Human muscle precursor cell regeneration in the mouse host is enhanced by growth factors.

The aim of this study was to optimize human muscle formation in vivo from implanted human muscle precursor cells. We transplanted donor muscle precursor cells (MPCs) prepared from postnatal or fetal human muscle into immunodeficient host mice and showed that irradiation of host muscle significantly enhanced muscle formation by donor cells. The amount of donor muscle formed in cryodamaged host muscle was increased by exposure of donor cells to growth factors before their implantation into injured host muscle. Insulin-like growth factor type I (IGF-I) significantly increased the amount of muscle formed by postnatal human muscle cells, but not by fetal human MPCs. However, treatment of fetal muscle cells with IGF-I, in combination with basic fibroblast growth factor and plasmin, significantly increased the amount of donor muscle formed. In vivo, human MPCs formed mosaic human-mouse muscle fibers, in which each human myonucleus was associated with a zone of human sarcolemmal protein spectrin.

Non-viral gene delivery in skeletal muscle: a protein factory.

Ever since the publication of the first reports in 1990 using skeletal muscle as a direct target for expressing foreign transgenes, an avalanche of papers has identified a variety of proteins that can be synthesized and correctly processed by skeletal muscle. The impetus to the development of such applications is not only amelioration of muscle diseases, but also a range of therapeutic applications, from immunization to delivery of therapeutic proteins, such as clotting factors and hormones. Although the most efficient way of introducing transgenes into muscle fibres has been by a variety of recombinant viral vectors, there are potential benefits in the use of non-viral vectors. In this review we assess the recent advances in construction and delivery of naked plasmid DNA to skeletal muscle and highlight the options available for further improvements to raise efficiency to therapeutic levels.

Cells that participate in regeneration of skeletal muscle.

Skeletal muscle is a post-mitotic tissue that is thought, conventionally to be maintained by repair and regeneration by a population of stem cell-like satellite cells. Recent findings have brought into question the extent to which these satellite cells represent a single homogeneous population and whether they are the only source of myogenic cells in mature muscle. It has been shown that myogenic cells can be derived from the bone marrow or from the supposedly post-mitotic nuclei of muscle fibres. However, neither of these sources has been demonstrated to generate more than a tiny proportion of muscle nuclei. It remains possible nonetheless that such mechanisms might be exploited for therapeutic uses in primary muscle disease.

Suppression of revertant fibers in mdx mice by expression of a functional dystrophin.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration that results from the absence of dystrophin. Despite null mutations in the dystrophin gene, many DMD patients display a low percentage of dystrophin-positive fibers. These "revertant fibers" are also present in the dystrophin-deficient mdx mouse and are believed to result from alternative splicing or second mutation events that bypass the mutation and restore an open reading frame. However, it is unclear what role dystrophin and the dystrophic pathology might play in revertant fiber formation and accumulation. We have analyzed the role of dystrophin expression and the dystrophic pathology in this process by monitoring revertant fibers in transgenic mdx mice that express truncated dystrophins. We found that newborn transgenic mice displayed approximately the same number of revertant fibers as newborn mdx mice, indicating that expression of a functional dystrophin does not suppress the initiation of revertant fiber formation. Surprisingly, when the transgene encoded a functional dystrophin, revertant fibers were not detected in adult or old mdx mice. In contrast, adult transgenic mice expressing a non-functional dystrophin accumulated increasing numbers of revertant fibers, similar to mdx mice, suggesting that positive selection is required for the persistence of revertant fibers. Finally, we provide evidence that the loss of revertant dystrophin in transgenic mdx muscle fibers overexpressing a functional dystrophin results from displacement of the revertant protein by the transgene-encoded dystrophin.

Transplanted primary neonatal myoblasts can give rise to functional satellite cells as identified using the Myf5nlacZl+ mouse.

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.

A new immunodeficient mouse model for human myoblast transplantation.

Design of efficient transplantation strategies for myoblast-based gene therapies in humans requires animal models in which xenografts are tolerated for long periods of time. In addition, such recipients should be able to withstand pretransplantation manipulations for enhancement of graft growth. Here we report that a newly developed immunodeficient mouse carrying two known mutations (the recombinase activating gene 2, RAG2, and the common cytokine receptor gamma, gammac) is a candidate fulfilling these requirements. Skeletal muscles from RAG2(-/-)/gammac(-/-) double mutant mice recover normally after myotoxin application or cryolesion, procedures commonly used to induce regeneration and improve transplantation efficiency. Well-differentiated donor-derived muscle tissue could be detected up to 9 weeks after transplantation of human myoblasts into RAG2(-/-)/gammac(-/-) muscles. These results suggest that the RAG2(-/-)/gammac(-/-) mouse model will provide new opportunities for human muscle research.

Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease arising from defects in the dystrophin gene, typically nonsense or frameshift mutations, that preclude the synthesis of a functional protein. A milder, allelic version of the disease, Becker muscular dystrophy, generally arises from in-frame deletions that allow synthesis of a shorter but still semifunctional protein. Therapies to introduce functional dystrophin into dystrophic tissue through either cell or gene replacement have not been successful to date. We report an alternative approach where 2'-O-methyl antisense oligoribonucleotides have been used to modify processing of the dystrophin pre-mRNA in the mdx mouse model of DMD. By targeting 2'-O-methyl antisense oligoribonucleotides to block motifs involved in normal dystrophin pre-mRNA splicing, we induced excision of exon 23, and the mdx nonsense mutation, without disrupting the reading frame. Exon 23 skipping was first optimized in vitro in transfected H-2K(b)-tsA58 mdx myoblasts and then induced in vivo. Immunohistochemical staining demonstrated the synthesis and correct subsarcolemmal localization of dystrophin and gamma-sarcoglycan in the mdx mouse after intramuscular delivery of antisense oligoribonucleotide:liposome complexes. This approach should reduce the severity of DMD by allowing a dystrophic gene transcript to be modified, such that it can be translated into a Becker-dystrophin-like protein.

Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite 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 Myf5(nlacZ/+) mice, all CD34(+ve) satellite cells also express beta-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.

Thymic myoid cells as a source of cells for myoblast transfer.

Transplantation of disaggregated myoblasts from normal donor to the muscles of a diseased host, or reimplantation of genetically modified host myoblasts, has been suggested as a possible route to therapy for inherited myopathies such as Duchenne muscular dystrophy, or to supply missing proteins that are required systemically in diseases such as hemophilia. With two exceptions, studies of myoblast transfer in the mouse have involved transplantation of donor myoblasts isolated from adult or neonatal skeletal muscle satellite cells. In this study we present evidence that thymic myoid cells are capable of participating in the regeneration of postnatal skeletal muscle, resulting in the expression of donor-derived proteins such as dystrophin and retrovirally encoded proteins such as beta-galactosidase within host muscles. This leads us to conclude that thymic myoid cells may provide an alternative to myoblasts derived from skeletal muscle as a source of myogenic cells for myoblast transfer.

Evidence for a myogenic stem cell that is exhausted in dystrophic muscle.

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.

Massive idiosyncratic exon skipping corrects the nonsense mutation in dystrophic mouse muscle and produces functional revertant fibers by clonal expansion.

Conventionally, nonsense mutations within a gene preclude synthesis of a full-length functional protein. Obviation of such a blockage is seen in the mdx mouse, where despite a nonsense mutation in exon 23 of the dystrophin gene, occasional so-called revertant muscle fibers are seen to contain near-normal levels of its protein product. Here, we show that reversion of dystrophin expression in mdx mice muscle involves unprecedented massive loss of up to 30 exons. We detected several alternatively processed transcripts that could account for some of the revertant dystrophins and could not detect genomic deletion from the region commonly skipped in revertant dystrophin. This, together with exon skipping in two noncontiguous regions, favors aberrant splicing as the mechanism for the restoration of dystrophin, but is hard to reconcile with the clonal idiosyncrasy of revertant dystrophins. Revertant dystrophins retain functional domains and mediate plasmalemmal assembly of the dystrophin-associated glycoprotein complex. Physiological function of revertant fibers is demonstrated by the clonal growth of revertant clusters with age, suggesting that revertant dystrophin could be used as a guide to the construction of dystrophin expression vectors for individual gene therapy. The dystrophin gene in the mdx mouse provides a favored system for study of exon skipping associated with nonsense mutations.

Forced MyHCIIB expression following targeted genetic manipulation of conditionally immortalized muscle precursor cells.

The ability to carry out gene targeting in somatic stem cells while maintaining their stem cell characteristics would have important implications for gene therapy and for the analysis of gene function. Using mouse myoblasts, we have explored this possibility by attempting to alter the promoter of a myosin heavy chain gene (MyHCIIB) characteristic of physiologically "fast" muscle so as to force its unscheduled expression in physiologically "slow" muscle fibers. Conditionally immortalized muscle precursor cells were transfected with a gene targeting construct designed to replace the MyHCIIB promoter with that for the carbonic anhydrase III gene (CAIII), which is highly expressed in slow muscle. A potentially targeted clone was isolated and differentiated in culture to form myotubes which expressed MyHCIIB. Cells from the same clone were injected into both slow and fast muscle of host mice, where they contributed to fiber formation. In slow muscle, the fibers derived from this clone did not express MyHCIIB; this may reflect an instability of the targeted MyHCIIB locus and/or a failure of the hybrid promoter to function in slow fibers in vivo. Nonetheless, we have demonstrated that a "promoter knock-in" gene targeting procedure can be used to generate unique MyHCIIB-expressing myotubes in culture and that conditionally immortalized myoblasts can be subjected to extensive passaging and genetic manipulation without losing their ability to form fibers in culture and in vivo.

Patterns of repair of dystrophic mouse muscle: studies on isolated fibers.

Repair of damaged skeletal muscle fibers by muscle precursor cells (MPC) is central to the regeneration that occurs after injury or disease of muscle and is vital to the success of myoblast transplantation to treat inherited myopathies. However, we lack a detailed knowledge of the mechanisms of this muscle repair. Here, we have used a novel combination of techniques to study this process, marking MPC with nuclear-localizing LacZ and tracing their contribution to regeneration of muscle fibers after grafting into preirradiated muscle of the mdx nu/nu mouse. In this model system, there is muscle degeneration, but little or no regeneration from endogenous MPC. Incorporation of donor MPC into injected muscles was analyzed by preparing single viable muscle fibers at various times after cell implantation. Fibers were either stained immediately for beta-gal, or cultured to allow their associated satellite cells to migrate from the fiber and then stained for beta-gal. Marked myonuclei were located in discrete segments of host muscle fibers and were not incorporated preferentially at the ends of the fibers. All branches on host fibers were also found to be composed of myonuclei carrying the beta-gal marker. There was no significant movement of donor myonuclei within myofibers for up to 7 weeks after MPC implantation. Although donor-derived dystrophin was usually located coincidentally with donor myonuclei, in some fibers, the dystrophin protein had spread further along the mosaic myofibers than had the myonuclei of donor origin. In addition to repairing segments of the host fiber, the implanted MPC also gave rise to satellite cells, which may contribute to future muscle repair.

Covert persistence of mdx mouse myopathy is revealed by acute and chronic effects of irradiation.

To compare muscle fiber loss in young and old mdx mice, we have blocked regeneration in one leg with a high dose (18 Gy) of X-rays administered at two ages; 16 days, just prior to the onset of the myopathy, and 15 weeks, when the myopathy is considered to be quiescent. Mice were examined 4 days after irradiation to look for acute effects, or after 6 weeks to look for cumulative effects. Tibial length, muscle weight, muscle fiber size, fiber number and histological changes were recorded. Signs of acute damage to muscle fibers, leakage of Procion Orange dye into fibers and loss of creatine kinase from the fibers were also examined. Irradiation caused no acute or chronic damage to muscle fibers; on the contrary, in the youngest mdx mice, irradiation delayed the onset of the disease. However, in mdx but not in normal mice, there was a loss of muscle mass and fiber number in irradiated by comparison with the non-irradiated contra-lateral muscles. This loss, attributed to fiber necrosis in the absence of regeneration, was as great in animals irradiated at 15 weeks as in those irradiated at 16 days. Such persistence of muscle fiber necrosis contradicts the standard view of the mdx mouse and establishes it as a closer model of Duchenne muscular dystrophy than is generally appreciated.

Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source.

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.

A new blocking method for application of murine monoclonal antibody to mouse tissue sections.

Antigen detection with primary antibody of the same species as the test tissue is complicated by high levels of background staining when indirect immunohistochemical detection methods are used. This severely limits the use of murine monoclonal antibodies on tissues of the mouse, the most widely used experimental model system; no method for blocking this is fully satisfactory. Here we show that background staining encountered in this system results largely from the binding of secondary antibodies via both Fc and Fab to endogenous immunoglobulins and other tissue components. A simple and efficient blocking strategy was established, employing papain-digested whole fragments of unlabeled secondary anti-mouse Igs enriched with Fc fragment of the same Igs. We have used this method to visualize dystrophin, an antigen expressed at low level, in revertant fibers of mdx mouse by both immunoperoxidase and immunofluorescence methods. In combination with the use of a biotin-streptavidin immunohistochemical detection protocol with biotinylated anti-mouse F(ab')2 as second layer, we eliminated the heavy background in this system and achieved strong signal amplification to demonstrate the specific antigen clearly. Double labeling with one mouse antibody and one antibody from another species was performed without signal interference. This principle can be adapted for wider applications, such as antibodies of other species on homologous tissues and perhaps where high background is found with heterologous antibodies. (J Histochem Cytochem 46:977-983, 1998)

Dual regulation of the AMP-activated protein kinase provides a novel mechanism for the control of creatine kinase in skeletal muscle.

The AMP-activated protein kinase (AMPK) is activated by a fall in the ATP:AMP ratio within the cell in response to metabolic stresses. Once activated, it phosphorylates and inhibits key enzymes in energy-consuming biosynthetic pathways, thereby conserving cellular ATP. The creatine kinase-phosphocreatine system plays a key role in the control of ATP levels in tissues that have a high and rapidly fluctuating energy requirement. In this study, we provide direct evidence that these two energy-regulating systems are linked in skeletal muscle. We show that the AMPK inhibits creatine kinase by phosphorylation in vitro and in differentiated muscle cells. AMPK is itself regulated by a novel mechanism involving phosphocreatine, creatine and pH. Our findings provide an explanation for the high expression, yet apparently low activity, of AMPK in skeletal muscle, and reveal a potential mechanism for the co-ordinated regulation of energy metabolism in this tissue. Previous evidence suggests that AMPK activates fatty acid oxidation, which provides a source of ATP, following continued muscle contraction. The novel regulation of AMPK described here provides a mechanism by which energy supply can meet energy demand following the utilization of the immediate energy reserve provided by the creatine kinase-phosphocreatine system.

Aging normal and dystrophic mouse muscle: analysis of myogenicity in cultures of living single fibers.

In the early stages of Duchenne muscular dystrophy, chronic muscle degeneration is counterbalanced by regeneration whose progressive failure beyond the fifth year is attributed to an accelerated senescence associated with excessive myogenic cell division. This idea was tested by counting the numbers of myogenic cells accumulating over 90 h around individual living fibers isolated from muscles of dystrophic (mdx) and normal mice, 14-550 days of age. In cultures of normal muscle fibers, the number of myogenic cells per fiber decreased with increasing age of the animal. Cultures from young mdx mice exhibited an age-related increase in myogenic cell number, attaining at 50 days three times the number in control cultures. Thereafter, myogenic cell number progressively declined with animal age to control values. Thus, there is no evidence that extensive myoblast proliferation in young dystrophic mice-and, by association, in Duchenne muscular dystrophy patients-depletes their myogenic responsiveness in later life when they become weak and wasted.

Selection and use of ligands for receptor-mediated gene delivery to myogenic cells.

Identification of myogenic cell targeting ligands is a critical step in the development of synthetic vectors for gene delivery to skeletal muscle. Here we describe the screening of six potential targeting ligands (insulin, insulin-like growth factor I, iron transferrin, gallium transferrin, alpha-bungarotoxin and carnitine) for their ability to bind dystrophin-deficient myotubes in vitro. Those ligands showing high levels of binding to myotubes were then tested on fully differentiated, isolated, viable myofibers. Of the ligands tested, transferrin showed the most promise based on high levels of binding to myogenic cells, high levels of receptor observed in regenerating fibers of patients with Duchenne muscular dystrophy and the ability to direct a large enzyme conjugate to the cytoplasm of myotubes. Finally, we show that incorporation of transferrin into an artificial virus consisting of poly-L-lysine-condensed DNA coated with a lipid shell (LPDII formulation) results in ligand-directed delivery of DNA to myogenic cells. This is the first report of gene transfer to myogenic cells using a ligand-directed synthetic vector. These results suggest that rational design of ligand-directed, fully synthetic, gene delivery vehicles is a viable approach to skeletal muscle vector development.

A dual-marker system for quantitative studies of myoblast transplantation in the mouse.

BACKGROUND: Myoblast transplantation (MT) is a potential approach for gene transfer into skeletal muscle, the efficiency of which depends upon the number of copies of donor genome incorporated into the host tissue. We have developed a system for quantitative studies of MT that measures amounts of donor-derived genome in host muscles and estimates the contributions of donor cell survival and proliferation in vivo. METHODS: [14C]thymidine-labeled, male myoblasts were transplanted into female muscles, providing two donor cell markers, Y chromosome and [14C]. The markers were measured in muscle extracts by slot blotting and scintillation counting, respectively. RESULTS: In each extract, the amount of Y chromosome was used to quantify donor-derived genome, whereas the radiolabel provided an estimate of cell survival. Furthermore, the different modes of inheritance of the markers meant that proliferation of surviving donor cells was detected as a change in marker ratio. CONCLUSIONS: This system provides a method for assessing potential improvements of MT.