Developmental behavior of embryonic myogenic progenitors transplanted into adult muscle as revealed by desmin LacZ recombinant gene.

We studied the behavior of myogenic progenitors from donor desmin(+/-) LacZ embryos after implantation into tibialis anterior muscle of 2-month-old mouse hosts. Myogenic progenitors were collected from 10-day post-coital mouse embryo somite dermomyotomes (DMs), forelimb buds (LBs), and trunks. The replacement of desmin by the LacZ coding sequence allowed specific monitoring of beta-galactosidase expression in donor myogenic cells. Immunostaining for myosin heavy chain and laminin expression was performed together with acetylcholine receptor histochemistry on sections of implanted muscle. Myogenic progenitors generated from DM, LB, and trunk were able to proliferate and adopt a myogenic pathway after transplantation into adult mouse muscle. Although their development appeared to be limited for DM and LB cell transplantation, the differentiation of myogenic progenitors occurred readily with trunk cell injection, suggesting that cell types associated with DM cells were involved in long-term myofiber differentiation (21 day). When neural tube/notochord (NTN) or sclerotomal (S) cells were co-transplanted with DM cells, myogenic nuclei were produced, indicating that both NTN and S are required for the differentiation of DMs grafted into adult muscle. These data are consistent with the differentiation of neural tissues and bone from NTN and S, respectively, and with the development of anatomic relations among all in vivo-differentiated tissues. These results suggest that embryonic trunk cells can be used to repair different types of injured tissues (especially skeletal muscle) under appropriate environmental conditions.     

Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing

Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin(+) cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34(+) cells and Bcl-2(+) cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34(+)Bcl-2(+) enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.     

Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle

Skeletal muscle regeneration in adults is thought to occur through the action of myogenic satellite cells located in close association with mature muscle fibers; however, these precursor cells have not been prospectively isolated, and recent studies have suggested that additional muscle progenitors, including cells of bone marrow or hematopoietic origin, may exist. To clarify the origin(s) of adult myogenic cells, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of myofiber-associated cells capable at the single cell level of generating myogenic colonies at high frequency. Importantly, although muscle-engrafted cells from marrow and/or circulation localized to the same anatomic compartment as myogenic satellite cells and expressed some though not all satellite cell markers, they displayed no intrinsic myogenicity. Together, these studies describe the clonal isolation of functional adult myogenic progenitors and demonstrate that these cells do not arise from hematopoietic or other bone marrow or circulating precursors.     

Human ES- and iPS-derived myogenic progenitors restore DYSTROPHIN and improve contractility upon transplantation in dystrophic mice

A major obstacle in the application of cell-based therapies for the treatment of neuromuscular disorders is obtaining the appropriate number of stem/progenitor cells to produce effective engraftment. The use of embryonic stem (ES) or induced pluripotent stem (iPS) cells could overcome this hurdle. However, to date, derivation of engraftable skeletal muscle precursors that can restore muscle function from human pluripotent cells has not been achieved. Here we applied conditional expression of PAX7 in human ES/iPS cells to successfully derive large quantities of myogenic precursors, which, upon transplantation into dystrophic muscle, are able to engraft efficiently, producing abundant human-derived DYSTROPHIN-positive myofibers that exhibit superior strength. Importantly, transplanted cells also seed the muscle satellite cell compartment, and engraftment is present over 11 months posttransplant. This study provides the proof of principle for the derivation of functional skeletal myogenic progenitors from human ES/iPS cells and highlights their potential for future therapeutic application in muscular dystrophies.     

Fetal muscle contains different CD34+ cell subsets that distinctly differentiate into adipogenic, angiogenic and myogenic lineages

We have previously demonstrated that CD34(+) cells isolated from fetal mouse muscles are an interesting source of myogenic progenitors. In the present work, we pinpoint the tissue location of these CD34(+) cells using cell surface and phenotype markers. In order to identify the myogenic population, we next purified different CD34(+) subsets, determined their expression of relevant lineage-related genes, and analyzed their differentiation capacities in vitro and in vivo. The CD34(+) population comprised a CD31(+)/CD45(-) cell subset exhibiting endothelial characteristics and only capable of forming microvessels in vivo. The CD34(+)/CD31(-)/CD45(-)/Sca1(+) subpopulation, which is restricted to the muscle epimysium, displayed adipogenic differentiation both in vitro and in vivo. CD34(+)/CD31(-)/CD45(-)/Sca1(-) cells, localized in the muscle interstitium, transcribed myogenic genes, but did not display the characteristics of adult satellite cells. These cells were distinct from pericytes and fibroblasts. They were myogenic in vitro, and efficiently contributed to skeletal muscle regeneration in vivo, although their myogenic potential was lower than that of the unfractionated CD34(+) cell population. Our results indicate that angiogenic and adipogenic cells grafted with myogenic cells enhance their contribution to myogenic regeneration, highlighting the fundamental role of the microenvironment on the fate of transplanted cells.     

Skeletal myogenic progenitors in the endothelium of lung and yolk sac

We previously showed that clonable skeletal myogenic cells can be derived from the embryonic aorta but become very rare in the more mature and structured fetal aorta. The aim of this study was to investigate whether, during fetal and postnatal development, these myogenic progenitors progressively disappear or may rather associate with the microvascular district, being thus distributed to virtually all tissues. To test this hypothesis, we used F1 embryos (or mice) from a transgenic line expressing a striated muscle-specific reporter gene (LacZ) crossed with a transgenic line expressing a different endothelial-specific reporter genes (GFP). Endothelial cells were isolated from yolk sac (at E11) and lung (at E11, E17, P1, P10, and P60), two organs embryologically unrelated to paraxial mesoderm, rich in vessels, and devoid of skeletal muscle. Endothelial cells, purified by magnetic bead selection (CD31/PECAM-1(+)) or cell sorting (Tie2-GFP(+)) were then challenged for their skeletal myogenic potential in vitro and in vivo.The results demonstrated that both yolk sac and lung contain progenitor cells, which express endothelial markers and are endowed with a skeletal myogenic potential that they reveal when in the presence of differentiating myoblasts, in vitro, and regenerating muscle, in vivo.The number (or potency to generate skeletal muscle) of these vessels associated cells decreases rapidly with age and is very low in mature animals, possibly correlating with reduced regenerative capacity of adult mammalian tissues.     

Osteogenic properties of human myogenic progenitor cells

Here, we identified human myogenic progenitor cells coexpressing Pax7, a marker of muscle satellite cells and bone-specific alkaline phosphatase, a marker of osteoblasts, in regenerating muscle. To determine whether human myogenic progenitor cells are able to act as osteoprogenitor cells, we cultured both primary and immortalized progenitor cells derived from the healthy muscle of a nondystrophic woman. The undifferentiated myogenic progenitors spontaneously expressed two osteoblast-specific proteins, bone-specific alkaline phosphatase and Runx2, and were able to undergo terminal osteogenic differentiation without exposure to an exogenous inductive agent such as bone morphogenetic proteins. They also expressed the muscle lineage-specific proteins Pax7 and MyoD, and lost their osteogenic characteristics in association with terminal muscle differentiation. Both myoblastic and osteoblastic properties are thus simultaneously expressed in the human myogenic cell lineage prior to commitment to muscle differentiation. In addition, C3 transferase, a specific inhibitor of Rho GTPase, blocked myogenic but not osteogenic differentiation of human myogenic progenitor cells. These data suggest that human myogenic progenitor cells retain the capacity to act as osteoprogenitor cells that form ectopic bone spontaneously, and that Rho signaling is involved in a critical switch between myogenesis and osteogenesis in the human myogenic cell lineage.     

Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells

Cells derived from blood vessels of human skeletal muscle can regenerate skeletal muscle, similarly to embryonic mesoangioblasts. However, adult cells do not express endothelial markers, but instead express markers of pericytes, such as NG2 proteoglycan and alkaline phosphatase (ALP), and can be prospectively isolated from freshly dissociated ALP(+) cells. Unlike canonical myogenic precursors (satellite cells), pericyte-derived cells express myogenic markers only in differentiated myotubes, which they form spontaneously with high efficiency. When transplanted into severe combined immune deficient-X-linked, mouse muscular dystrophy (scid-mdx) mice, pericyte-derived cells colonize host muscle and generate numerous fibres expressing human dystrophin. Similar cells isolated from Duchenne patients, and engineered to express human mini-dystrophin, also give rise to many dystrophin-positive fibres in vivo. These data show that myogenic precursors, distinct from satellite cells, are associated with microvascular walls in the human skeletal muscle, may represent a correlate of embryonic 'mesoangioblasts' present after birth and may be a promising candidate for future cell-therapy protocols in patients.     

Induced Wnt5a expression perturbs embryonic outgrowth and intestinal elongation, but is well-tolerated in adult mice

Embryonic mesoangioblasts are the in vitro counterpart of vessel-associated progenitors, able to differentiate into different mesoderm cell types. To investigate signals recruiting these progenitors to a skeletal myogenic fate, we developed an in vitro assay, based upon co-culture of E11.5 dorsal aorta (from MLC3F-nLacZ transgenic embryos, expressing nuclear beta galactosidase only in striated muscle) with differentiating C2C12 or primary myoblasts. Under these conditions muscle differentiation from cells originating from the vessel can be quantified by counting the number of beta gal+nuclei. Results indicated that Noggin (but not Follistatin, Chordin or Gremlin) stimulates while BMP2/4 inhibits myogenesis from dorsal aorta progenitors; neutralizing antibodies and shRNA greatly reduce these effects. In contrast, TGF-β1, VEGF, Wnt7A, Wnt3A, bFGF, PDGF-BB and IGF1 have no effect. Sorting experiments indicated that the majority of these myogenic progenitors express the pericyte marker NG2. Moreover they are abundant in the thoracic segment at E10.5 and in the iliac bifurcation at E11.5 suggesting the occurrence of a cranio-caudal wave of competent cells along the aorta. BMP2 is expressed in the dorsal aorta and Noggin in newly formed muscle fibers suggesting that these two tissues compete to recruit mesoderm cells to a myogenic or to a perithelial fate in the developing fetal muscle.     

Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration

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.     

A new look at the origin, function, and "stem-cell" status of muscle satellite cells

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.     

Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells

Differentiation of embryonic and adult myogenic progenitors undergoes a complex series of cell rearrangements and specification events which are controlled by distinct gene regulatory networks. Delineation of the molecular mechanisms that regulate skeletal muscle specification and formation should be important for understanding congenital myopathies and muscular degenerative diseases. Retinoic acid (RA) signaling plays an important role in development. However, the role of RA signaling in adult myogenic progenitors is poorly understood. Here, we investigate the role of RA signaling in regulating myogenic differentiation of myoblastic progenitor cells. Using the mouse myoblast progenitor C2C12 line as a model, we have found that the endogenous expression of most RAR and RXR isotypes is readily detected. While the nuclear receptor co-repressors are highly expressed, two of the three nuclear receptor co-activators and the enzymes involved in RA synthesis are expressed at low level or undetectable, suggesting that the RA signaling pathway may be repressed in myogenic progenitors. Using the α-myosin heavy chain promoter-driven reporter (MyHC-GLuc), we have demonstrated that either ATRA or 9CRA is able to effectively induce myogenic differentiation, which can be synergistically enhanced when both ATRA and 9CRA are used. Upon ATRA and 9CRA treatment of C2C12 cells the expression of late myogenic markers significantly increases. We have further shown that adenovirus-mediated exogenous expression of RARα and/or RXRα is able to effectively induce myogenic differentiation in a ligand-independent fashion. Morphologically, ATRA- and 9CRA-treated C2C12 cells exhibit elongated cell body and become multi-nucleated myoblasts, and even form myoblast fusion. Ultrastructural analysis under transmission electron microscope reveals that RA-treated myogenic progenitor cells exhibit an abundant presence of muscle fibers. Therefore, our results strongly suggest that RA signaling may play an important role in regulating myogenic differentiation.     

Intramuscular adipose is derived from a non-Pax3 lineage and required for efficient regeneration of skeletal muscles

Ectopic accumulation of adipose in the skeletal muscle is associated with muscle wasting, insulin resistance and diabetes. However, the developmental origin of postnatal intramuscular adipose and its interaction with muscle tissue are unclear. We report here that compared to the fast EDL muscles, slow SOL muscles are more enriched with adipogenic progenitors and have higher propensity to form adipose. Using Cre/LoxP mediated lineage tracing in mice, we show that intramuscular adipose in both EDL and SOL muscles is exclusively derived from a Pax3(-) non-myogenic lineage. In contrast, inter-scapular brown adipose is derived from the Pax3(+) lineage. To dissect the interaction between adipose and skeletal muscle tissues, we used Myf5-Cre and aP2-Cre mice in combination with ROSA26-iDTR mice to genetically ablate myogenic and adipogenic cell lineages, respectively. Whereas ablation of the myogenic cell lineage facilitated adipogenic differentiation, ablation of the adipogenic cell lineage surprisingly impaired the regeneration of acutely injured skeletal muscles. These results reveal striking heterogeneity of tissue-specific adipose and a previously unappreciated role of intramuscular adipose in skeletal muscle regeneration.     

Bone and cartilage formation by skeletal muscle derived cells

In adult individuals when most tissues have progressively lost the ability to regenerate, bone maintains the potential for a continuous self remodeling. The bone marrow has been so far the main recognized source of osteoprogenitor cells that contribute to the turnover of the skeletal scaffold. The possibility though exists that a pool of osteoprogenitor cells resides within other adult tissues and in particular, as reported previously, in other connective tissues such as fat and skeletal muscle. In an attempt to identify an alternative source of osteoprogenitor cells other than bone marrow we looked into the skeletal muscle. A plastic adhering cell population, from now on referred to as skeletal muscle derived cells (SMDCs), was obtained from biopsies of human skeletal muscle. SMDCs were clonogenic and displayed a fibroblast-like morphology. The isolated cell population had a mesenchymal origin as indicated by abundant expression of type I collagen, fibronectin, and vimentin and appeared heterogeneous. SMDCs were positive for alpha smooth actin, and to a lesser extent for desmin and alpha sarcomeric myosin, two specific markers of the myogenic phenotype. Surprisingly though SMDCs expressed early markers of an osteogenic commitment as indicated by positive staining for alkaline phosphatase, osteopontin, and osteonectin. Under the appropriate stimuli, these cells deposited in vitro a mineralized bone matrix and a proteoglycan rich matrix. In addition, SMDCs cultured in the presence of low serum and insulin differentiated towards adipocytes developing abundant lipid droplets in the cytoplasm. Furthermore SMDCs formed three-dimensional bone tissue in vivo when implanted in an immunodeficient mouse, and a mature cartilage rudiment when maintained as a pellet culture. In summary, we report the isolation and characterization of a cell population from the human skeletal muscle not only able to express in vitro specific markers of distinct mesenchymal lineages (adipogenic, chondrogenic, and osteogenic), but most importantly, able to complete the differentiation pathway leading to the formation of bone and cartilage. In this respect SMDCs resemble bone marrow stromal cells (BMSCs).     

The skeletal muscle satellite cell: stem cell or son of stem cell?

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.     

??4 Integrin Marks Interstitial Myogenic Progenitor Cells in Adult Murine Skeletal Muscle

Skeletal muscle growth and its regeneration following injury rely on myogenic progenitor cells, a heterogeneous population that includes the satellite cells and other interstitial progenitors. The present study demonstrates that surface expression of β4 integrin marks a population of vessel-associated interstitial muscle progenitor cells. Muscle β4 integrin–positive cells do not express myogenic markers upon isolation. However, they are capable of undergoing myogenic specification in vitro and in vivo: β4 integrin cells differentiate into multinucleated myotubes in culture dishes and contribute to muscle regeneration upon delivery into diseased mice. Subfractionation of β4 integrin–expressing cells based on CD31 expression does not further enrich for myogenic precursors. These findings support the expression of β4 integrin in interstitial, vessel-associated cells with myogenic activity within adult skeletal muscle.