce downstream myogenic gene expression and differentiation. In this manner, a better understanding of early muscle differentiation might be achieved and applied to the use of stem cells in transplantation studies using muscular dystrophy models.
Skeletal muscle tissue engineering is one of the important ways for regenerating functionally defective muscles. Among the myopathies, the Duchenne muscular dystrophy (DMD) is a progressive disease due to mutations of the dystrophin gene leading to progressive myofiber degeneration with severe symptoms. Although current therapies in muscular dystrophy are still very challenging, important progress has been made in materials science and in cellular technologies with the use of stem cells. It is therefore useful to review these advances and the results obtained in a clinical point of view. This article focuses on the differentiation of stem cells into myoblasts, and their application in muscular dystrophy. After an overview of the different stem cells that can be induced to differentiate into the myogenic lineage, we introduce scaffolding materials used for muscular tissue engineering. We then described some widely used methods to differentiate different types of stem cell into myoblasts. We highlight recent insights obtained in therapies for muscular dystrophy. Finally, we conclude with a discussion on stem cell technology. We discussed in parallel the benefits brought by the evolution of the materials and by the expansion of cell sources which can differentiate into myoblasts. We also discussed on future challenges for clinical applications and how to accelerate the translation from the research to the clinic in the frame of DMD.
According to the cancer stem cell theory, a small subpopulation of cancer cells, known as cancer stem cells (CSCs), exist that are self-renewing and are involved in tumor invasion, metastasis and recurrence. A number of studies have reported that certain cancer cells are able to efflux the Hoechst 33342 dye. These cells are termed side population (SP) cells and share characteristic features of CSCs. The results of the present study revealed that 2.7% of primary head and neck squamous cell carcinoma (HNSCC) cells were SP cells. This was reduced to 0.7% following treatment with verapamil. The immunofluorescence and reverse transcription polymerase chain reaction analysis revealed that SP cells have an enhanced expression of the ATP-binding cassette (ABC) transporter protein ABC subfamily G, member 2 (ABCG2), which has been identified to be actively involved in drug exclusion. Similarly, the mRNA level of the oncogene B lymphoma Mo-MLV insertion region-1 and the stem cell surface proteins nestin and octamer-binding transcription factor-4 were highly expressed in the SP cells compared with the non-SP cells. In addition, it was demonstrated that HNSCC SP cells exhibited increased proliferation and were highly resistant to multiple drugs. These findings suggest that the presence of CSCs, such as SP cells, may be responsible for chemotherapy failure and tumor relapse in patients with HNSCC. Therefore, the identification of a novel therapeutic drug that could effectively target CSCs may help to eradicate refractory tumors.
Satellite cells are considered the major source of muscle progenitors, however, other populations with myogenic popential have been discovered. We have identified a new muscle-resident non-satellite cell population, termed PICs, which can differentiate into three different lineages, skeletal muscle, smooth muscle and fat. PICs rescue satellite cells from myostatin inhibition in vitro through follistatin release. When myostatin is inactivated in vivo, PICs number is markedly increased and mice display hypertrophied muscles. While recent studies have demonstrated that muscle regeneration cannot occur without satellite cells, we show that muscle regeneration is restored when mice have been previously treated with a myostatin inhibitor. We postulate that PICs have participated in muscle repair rescue, and thus constitute an interesting population to be targeted for pharmaceutical strategies aimed at improving skeletal muscle mass and function
All animals have evolved means of dealing with tissue damage due to injury or disease. Regenerative phenomena can be classified by their scale: First, some invertebrates are capable of regenerating the entire body from pieces of tissue or even dissociated cells. Secondly, the restoration of the complex architecture of damaged organs or severed appendages is possible in many organisms. Finally, tissue regeneration would describe restoration of a tissue within an otherwise largely intact organ, for example, skeletal muscle regeneration and repair of the epithelia of kidney or lung, all of which efficiently occur also in mammals. This chapter discusses select, fairly well-characterized phenomena from all three scales, namely, planarian whole-body regeneration, lower vertebrate appendage and vertebrate liver regeneration, and finally skeletal muscle and kidney epithelia regeneration. It concentrates on these regeneration systems where significant progress has been made into a deeper understanding of Wnt signaling function.
Muscle regeneration recapitulates many aspects of embryonic myogenesis and is an important homeostatic process of the adult skeletal muscle, which, after development, retains the capacity to regenerate in response to appropriate stimuli, activating the muscle compartment of stem cells, namely, satellite cells, as well as other precursor cells. Moreover, significant evidence suggests that while stem cells represent an important determinant for tissue regeneration, a “qualified” environment is necessary to guarantee and achieve functional results. It is therefore plausible that the loss of control over these cell fate decisions could lead to a pathological transdifferentiation, leading to pathologic defects in the regenerative process. This review provides an overview about the general aspects of muscle development and discusses the cellular and molecular aspects that characterize the five interrelated and time-dependent phases of muscle regeneration, namely, degeneration, inflammation, regeneration, remodeling, and maturation/functional repair.
1. Muscle Regeneration Recapitulates Many Aspects of Development
Regenerative potential, robust in lower vertebrates, is gradually lost in higher vertebrates such as mammals [1–5]. Nevertheless, mammalian tissues, including skeletal muscle, are capable of homeostasis and regeneration, partially recapitulating the embryonic developmental program.
Muscle development and regeneration share common features because the molecular program that underlines prenatal development is reactivated for tissue reconstruction after injury [6–8] (Figure 1). Regenerative medicine has therefore gained important insights through the study of developmental biology.
Previously, in an attempt to isolate stem cells that would be capable of regenerating injured skeletal muscle, we cultured cells derived from muscle, non-adherently, in serum-free media. As a result of the culture conditions used, these cells formed spheres, and thus were referred to as myospheres. It was found that myosphere-derived cells expressed Sca-1, a marker that is not typically associated with myogenic cells, and as a result has generated some questions as to the origin of these cells. The goal of this study was to clearly determine the origin of myosphere-derived cells, and in particular to answer the question of whether myospheres contain myogenic cells. To determine if myospheres were composed of myogenic cells without altering the structure of myospheres or the culture conditions used to maintain myospheres, I isolated these cells from yellow fluorescent protein (YFP)-Myf5, YFP-MyoD, and ZsGreen-Pax7 lineage-tracing mice and monitored their growth over time. I found that myospheres do contain myogenic cells, but that these cells are gradually lost over time (within 2 months). Additionally, the use of the lineage-tracing mice gave an interesting perspective into the composition of myospheres. I found that myospheres were composed of two distinct cell types, one that is myogenic (α7 integrin+) and contains cells expressing Myf5, MyoD, and Pax7, and a second that is non-myogenic (α7 integrin-) expressing platelet-derived growth factor receptor alpha (PDGFRα) and Sca-1, both of which have been associated with fibro/adipocyte mesenchymal cells.
The satellite cells are long regarded as heterogeneous cell population, which is intimately linked to the processes of muscular recovery. The heterogeneous cell population may be classified by specific markers. In spite of the significant amount of variation amongst the satellite cell populations, it seems that their activity is tightly bound to the paired box 7 transcription factor expression, which is, therefore, used as a canonical marker for these cells. Muscular dystrophic diseases, such as Duchenne muscular dystrophy, elicit severe tissue injuries leading those patients to display a very specific pattern of muscular recovery abnormalities. There have been works on the application of precursors cells as a therapeutic alternative for Duchenne muscular dystrophy and initial attempts have proven the cells inefficient; however later endeavours have proposed solutions for the experiments improving significantly the results. The presence of a range of satellite cells populations indicates the existence of specific cells with enhanced capability of muscular recovery in afflicted muscles.
The use of stem cells represents a promising approach for the treatment of bone defects. However, successful treatments rely upon the availability of cells that are easily obtained and that appropriately differentiate into osteoblasts. The tongue potentially represents a source of autologous cells for such purposes. In the present study, the ability of stem cell antigen‑1 (Sca‑1) positive cells derived from tongue muscle to differentiate into osteoblasts was investigated. The tongue muscles were excised from Jcl‑ICR mice and tongue muscle‑derived Sca‑1‑positive cells (TDSCs) were isolated from the tongue muscle using a magnetic cell separation system with microbeads. TDSCs were cultured in plastic dishes or gelatin sponges of β‑tricalcium phosphate (β‑TCP) with bone differentiation‑inducing medium. The expression of osteogenic markers (Runx2, osterix, alkaline phosphatase, fibronectin, osteocalcin, osteonectin and osteopontin) was investigated in cultured TDSCs by western blot analysis. The formation of mineralized matrices was examined using alizarin red S and Von Kossa staining. Bone formation was investigated in cultured TDSCs by hematoxylin‑eosin staining and immunohistochemstry. In the present study, the expression of Sca‑1 in mouse tongue muscle was demonstrated and TDSCs were isolated at high purity. TDSCs differentiated into cells of osteoblast lineage, as demonstrated by the upregulation of osteoblastic marker expression. The formation of mineralized matrices was confirmed by alizarin red S or Von Kossa staining in vitro. Bone formation was observed in the gelatin sponges of β‑TCP, which were subsequently implanted under the skin of the backs of nude mice. These results suggested that TDSCs retain their osteogenic differentiation potential and therefore the tongue muscle may be used as a source of stem cells for bone regeneration.
Regenerating muscle fibers emerge from quiescent satellite cells, which differentiate into mature multinuclear myofibers upon activation. It has recently been found that ATOH8, a bHLH transcription factor, is regulated during myogenic differentiation. In this study, expression and localization of ATOH8, the other well-described regeneration markers, vimentin, nestin and neonatal myosin, and the satellite cell marker Pax7 were analyzed on protein level in human myopathy samples by immunofluorescence studies. On mRNA level, expression levels of ATOH8 and vimentin were studied by quantitative real-time PCR. ATOH8 is expressed in activated satellite cells and proliferating myoblasts of human skeletal muscle tissue. Quantitative analyses of ATOH8+, Pax7+, vimentin+, nestin+ and neonatal myosin+ muscle fibers showed the highest amount of regenerating muscle fibers in inflammatory myopathies, followed by muscular dystrophy. The relative co-expression of ATOH8 with the above-mentioned markers did not vary among the disorders. These results show that the novel regeneration marker ATOH8 contributes to muscle cell differentiation in healthy and diseased human muscle tissue.
An increase in intramuscular adipocyte tissue (IMAT) is associated with glucose dysregulation, decreased muscle strength, and increased risk of disability. Unfortunately, the mechanisms stimulating intramuscular adipogenesis remain unclear. We found that dexamethasone (Dex) administration to mice with injured muscles stimulates the accumulation of IMAT. To identify precursors of these adipocytes, we isolated satellite cells and fibro/adipogenic progenitors (FAPs) from muscle; satellite cells did not differentiate into adipocytes even following Dex treatment. In contrast, Dex stimulated FAP differentiation into adipocytes. In vivo, we transplanted purified FAPs from transgenic, EGFP mice into the injured muscles of C57/BL6 mice and found that Dex administration stimulated adipogenesis from FAP-EGFP. The increase in adipogenesis depended on Dex-induced inhibition of interleukin-4 (IL-4). In the injured muscle of IL-4-knockout mice, the levels of adipocytes were increased, while in the injured muscles of Dex-treated mice with IL-4 injections, adipogenesis was suppressed. In cultured FAPs, IL-4 inhibited Dex-induced conversion of FAPs into adipocytes; this did not occur in FAPs expressing knockdown of the IL-4 receptor. Thus, we concluded that glucocorticoids stimulate FAPs to differentiate into adipocytes in injured muscles. This process is blocked by IL-4, suggesting that interfering with IL-4 signaling could prevent adipogenesis in muscle.-Dong, Y., Silva, K. A. S., Dong, Y., Zhang, L. Glucocorticoids increase adipocytes in muscle by affecting IL-4 regulated FAP activity.
The article describes myogenic satellite cells, their markers, quantification and distribution, growth factors and hormones involved in their regulation, interaction with macrophages and monocytes, functional answers to physiologic stimuli/disease states, genetic models of myopathies and muscular regeneration, ectopic muscle formation, muscle formation and precursor cell, satellite cell origin, peripheral cells, extraocular muscles and satellite cells.
Myocyte loss in the ischemically injured mammalian heart often leads to irreversible deficits in cardiac function. To identify a source of stem cells capable of restoring damaged cardiac tissue, we transplanted highly enriched hematopoietic stem cells, the so-called side population (SP) cells, into lethally irradiated mice subsequently rendered ischemic by coronary artery occlusion for 60 minutes followed by reperfusion. The engrafted SP cells (CD34–/low, c-Kit+, Sca-1+) or their progeny migrated into ischemic cardiac muscle and blood vessels, differentiated to cardiomyocytes and endothelial cells, and contributed to the formation of functional tissue. SP cells were purified from Rosa26 transgenic mice, which express lacZ widely. Donor-derived cardiomyocytes were found primarily in the peri-infarct region at a prevalence of around 0.02% and were identified by expression of lacZ and α-actinin, and lack of expression of CD45. Donor-derived endothelial cells were identified by expression of lacZ and Flt-1, an endothelial marker shown to be absent on SP cells. Endothelial engraftment was found at a prevalence of around 3.3%, primarily in small vessels adjacent to the infarct. Our results demonstrate the cardiomyogenic potential of hematopoietic stem cells and suggest a therapeutic strategy that eventually could benefit patients with myocardial infarction.
The activation of mononuclear muscle precursor cells after crush injury to mouse tibialis anterior muscles was monitored in vivo by in situ hybridization with MyoD1 and myogenin probes. These genes are early markers of skeletal muscle differentiation and have been extensively studied in vitro. The role in vivo of these regulatory proteins during myogenesis of mature muscle has not been studied previously. MyoD1 and myogenin mRNA were present in occasional mononuclear cells of uninjured muscle. Increased MyoD1 and myogenin mRNA sequences in mononuclear cells were detected as early as 6 h after injury, peaked between 24 and 48 h, and thereafter declined to pre-injury levels at about 8 days. The mRNAs were detected in mononuclear cells throughout the muscle, with the majority of cells located some distance from the site of crush injury. The presence of MyoD1 and myogenin mRNA at 6 to 48 h indicates that transcription of these genes is occurring at the same time as replication of muscle precursor cells in vivo. At no time were significant levels of mRNA for these genes detected in myotubes. MyoD1 and myogenin provide precise markers for the very early identification and study of mononuclear skeletal muscle precursor cells in muscle regenerating in vivo.
We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and
carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression
when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium
butyrate to further increase expression of each component, we achieved helper-free viral stocks of ∼107 Infectious units/ml by 48 h after transient co-transfectlon with the three plasmid components. This system can be used both
for the generation of high titer retroviral stocks for transductlon and for the rapid screening of a large number of MLV gag-pol or env mutants.
The ROSA beta geo26 (ROSA26) mouse strain was produced by random retroviral gene trapping in embryonic stem cells. Staining of ROSA26 tissues and fluorescence-activated cell sorter-Gal analysis of hematopoietic cells demonstrates ubiquitous expression of the proviral beta geo reporter gene, and bone marrow transfer experiments illustrate the general utility of this strain for chimera and transplantation studies. The gene trap vector has integrated into a region that produces three transcripts. Two transcripts, lost in ROSA26 homozygous animals, originate from a common promoter and share identical 5' ends, but neither contains a significant ORF. The third transcript, originating from the reverse strand, shares antisense sequences with one of the noncoding transcripts. This third transcript potentially encodes a novel protein of at least 505 amino acids that is conserved in humans and in Caenorhabditis elegans.
Green fluorescent protein (GFP) and its variants currently represent the only non-invasive markers available for labeling mammalian cells in culture or in a multicellular organism through transgenesis. To date this marker gene has been widely used in the study of many organisms, but as yet has not found large-scale application in mammals due to problems encountered with weak fluorescence and instability of the wild-type protein at higher temperatures. Recently, though, several mutants have been made in the wild-type (wt) GFP so as to improve its thermostability and fluorescence. EGFP (enhanced GFP) is one such wtGFP variant. As a first step in assessing the use of EGFP in ES cell-mediated strategies, we have established a mouse embryonic stem (ES) cell lines expressing EGFP, which can be propagated in culture, reintroduced into mice. or induced to differentiate in vitro, while still maintaining ubiquitous EGFP expression. From the results presented we can suggest that: 1) possible improvements in the efficiency of transgenic regimes requiring the germline transmission of ES cells by aggregation chimeras can be made by the preselection chimeric embryos at the blastocyst stage: (2) the expression of a noninvasive marker, driven by a promoter that is active during early postimplantation development, allows access to embryos during a window of embryonic development that has previously been difficult to investigate (3) the behavior of mutant ES cells can be followed with simple microscopic observation of chimeric embryos or adult animals comprising green fluorescent cells/tissues. and (4) intercrosses of F1 mice and subsequent generations of animals show that progeny can be genotyped by UV light, such that mice homozygous for the transgene can be distinguished from hemizygotes due to their increased fluorescence.
To gain insight into the regeneration deficit of MyoD-/- muscle, we investigated the growth and differentiation of cultured MyoD-/- myogenic cells. Primary MyoD-/- myogenic cells exhibited a stellate morphology distinct from the compact morphology of wild-type myoblasts, and expressed c-met, a receptor tyrosine kinase expressed in satellite cells. However, MyoD-/- myogenic cells did not express desmin, an intermediate filament protein typically expressed in cultured myoblasts in vitro and myogenic precursor cells in vivo. Northern analysis indicated that proliferating MyoD-/- myogenic cells expressed fourfold higher levels of Myf-5 and sixfold higher levels of PEA3, an ETS-domain transcription factor expressed in newly activated satellite cells. Under conditions that normally induce differentiation, MyoD-/- cells continued to proliferate and with delayed kinetics yielded reduced numbers of predominantly mononuclear myocytes. Northern analysis revealed delayed induction of myogenin, MRF4, and other differentiation-specific markers although p21 was upregulated normally. Expression of M-cadherin mRNA was severely decreased whereas expression of IGF-1 was markedly increased in MyoD-/- myogenic cells. Mixing of lacZ-labeled MyoD-/- cells and wild-type myoblasts revealed a strict autonomy in differentiation potential. Transfection of a MyoD-expression cassette restored cytomorphology and rescued the differentiation deficit. We interpret these data to suggest that MyoD-/- myogenic cells represent an intermediate stage between a quiescent satellite cell and a myogenic precursor cell.
Myoblasts, the precursors of skeletal muscle fibers, can be induced to withdraw from the cell cycle and differentiate in vitro. Recent studies have also identified undifferentiated subpopulations that can self-renew and generate myogenic cells (Baroffio, A., M. Hamann, L. Bernheim, M.-L. Bochaton-Pillat, G. Gabbiani, and C.R. Bader. 1996. Differentiation. 60:47-57; Yoshida, N., S. Yoshida, K. Koishi, K. Masuda, and Y. Nabeshima. 1998. J. Cell Sci. 111:769-779). Cultured myoblasts can also differentiate and contribute to repair and new muscle formation in vivo, a capacity exploited in attempts to develop myoblast transplantation (MT) for genetic modification of adult muscle. Our studies of the dynamics of MT demonstrate that cultures of myoblasts contain distinct subpopulations defined by their behavior in vitro and divergent responses to grafting. By comparing a genomic and a semiconserved marker, we have followed the fate of myoblasts transplanted into muscles of dystrophic mice, finding that the majority of the grafted cells quickly die and only a minority are responsible for new muscle formation. This minority is behaviorally distinct, slowly dividing in tissue culture, but rapidly proliferative after grafting, suggesting a subpopulation with stem cell-like characteristics.
Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as
undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage,
fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated
from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro.
These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages.
Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.
Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met−/− embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells.
The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system.
We have discovered that cells derived from the skeletal muscle of adult mice contain a remarkable capacity for hematopoietic differentiation. Cells prepared from muscle by enzymatic digestion and 5-day in vitro culture were harvested, and 18 x 10(3) cells were introduced into each of six lethally irradiated recipients together with 200 x 10(3) distinguishable whole bone marrow cells. After 6 or 12 weeks, all recipients showed high-level engraftment of muscle-derived cells representing all major adult blood lineages. The mean total contribution of muscle cell progeny to peripheral blood was 56 +/- 20% (SD), indicating that the cultured muscle cells generated approximately 10- to 14-fold more hematopoietic activity than whole bone marrow. When bone marrow from one mouse was harvested and transplanted into secondary recipients, all recipients showed high-level multilineage engraftment (mean 40%), establishing the extremely primitive nature of these stem cells. We also show that muscle contains a population of cells with several characteristics of bone marrow-derived hematopoietic stem cells, including high efflux of the fluorescent dye Hoechst 33342 and expression of the stem cell antigens Sca-1 and c-Kit, although the cells lack the hematopoietic marker CD45. We propose that this population accounts for the hematopoietic activity generated by cultured skeletal muscle. These putative stem cells may be identical to muscle satellite cells, some of which lack myogenic regulators and could be expected to respond to hematopoietic signals.
Injection of the myotoxin notexin, was found to induce regeneration in muscles that had been subjected to 18 Gy of radiation. This finding was unexpected as irradiation doses of this magnitude are known to block regeneration in dystrophic (mdx) mouse muscle. To investigate this phenomenon further we subjected mdx and normal (C57Bl/10) muscle to irradiation and notexin treatment and analysed them in two ways. First by counting the number of newly regenerated myofibres expressing developmental myosin in cryosections of damaged muscles. Second, by isolating single myofibres from treated muscles and counting the number of muscle precursor cells issuing from these over 2 day and 5 day periods. After irradiation neither normal nor dystrophic muscles regenerate to any significant extent. Moreover, single myofibres cultured from such muscles produce very few muscle precursor cells and these undergo little or no proliferation. However, when irradiated normal and mdx muscles were subsequently treated with notexin, regeneration was observed. In addition, some of the single myofibres produced rapidly proliferative muscle precursor cells when cultured. This occurred more frequently, and the myogenic cells proliferated more extensively, with fibres cultured from normal compared with dystrophic muscles. Even after 25 Gy, notexin induced some regeneration but no proliferative myogenic cells remained associated with the muscle fibres. Thus, skeletal muscles contain a number of functionally distinct populations of myogenic cells. Most are radiation sensitive. However, some survive 18 Gy as proliferative myogenic cells that can be evoked by extreme conditions of muscle damage; this population is markedly diminished in muscles of the mdx mouse. A small third population survives 25 Gy and forms muscle but not proliferative myogenic cells.
Distinct cell lineages established early in development are usually maintained throughout adulthood. Thus, adult stem cells have been thought to generate differentiated cells specific to the tissue in which they reside. This view has been challenged; for example, neural stem cells can generate cells that normally originate from a different germ layer. Here we show that acutely isolated and clonally derived neural stem cells from mice and humans could produce skeletal myotubes in vitro and in vivo, the latter following transplantation into adult animals. Myogenic conversion in vitro required direct exposure to myoblasts, and was blocked if neural cells were clustered. Thus, a community effect between neural cells may override such myogenic induction. We conclude that neural stem cells, which generate neurons, glia and blood cells, can also produce skeletal muscle cells, and can undergo various patterns of differentiation depending on exposure to appropriate epigenetic signals in mature tissues.
The characterization of hepatic progenitor cells is of great scientific and clinical interest. Here we report that intravenous injection of adult bone marrow cells in the FAH(-/-) mouse, an animal model of tyrosinemia type I, rescued the mouse and restored the biochemical function of its liver. Moreover, within bone marrow, only rigorously purified hematopoietic stem cells gave rise to donor-derived hematopoietic and hepatic regeneration. This result seems to contradict the conventional assumptions of the germ layer origins of tissues such as the liver, and raises the question of whether the cells of the hematopoietic stem cell phenotype are pluripotent hematopoietic cells that retain the ability to transdifferentiate, or whether they are more primitive multipotent cells.
Skeletal muscle is one of a several adult post-mitotic tissues that retain the capacity to regenerate. This relies on a population of quiescent precursors, termed satellite cells. Here we describe two novel markers of quiescent satellite cells: CD34, an established marker of hematopoietic stem cells, and Myf5, the earliest marker of myogenic commitment. CD34+ve myoblasts can be detected in proliferating C2C12 cultures. In differentiating cultures, CD34+ve cells do not fuse into myotubes, nor express MyoD. Using isolated myofibers as a model of synchronous precursor cell activation, we show that quiescent satellite cells express CD34. An early feature of their activation is alternate splicing followed by complete transcriptional shutdown of CD34. This data implicates CD34 in the maintenance of satellite cell quiescence.
In heterozygous Myf5nlacZ/+ mice, all CD34+ve satellite cells also express β-galactosidase, a marker of activation of Myf5, showing that quiescent satellite cells are committed to myogenesis. All such cells are positive for the accepted satellite cell marker, M-cadherin. We also show that satellite cells can be identified on isolated myofibers of the myosin light chain 3F-nlacZ-2E mouse as those that do not express the transgene. The numbers of satellite cells detected in this way are significantly greater than those identified by the other three markers.
We conclude that the expression of CD34, Myf5, and M-cadherin defines quiescent, committed precursors and speculate that the CD34−ve, Myf5−ve minority may be involved in maintaining the lineage-committed majority.
Duchenne muscular dystrophy is a lethal recessive disease characterized by widespread muscle damage throughout the body. This increases the difficulty of cell or gene therapy based on direct injections into muscles. One way to circumvent this obstacle would be to use circulating cells capable of homing to the sites of lesions. Here, we showed that stem cell antigen 1 (Sca-1), CD34 double-positive cells purified from the muscle tissues of newborn mice are multipotent in vitro and can undergo both myogenic and multimyeloid differentiation. These muscle-derived stem cells were isolated from newborn mice expressing the LacZ gene under the control of the muscle-specific desmin or troponin I promoter and injected into arterial circulation of the hindlimb of mdx mice. The ability of these cells to interact and firmly adhere to endothelium in mdx muscles microcirculation was demonstrated by intravital microscopy after an intraarterial injection. Donor Sca-1, CD34 muscle-derived stem cells were able to migrate from the circulation into host muscle tissues. Histochemical analysis showed colocalization of LacZ and dystrophin expression in all muscles of the injected hindlimb in all of five out of five 8-wk-old treated mdx mice. Their participation in the formation of muscle fibers was significantly increased by muscle damage done 48 h after their intraarterial injection, as indicated by the presence of 12% beta-galactosidase-positive fibers in muscle cross sections. Normal dystrophin transcripts detected enzymes in the muscles of the hind limb injected intraarterially by the mdx reverse transcription polymerase chain reaction method, which differentiates between normal and mdx message. Our results showed that the muscle-derived stem cells first attach to the capillaries of the muscles and then participate in regeneration after muscle damage.
Purification of rare hematopoietic stem cell(s) (HSC) to homogeneity is required to study their self-renewal, differentiation, phenotype, and homing. Long-term repopulation (LTR) of irradiated hosts and serial transplantation to secondary hosts represent the gold standard for demonstrating self-renewal and differentiation, the defining properties of HSC. We show that rare cells that home to bone marrow can LTR primary and secondary recipients. During the homing, CD34 and SCA-1 expression increases uniquely on cells that home to marrow. These adult bone marrow cells have tremendous differentiative capacity as they can also differentiate into epithelial cells of the liver, lung, GI tract, and skin. This finding may contribute to clinical treatment of genetic disease or tissue repair.
Myoblast transplantation is a potential therapeutic approach for the genetic modification of host skeletal muscle tissue. To be considered an effective, long-lived method of delivery, however, it is essential that at least a proportion of the transplanted cells also retain their proliferative potential. We sought to investigate whether transplanted neonatal myoblasts can contribute to the satellite cell compartment of adult skeletal muscle by using the Myf5nlacZ/+ mouse. The Myf5nlacZ/+ mouse has nlacZ targeted to the Myf5 locus resulting in beta-galactosidase activity in quiescent satellite cells. Following transplantation, beta-galactosidase-labelled nuclei were detected in host muscles, showing that donor cells had been incorporated. Significantly, beta-galactosidase-positive, and therefore donor-derived, satellite cells were detected. When placed in culture, beta-galactosidase marked myogenic cells emanated from the parent fibre. These observations demonstrate that cell transplantation not only results in the incorporation of donor nuclei into the host muscle syncytia, but also that the donor cells can become functional satellite cells. The Myf5nlacZ/+ mouse therefore provides a novel and specific marker for determining the contribution of transplanted cells to the satellite cell pool.
Muscle satellite cells are believed to represent a committed stem cell population that is responsible for the postnatal growth and regeneration of skeletal muscle. However, the observation that cultured myoblasts differentiate into osteocytes or adipocytes following treatment with bone morphogenetic proteins (BMPs) or adipogenic inducers, respectively, suggests some degree of plasticity within the mesenchymal lineage. To further investigate this phenomenon, we explore the osteogenic and adipogenic potential of satellite cells isolated from adult mice. Our experiments clearly demonstrate that satellite cell-derived primary myoblasts, expressing myogenic markers such as MyoD, Myf5, Pax7 and desmin, differentiated only into osteocytes or adipocytes following treatment with BMPs or adipogenic inducers, respectively However, satellite cells on isolated muscle fibers cultured in Matrigel readily differentiated into myocytes as well as osteogenic and adipogenic lineages, whereas primary myoblasts did not. Satellite cell-derived primary myoblasts isolated from mice lacking the myogenic transcription factor MyoD (MyoD-/-) differentiate into myocytes poorly in vivo and in vitro (Megeney et al., Genes Dev. 1996; Sabourin et. al, J. Cell Biol., 1999). Therefore, we tested whether MyoD-/- primary myoblasts display increased plasticity relative to wild type cells. Unexpectedly, the osteogenic or adipogenic differentiation potential of MyoD-/- primary myoblasts did not increase compared to wild-type cells. Taken together, these results strongly suggest that muscle satellite cells possess multipotential mesenchymal stem cell activity and are capable of forming osteocytes and adipocytes as well as myocytes.
It has recently been shown that mononuclear cells from murine skeletal muscle contain the potential to repopulate all major peripheral blood lineages in lethally irradiated mice, but the origin of this activity is unknown. We have fractionated muscle cells on the basis of hematopoietic markers to show that the active population exclusively expresses the hematopoietic stem cell antigens Sca-1 and CD45. Muscle cells obtained from 6- to 8-week-old C57BL/6-CD45.1 mice and enriched for cells expressing Sca-1 and CD45 were able to generate hematopoietic but not myogenic colonies in vitro and repopulated multiple hematopoietic lineages of lethally irradiated C57BL/6-CD45.2 mice. These data show that muscle-derived hematopoietic stem cells are likely derived from the hematopoietic system and are a result not of transdifferentiation of myogenic stem cells but instead of the presence of substantial numbers of hematopoietic stem cells in the muscle. Although CD45-negative cells were highly myogenic in vitro and in vivo, CD45-positive muscle-derived cells displayed only very limited myogenic activity and only in vivo.
Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1, and mostly negative (<3% positive) for CD14, 31, 49, 144, c-kit, and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting, CD34+/45- cells expressed only c-met mRNA, and did not express any other myogenic cell-related markers such as MyoD, myf-5, myf-6, myogenin, M-cadherin, Pax-3, and Pax-7. However, after 3 d of culture, these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells, as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al., 2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles, and that they can potentially contribute to postnatal skeletal muscle growth.
Three populations of myogenic cells were isolated from normal mouse skeletal muscle based on their adhesion characteristics and proliferation behaviors. Although two of these populations displayed satellite cell characteristics, a third population of long-time proliferating cells expressing hematopoietic stem cell markers was also identified. This third population comprises cells that retain their phenotype for more than 30 passages with normal karyotype and can differentiate into muscle, neural, and endothelial lineages both in vitro and in vivo. In contrast to the other two populations of myogenic cells, the transplantation of the long-time proliferating cells improved the efficiency of muscle regeneration and dystrophin delivery to dystrophic muscle. The long-time proliferating cells' ability to proliferate in vivo for an extended period of time, combined with their strong capacity for self-renewal, their multipotent differentiation, and their immune-privileged behavior, reveals, at least in part, the basis for the improvement of cell transplantation. Our results suggest that this novel population of muscle-derived stem cells will significantly improve muscle cell-mediated therapies.
The development of cell or gene therapies for diseases involving cells that are widely distributed throughout the body has been severely hampered by the inability to achieve the disseminated delivery of cells or genes to the affected tissues or organ. Here we report the results of bone marrow transplantation studies in the mdx mouse, an animal model of Duchenne's muscular dystrophy, which indicate that the intravenous injection of either normal haematopoietic stem cells or a novel population of muscle-derived stem cells into irradiated animals results in the reconstitution of the haematopoietic compartment of the transplanted recipients, the incorporation of donor-derived nuclei into muscle, and the partial restoration of dystrophin expression in the affected muscle. These results suggest that the transplantation of different stem cell populations, using the procedures of bone marrow transplantation, might provide an unanticipated avenue for treating muscular dystrophy as well as other diseases where the systemic delivery of therapeutic cells to sites throughout the body is critical. Our studies also suggest that the inherent developmental potential of stem cells isolated from diverse tissues or organs may be more similar than previously anticipated.
We have introduced the nlacZ reporter gene into the locus of the myogenic factor gene myf-5 by homologous recombination in embryonic stem (ES) cells. Targeted ES clones were injected into precompaction morula, and the β-galactosidase expression pattern was monitored. These mice permit the sensitive visualization of myf-5 expression throughout the embryo, and provide a standard for comparing it with that seen with different myf-5/nlacZ transgenes. Thus, in a comparison using ES cells in chiameric embryos containing the targeted or randomly integrated myf-5/nlacZ construct, we demonstrate that 5.5 kbp of myf-5 upstream flanking sequence including exon1 and most of intron1 directs some skeletal muscle expression, but this is neither qualitatively nor quantitatively equivalent to that of the endogenous gene. Myf-5 is expressed early, before terminal myogenesis takes place in the medial half of the somite, and subsequently it is a major myogenic factor as skeletal muscle forms. All skeletal muscle shows β-galactosidase activity, even after birth, indicating that myf-5 expression is not confined to primary myotubes, which are derived from embryonic myoblasts, but is also present in muscles containing different adult fibre types. The presence of myf-5 transcripts from the endogenous gene in older muscle was confirmed by in situ hybridization. These results suggest that the myf-5 gene is not activated in only a subset of muscle cells and are consistent with the results on the MyoD knockout mice.
The development of cell or gene therapies for diseases involving cells that are widely distributed throughout the body has been severely hampered by the inability to achieve the disseminated delivery of cells or genes to the affected tissues or organ. Here we report the results of bone marrow transplantation studies in the mdx mouse, an animal model of Duchenne's muscular dystrophy, which indicate that the intravenous injection of either normal haematopoietic stem cells or a novel population of muscle-derived stem cells into irradiated animals results in the reconstitution of the haematopoietic compartment of the transplanted recipients, the incorporation of donor-derived nuclei into muscle, and the partial restoration of dystrophin expression in the affected muscle. These results suggest that the transplantation of different stem cell populations, using the procedures of bone marrow transplantation, might provide an unanticipated avenue for treating muscular dystrophy as well as other diseases where the systemic delivery of therapeutic cells to sites throughout the body is critical. Our studies also suggest that the inherent developmental potential of stem cells isolated from diverse tissues or organs may be more similar than previously anticipated.
The myoD gene converts many differentiated cell types into muscle. MyoD is a member of the basic-helix-loop-helix family of
proteins; this 68-amino acid domain in MyoD is necessary and sufficient for myogenesis. MyoD binds cooperatively to muscle-specific
enhancers and activates transcription. The helix-loop-helix motif is responsible for dimerization, and, depending on its dimerization
partner, MyoD activity can be controlled. MyoD senses and integrates many facets of cell state. MyoD is expressed only in
skeletal muscle and its precursors; in nonmuscle cells myoD is repressed by specific genes. MyoD activates its own transcription;
this may stabilize commitment to myogenesis.
To analyze the transcriptional regulatory mechanisms of the myoD gene, we generated transgenic mice bearing a lacZ gene driven by a 6-kb 5'-flanking sequence of the mouse myoD gene including a proximal regulatory region (PRR) and a distal regulatory region (DRR), which are sufficient for activation of muscle-specific transcription in vitro. The expression of the reporter lacZ gene was detected from 10.5 days post coitum in the myotomes, consistent with endogenous myoD mRNA. However, in limb buds and branchial arches, the appearance of the lacZ-positive cells was delayed for one day compared with the endogenous myoD mRNA, suggesting the existence of a different control mechanism among muscle cell lineages. Further, a subset of cells in the central nervous system (CNS), where endogenous myoD mRNA was not detected, expressed the transgene transiently. The same 6-kb MyoD-lacZ gene injected into Xenopus embryos was expressed in the myotomes and in CNS cells at the tailbud stage. Deletion analyses in both transgenic mice and Xenopus embryos indicated that the DRR and PRR were together sufficient for the expression pattern in skeletal muscle. In addition, analysis in Xenopus indicated that a third enhancer region between -1.3 kb and -275 bp could substitute for the DRR. The functional conservation of the regulatory region of the mouse myoD gene in Xenopus embryos suggests that the regulatory pathway that activates myoD gene transcription in axial muscles is conserved among vertebrates.
The objective of ex vivo expansion of primitive hematopoietic progenitor cells (HPC) is to increase the number of progeny cells possessing hematopoietic potential similar to the original HPC. In the context of bone marrow (BM) transplantation in mice, this implies that expanding a number of HPC sufficient for long-term rescue of one lethally irradiated animal should generate enough cells to rescue more than one lethally irradiated recipient. In the present study, Sca-1+Lin- cells from male C57Bl/6 mice were expanded in vitro with stem cell factor (SCF), interleukin-1alpha (IL-1alpha), IL-3, and IL-6 and used to transplant lethally irradiated syngeneic female recipients. Expanded cells were tracked in vitro with the fluorescent membrane dye PKH2, which becomes evenly distributed among dividing daughter cells, and fractionated on day 7 into Sca-1+ cells which did not divide (Sca-1+PKH2bright), those which had divided 1 to 2 times (Sca-1+PKH2moderate), or those which had divided four or more times (Sca-1+PKH2dim). Grafts of expanded cells consisted of either the same number of fresh cells proven to rescue lethally irradiated animals [3X10(3) cells; referred to as one repopulating dose (1 RD)] or the expansion equivalent (EE) of these cells. One EE of cells represented 3X10(3) multiplied by the fold increase in the number of cultured cells on day 7. All animals transplanted with 3X10(3) freshly isolated Sca-1+Lin- cells survived long-term. Only 53% of animals receiving 1 EE of all cultured day-7 cells survived. One RD from all three PKH2 fractions (bright, moderate, and dim) of day-7 cultured Sca-1+ cells failed to rescue more than 30% of lethally irradiated recipients. Comparable survival rates were obtained when 1 EE of Sca-1+PKH2dim or only 4 RD of Sca-1+PKH2bright cells were used as grafts, suggesting that a larger frequency of long-term repopulating cells may have been retained within the fraction of Sca-1+ cells undergoing minimal or no proliferation in culture. Engraftment of male ex vivo expanded cells in recipients was confirmed by polymerase chain reaction (PCR) analysis with Y chromosome-specific primers. When analyzed for their cell cycle status, Sca-1+PKH2bright cells were mostly quiescent, whereas a higher percentage of Sca-1+PKH2dim cells were in active phases of cell cycle. These data suggest that ex vivo expansion does not augment the number of BM repopulating HPC and that ex vivo expansion generates classes of progenitor cells with different BM repopulating potentials depending on their proliferative history. These studies also suggest that the cell cycle status of graft cells may affect the ability of these cells to engraft in myeloablated hosts.
We have introduced the nlacZ reporter gene into the locus of the myogenic factor gene myf-5 by homologous recombination in embryonic stem (ES) cells. Targeted ES clones were injected into precompaction morula, and the beta-galactosidase expression pattern was monitored. These mice permit the sensitive visualization of myf-5 expression throughout the embryo, and provide a standard for comparing it with that seen with different myf-5/nlacZ transgenes. Thus, in a comparison using ES cells in chimaeric embryos containing the targeted or randomly integrated myf-5/nlacZ construct, we demonstrate that 5.5 kbp of myf-5 upstream flanking sequence including exon1 and most of intron1 directs some skeletal muscle expression, but this is neither qualitatively nor quantitatively equivalent to that of the endogenous gene. Myf-5 is expressed early, before terminal myogenesis takes place in the medial half of the somite, and subsequently it is a major myogenic factor as skeletal muscle forms. All skeletal muscle shows beta-galactosidase activity, even after birth, indicating that myf-5 expression is not confined to primary myotubes, which are derived from embryonic myoblasts, but is also present in muscles containing different adult fibre types. The presence of myf-5 transcripts from the endogenous gene in older muscle was confirmed by in situ hybridization. These results suggest that the myf-5 gene is not activated in only a subset of muscle cells and are consistent with the results on the MyoD knockout mice.
Growth and repair of skeletal muscle are normally mediated by the satellite cells that surround muscle fibers. In regenerating muscle, however, the number of myogenic precursors exceeds that of resident satellite cells, implying migration or recruitment of undifferentiated progenitors from other sources. Transplantation of genetically marked bone marrow into immunodeficient mice revealed that marrow-derived cells migrate into areas of induced muscle degeneration, undergo myogenic differentiation, and participate in the regeneration of the damaged fibers. Genetically modified, marrow-derived myogenic progenitors could potentially be used to target therapeutic genes to muscle tissue, providing an alternative strategy for treatment of muscular dystrophies.
Muscle satellite cells have long been considered a distinct myogenic lineage responsible for postnatal growth, repair, and maintenance of skeletal muscle. Recent studies in mice, however, have revealed the potential for highly purified hematopoietic stem cells from bone marrow to participate in muscle regeneration. Perhaps more significantly, a population of putative stem cells isolated directly from skeletal muscle efficiently reconstitutes the hematopoietic compartment and participates in muscle regeneration following intravenous injection in mice. The plasticity of muscle stem cells has raised important questions regarding the relationship between the muscle-derived stem cells and the skeletal muscle satellite cells. Furthermore, the ability of hematopoietic cells to undergo myogenesis has prompted new investigations into the embryonic origin of satellite cells. Recent developmental studies suggest that a population of satellite cells is derived from progenitors in the embryonic vasculature. Taken together, these studies provide the first evidence that pluripotential stem cells are present within adult skeletal muscle. Tissue-specific stem cells, including satellite cells, may share a common embryonic origin and possess the capacity to activate diverse genetic programs in response to environmental stimuli. Manipulation of such tissue-specific stem cells may eventually revolutionize therapies for degenerative diseases, including muscular dystrophy.
The paired box transcription factor Pax7 was isolated by representational difference analysis as a gene specifically expressed in cultured satellite cell-derived myoblasts. In situ hybridization revealed that Pax7 was also expressed in satellite cells residing in adult muscle. Cell culture and electron microscopic analysis revealed a complete absence of satellite cells in Pax7(-/-) skeletal muscle. Surprisingly, fluorescence-activated cell sorting analysis indicated that the proportion of muscle-derived stem cells was unaffected. Importantly, stem cells from Pax7(-/-) muscle displayed almost a 10-fold increase in their ability to form hematopoietic colonies. These results demonstrate that satellite cells and muscle-derived stem cells represent distinct cell populations. Together these studies suggest that induction of Pax7 in muscle-derived stem cells induces satellite cell specification by restricting alternate developmental programs.
We investigated hematopoietic capabilities of murine skeletal muscle using methylcellulose culture and transplantation into lethally irradiated mice. Muscle mononuclear cells (MNC) contained colony-forming cells and long-term engrafting cells. Studies using chimeric mice indicated a bone marrow origin of the hematopoietic cells in the muscle. We then separated muscle MNC by FACS sorting into Ly-5-positive cells and Ly-5-negative cells and analyzed their hematopoietic capability in vitro and in vivo. The hematopoietic progenitors and stem cells were present only in the Ly-5-positive fraction.
It has been reported that mononuclear cells harvested from murine skeletal muscle are capable of hematopoietic reconstitution of lethally irradiated mice. First, the nature of the hematopoietic progenitors in the muscle of C57BL/6-Ly-5.1 mice was examined by means of methylcellulose culture. The types and incidences of colonies grown from muscle mononuclear cells were different from those cultured from bone marrow (BM) or peripheral blood mononuclear cells. The next step was to examine the origin of the hematopoietic progenitors and stem cells in the muscle with the use of Ly-5.2 mice that had been made chimeric by transplantation of Ly-5.1 BM cells. The percentages of Ly-5.1 cells cultured from the muscle of the chimeric mice correlated with those cultured from BM, indicating BM origin of hematopoietic progenitors in the muscle. Long-term hematopoietic engrafting cells in the muscle of the chimeric mice were also derived from BM. However, mobilization of progenitors into circulation by granulocyte colony-stimulating factor did not change the population of hematopoietic progenitors in the muscle. It is proposed that hematopoietic progenitors and stem cells in the muscle tissue are of BM origin but their transition from BM to muscle may be a slow process.
Skeletal muscle contains two types of stem cells: satellite cells, which function as myogenic precursors, and a population of multipotent adult stem cells. Satellite cells are believed to form a stable, self-renewing pool of stem cells in adult muscle where they function in tissue growth and repair. An additional stem cell population in adult muscle displays a remarkable capacity to differentiate into hematopoietic cells as well as muscle following transplantation. This article discusses the characteristics and properties of these cell populations, the relationship between them, and the potential for stem cell-based muscle therapeutics.
The concept of the adult tissue stem cell is fundamental to models of persistent renewal in functionally post-mitotic tissues. Although relatively ignored by stem cell biology, skeletal muscle is a prime example of an adult tissue that can generate terminally differentiated cells uniquely specialized to carry out tissue-specific functions. This capacity is attributed to satellite cells, a population of undifferentiated, quiescent precursors that become activated to divide and differentiate in response to the demands of growth or damage. The aim of this review is to discuss the role of the satellite cell as an adult tissue-specific stem cell. We examine evidence for the presence of behaviourally and phenotypically distinct subpopulations of precursor within the satellite cell pool. Further, we speculate on the possible identity, origins and relevance of multipotent muscle stem cells, a population with both myogenic and hematopoietic potentials that has been isolated from whole muscle. Taken together, current evidence suggests the possibility that the regenerative compartment of adult skeletal muscle may conform to an archetypal stem cell-based hierarchy, maintained within a stem cell niche. It therefore remains to be seen whether all satellite cells are skeletal muscle-specific stem cells, or whether some or all are the progeny of an as yet unidentified muscle stem cell.
The myogenic potential of bone marrow and fetal liver cells was examined using donor cells from green fluorescent protein (GFP)-gene transgenic mice transferred into chimeric mice. Lethally irradiated X-chromosome-linked muscular dystrophy (mdx) mice receiving bone marrow cells from the transgenic mice exhibited significant numbers of fluorescence(+) and dystrophin(+) muscle fibres. In order to compare the generating capacity of fetal liver cells with bone marrow cells in neonatal chimeras, these two cell types from the transgenic mice were injected into busulfantreated normal or mdx neonatal mice, and muscular generation in the chimeras was examined. Cardiotoxin-induced (or -uninduced, for mdx recipients) muscle regeneration in chimeras also produced fluorescence(+) muscle fibres. The muscle reconstitution efficiency of the bone marrow cells was almost equal to that of fetal liver cells. However, the myogenic cell frequency was higher in fetal livers than in bone marrow. Among the neonatal chimeras of normal recipients, several fibres expressed the fluorescence in the cardiotoxin-untreated muscle. Moreover, fluorescence(+) mononuclear cells were observed beneath the basal lamina of the cardiotoxin-untreated muscle of chimeras, a position where satellite cells are localizing. It was also found that mononuclear fluorescence(+) and desmin(+) cells were observed in the explantation cultures of untreated muscles of neonatal chimeras. The fluorescence(+) muscle fibres were generated in the second recipient mice receiving muscle single cells from the cardiotoxin-untreated neonatal chimeras. The results suggest that both bone marrow and fetal liver cells may have the potential to differentiate into muscle satellite cells and participate in muscle regeneration after muscle damage as well as in physiological muscle generation.
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