Disciplining the Stem Cell into Myogenesis
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《新英格兰医药杂志》
Naiveté is a much lauded but equivocal virtue of the stem cell that implies both versatility and — if this quality is to be exploited to clinical advantage — the need for firm discipline. A report by Dezawa and colleagues1 describes the systematic induction of differentiation of adult mesenchymal stem cells from the bone marrow of rats and humans into myoblasts.
The authors used a fairly straightforward protocol of exposing the cells to a cocktail of cytokines known to be promyogenic and then transfecting the cells with the active part of the Notch gene, which is involved in decision making during cell differentiation. The result was large numbers of cells that were highly myogenic in vivo (Figure 1). This is a major step after nearly continuous attempts to apply a cell-based therapy to skeletal muscle. It provides, in principle, solutions to two separate obstacles to this type of therapy: the need for a readily available supply of highly myogenic cells and a means of distributing and integrating them into the large bulk of widely dispersed skeletal muscle in the body.
Figure 1. Stem Cells — From Marrow to Muscle.
In a recent study by Dezawa et al.1 that showed the efficient conversion of stem cells from bone marrow to skeletal-muscle cells, such stem cells from humans were exposed to a combination of growth factors and then transfected with a gene construct that appears to commit the cells to the muscle lineage. The cells were observed to fuse in tissue culture, forming elongated, multinucleate cells that resemble myotubes and produce muscle-specific proteins. Before the cells fused, they were labeled with a gene that produces a green fluorescent protein and then were injected into the gastrocnemius muscle of a murine model of muscular dystrophy (one lacking dystrophin) that was selected for immunodeficiency and had previously been treated with a muscle-damaging agent, cardiotoxin. Musclebiopsy specimens obtained two weeks after injection showed muscle fibers made up of cells that contained the green fluorescent protein and produced human dystrophin (shown in red). In this study, similar results were obtained when labeled human cells were injected intravenously into a murine model of muscular dystrophy. These experiments showed that the damaged muscle was regenerated by the transplanted cells.
Early attempts to apply a cell-based therapy to skeletal muscle involved the use of myoblasts derived from endogenous satellite cells, stem cells that adhere to each muscle fiber. Myoblasts accomplish a remarkably rapid and complete regeneration of severely damaged muscle by proliferating and fusing with one another and with the damaged fibers. By analogy with bone marrow transplantation, in which a small number of stem cells fully reconstitute a hematopoietic system, it has been proposed that myogenic cells be used to rebuild damaged muscle in patients with genetic diseases such as Duchenne's muscular dystrophy. Myogenic cells from healthy donors or autologous cells that have been corrected genetically to express the missing gene could act as vectors to introduce copies of the normal gene into the damaged, multinucleated muscle fibers as they fuse with the fibers during the process of repairing them.
This general procedure was validated some years ago2 by the grafting of myogenic cells from skeletal muscle into muscles in a murine model of the most common form of muscular dystrophy in humans, Duchenne's muscular dystrophy, which is characterized by a lack of the muscle protein dystrophin. Dystrophin was shown to be present in the regenerated muscle fibers. However, the effect was local, and it became clear that endogenous myogenic cells, like the cells in most other solid tissues, do not migrate over distances of more than a few millimeters from the injection site. Disappointingly, despite numerous efforts to improve the dispersion of myoblasts, this has remained a refractory problem that is evident even in the most recent trials of myoblast transplantation in human volunteers.3
The difficulty of obtaining sufficient cells to graft is exacerbated by the rapid and unexplained death of the vast majority of grafted myoblasts within the recipient muscle; the donor muscle fibers derive from a rapidly proliferating minority of surviving cells.4 In cases in which it is possible to calculate the efficiency of the graft, the total yield of muscle from grafted myoblasts is usually less than would be gained from the direct conversion of the grafted cells into skeletal-muscle fibers. This inefficiency is a major problem with the use of primary muscle cells; their extensive expansion in tissue culture seems to be at least partly to blame.
Recently, attention has turned to the adult mesenchymal stem cell. A variety of protocols have been developed to isolate the rare cells that are capable of many cell divisions and of generating several alternative types of cells. One type of these stem cells, called the mesoangioblast because of its apparent derivation from endothelial cells, has generated particular interest, because it offers both a means of dispersing myogenic cells and a total yield of muscle sufficient for use in therapy. When injected intraarterially, mesoangioblasts become lodged in capillary beds downstream in the muscle. From these diffuse sites, the cells invade and progressively repair fibers in large, disparate regions of muscle. This procedure provides hope for the broad distribution of myogenic cells, but the intraarterial delivery must be repeated in order to treat a chronic disease such as muscular dystrophy.5
The methods of Dezawa et al.1 improve on this procedure in two ways. First, large numbers of cells committed to myogenesis can be produced by a simple conversion of cells from a readily accessible source. Second, when injected intravenously, appreciable numbers of these cells can reach damaged muscle, a finding that implies that they can pass fairly efficiently through the microvascular beds and that they are not filtered out by the lungs, liver, or spleen. What is not yet clear is how long these cells remain within the vasculature and for how long they are able to move into regions of damaged muscle. Ideally, the cells would circulate perpetually to combat the chronic muscle damage that characterizes most types of muscular dystrophy. There also remains the question of whether the minority of modified stem cells that are not myogenic are subject to the normal constraints on proliferation; usually, only a single rogue cell is needed to give rise to a tumor.
No potential conflict of interest relevant to this article was reported.
Source Information
From Généthon, Evry, France.
References
Dezawa M, Ishikawa H, Itokazu Y, et al. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science 2005;309:314-317.
Partridge TA, Morgan JE, Coulton GR, Hoffman EP, Kunkel LM. Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts. Nature 1989;337:176-179.
Skuk D, Roy B, Goulet M, et al. Dystrophin expression in myofibers of Duchenne muscular dystrophy patients following intramuscular injections of normal myogenic cells. Mol Ther 2004;9:475-482.
Beauchamp JR, Morgan JE, Pagel CN, Partridge TA. Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source. J Cell Biol 1999;144:1113-1122.
Sampaolesi M, Torrente Y, Innocenzi A, et al. Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science 2003;301:487-492.(Terry Partridge, Ph.D.)
The authors used a fairly straightforward protocol of exposing the cells to a cocktail of cytokines known to be promyogenic and then transfecting the cells with the active part of the Notch gene, which is involved in decision making during cell differentiation. The result was large numbers of cells that were highly myogenic in vivo (Figure 1). This is a major step after nearly continuous attempts to apply a cell-based therapy to skeletal muscle. It provides, in principle, solutions to two separate obstacles to this type of therapy: the need for a readily available supply of highly myogenic cells and a means of distributing and integrating them into the large bulk of widely dispersed skeletal muscle in the body.
Figure 1. Stem Cells — From Marrow to Muscle.
In a recent study by Dezawa et al.1 that showed the efficient conversion of stem cells from bone marrow to skeletal-muscle cells, such stem cells from humans were exposed to a combination of growth factors and then transfected with a gene construct that appears to commit the cells to the muscle lineage. The cells were observed to fuse in tissue culture, forming elongated, multinucleate cells that resemble myotubes and produce muscle-specific proteins. Before the cells fused, they were labeled with a gene that produces a green fluorescent protein and then were injected into the gastrocnemius muscle of a murine model of muscular dystrophy (one lacking dystrophin) that was selected for immunodeficiency and had previously been treated with a muscle-damaging agent, cardiotoxin. Musclebiopsy specimens obtained two weeks after injection showed muscle fibers made up of cells that contained the green fluorescent protein and produced human dystrophin (shown in red). In this study, similar results were obtained when labeled human cells were injected intravenously into a murine model of muscular dystrophy. These experiments showed that the damaged muscle was regenerated by the transplanted cells.
Early attempts to apply a cell-based therapy to skeletal muscle involved the use of myoblasts derived from endogenous satellite cells, stem cells that adhere to each muscle fiber. Myoblasts accomplish a remarkably rapid and complete regeneration of severely damaged muscle by proliferating and fusing with one another and with the damaged fibers. By analogy with bone marrow transplantation, in which a small number of stem cells fully reconstitute a hematopoietic system, it has been proposed that myogenic cells be used to rebuild damaged muscle in patients with genetic diseases such as Duchenne's muscular dystrophy. Myogenic cells from healthy donors or autologous cells that have been corrected genetically to express the missing gene could act as vectors to introduce copies of the normal gene into the damaged, multinucleated muscle fibers as they fuse with the fibers during the process of repairing them.
This general procedure was validated some years ago2 by the grafting of myogenic cells from skeletal muscle into muscles in a murine model of the most common form of muscular dystrophy in humans, Duchenne's muscular dystrophy, which is characterized by a lack of the muscle protein dystrophin. Dystrophin was shown to be present in the regenerated muscle fibers. However, the effect was local, and it became clear that endogenous myogenic cells, like the cells in most other solid tissues, do not migrate over distances of more than a few millimeters from the injection site. Disappointingly, despite numerous efforts to improve the dispersion of myoblasts, this has remained a refractory problem that is evident even in the most recent trials of myoblast transplantation in human volunteers.3
The difficulty of obtaining sufficient cells to graft is exacerbated by the rapid and unexplained death of the vast majority of grafted myoblasts within the recipient muscle; the donor muscle fibers derive from a rapidly proliferating minority of surviving cells.4 In cases in which it is possible to calculate the efficiency of the graft, the total yield of muscle from grafted myoblasts is usually less than would be gained from the direct conversion of the grafted cells into skeletal-muscle fibers. This inefficiency is a major problem with the use of primary muscle cells; their extensive expansion in tissue culture seems to be at least partly to blame.
Recently, attention has turned to the adult mesenchymal stem cell. A variety of protocols have been developed to isolate the rare cells that are capable of many cell divisions and of generating several alternative types of cells. One type of these stem cells, called the mesoangioblast because of its apparent derivation from endothelial cells, has generated particular interest, because it offers both a means of dispersing myogenic cells and a total yield of muscle sufficient for use in therapy. When injected intraarterially, mesoangioblasts become lodged in capillary beds downstream in the muscle. From these diffuse sites, the cells invade and progressively repair fibers in large, disparate regions of muscle. This procedure provides hope for the broad distribution of myogenic cells, but the intraarterial delivery must be repeated in order to treat a chronic disease such as muscular dystrophy.5
The methods of Dezawa et al.1 improve on this procedure in two ways. First, large numbers of cells committed to myogenesis can be produced by a simple conversion of cells from a readily accessible source. Second, when injected intravenously, appreciable numbers of these cells can reach damaged muscle, a finding that implies that they can pass fairly efficiently through the microvascular beds and that they are not filtered out by the lungs, liver, or spleen. What is not yet clear is how long these cells remain within the vasculature and for how long they are able to move into regions of damaged muscle. Ideally, the cells would circulate perpetually to combat the chronic muscle damage that characterizes most types of muscular dystrophy. There also remains the question of whether the minority of modified stem cells that are not myogenic are subject to the normal constraints on proliferation; usually, only a single rogue cell is needed to give rise to a tumor.
No potential conflict of interest relevant to this article was reported.
Source Information
From Généthon, Evry, France.
References
Dezawa M, Ishikawa H, Itokazu Y, et al. Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science 2005;309:314-317.
Partridge TA, Morgan JE, Coulton GR, Hoffman EP, Kunkel LM. Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts. Nature 1989;337:176-179.
Skuk D, Roy B, Goulet M, et al. Dystrophin expression in myofibers of Duchenne muscular dystrophy patients following intramuscular injections of normal myogenic cells. Mol Ther 2004;9:475-482.
Beauchamp JR, Morgan JE, Pagel CN, Partridge TA. Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source. J Cell Biol 1999;144:1113-1122.
Sampaolesi M, Torrente Y, Innocenzi A, et al. Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science 2003;301:487-492.(Terry Partridge, Ph.D.)