Jennifer E. Morgan

Muscle satellite cells.

Skeletal muscle satellite cells are quiescent mononucleated myogenic cells, located between the sarcolemma and basement membrane of terminally-differentiated muscle fibres. These are normally quiescent in adult muscle, but act as a reserve population of cells, able to proliferate in response to injury and give rise to regenerated muscle and to more satellite cells. The recent discovery of a number of markers expressed by satellite cells has provided evidence that satellite cells, which had long been presumed to be a homogeneous population of muscle stem cells, may not be equivalent. It is possible that a sub-population of satellite cells may be derived from a more primitive stem cell. Satellite cell-derived muscle precursor cells may be used to repair and regenerate damaged or myopathic skeletal muscle, or to act as vectors for gene therapy. CELL FACTS: (1) Number of cells in body: 2 x 10(7) to 3 x 10(7) myonuclei/g, 20-25 kg muscle in average man; 2 x 10(5) to 10 x 10(5) satellite cells/g, i.e. approximately 1 x 10(10) to 2 x 10(10) satellite cells per person. (2) Main functions: repair and maintenance of skeletal muscle. (3) Turnover rate: close to zero in non-traumatic conditions-high in disease or severe trauma.

A population of myogenic stem cells that survives skeletal muscle aging

Age‐related decline in integrity and function of differentiated adult tissues is widely attributed to reduction in number or regenerative potential of resident stem cells. The satellite cell, resident beneath the basal lamina of skeletal muscle myofibers, is the principal myogenic stem cell. Here we have explored the capacity of satellite cells within aged mouse muscle to regenerate skeletal muscle and to self‐renew using isolated myofibers in tissue culture and in vivo. Satellite cells expressing Pax7 were depleted from aged muscles, and when aged myofibers were placed in culture, satellite cell myogenic progression resulted in apoptosis and fewer total differentiated progeny. However, a minority of cultured aged satellite cells generated large clusters of progeny containing both differentiated cells and new cells of a quiescent satellite‐cell‐like phenotype characteristic of self‐renewal. Parallel in vivo engraftment assays showed that, despite the reduction in Pax7+ cells, the satellite cell population associated with individual aged myofibers could regenerate muscle and self‐renew as effectively as the larger population of satellite cells associated with young myofibers. We conclude that a minority of satellite cells is responsible for adult muscle regeneration, and that these stem cells survive the effects of aging to retain their intrinsic potential throughout life. Thus, the effectiveness of stem‐cell‐mediated muscle regeneration is determined by both extrinsic environmental influences and diversity in intrinsic potential of the stem cells themselves.

Human Fetal Mesenchymal Stem Cells as Vehicles for Gene Delivery

First‐trimester fetal blood contains a readily expandable population of stem cells, human fetal mesenchymal stem cells (hfMSCs), which might be exploited for autologous intrauterine gene therapy. We investigated the self‐renewal and differentiation of hfMSCs after transduction with onco‐retroviral and lentiviral vectors. After transduction with either a MoMuLV retrovirus or an HIV‐1‐based lentiviral vector carrying the β‐galactosidase and green fluorescent reporter gene, respectively, transgene expression, self‐renewal, and differentiation capabilities were assessed 2 and 14 weeks later. Transduction with the lentiviral vector resulted in higher efficiencies than with the MoMuLV‐based vector (mean, 97.7 ± 1.4% versus 80.2 ± 5.4%; p = .02). Transgene expression was maintained with lentiviral‐transduced cells (94.6 ± 2.6%) but decreased over 14 weeks in culture with onco‐retroviral‐transduced cells (48.3 ± 3.9%). The self‐renewal capability of these cells and their ability to undergo osteogenic, adipogenic, and myogenic differentiation was unimpaired after transduction with either vector. Finally, clonal expansion of lentivirally modified cells was expanded over 20 population doublings with maintenance of multiline age differentiation capacity. These results suggest that hfMSCs may be suitable targets for ex vivo genetic manipulation with onco‐retroviral or lentiviral vectors without affecting their stem cell properties.

Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration

Cell therapy for degenerative muscle diseases such as the muscular dystrophies requires a source of cells with the capacity to participate in the formation of new muscle fibers. We investigated the myogenic potential of human fetal mesenchymal stem cells (hfMSCs) using a variety of stimuli. The use of 5‐azacytidine or steroids did not produce skeletal muscle differentiation, whereas myoblast‐conditioned medium resulted in only 1%–2% of hfMSCs undergoing muscle differentiation. However, in the presence of galectin‐1, 66.1% ± 5.7% of hfMSCs, but not adult bone marrow‐derived mesenchymal stem cells, assumed a muscle phenotype, forming long, multinucleated fibers expressing both desmin and sarcomeric myosin via activation of muscle regulatory factors. Continuous exposure to galectin‐1 resulted in more efficient muscle differentiation than pulsed exposure (62.3% vs. 39.1%; p < .001). When transplanted into regenerating murine muscle, galectin‐1‐exposed hfMSCs formed fourfold more human muscle fibers than nonstimulated hfMSCs (p = .008), with similar results obtained in a scid/mdx dystrophic mouse model. These data suggest that hfMSCs readily undergo muscle differentiation in response to galectin‐1 through a stepwise progression similar to that which occurs during embryonic myogenesis. The high degree of myogenic conversion achieved by this method has relevance for the development of therapies for muscular dystrophies.

Age-related changes in replication of myogenic cells in mdx mice : quantitative autoradiographic studies

Cell replication in muscle was measured by tritiated thymidine (3H-TdR) incorporation and autoradiography, in mdx mice from 2-44 weeks of age. Pre-mitotic labelling (within 1 h of 3H-TdR injection) was determined in 16 mice aged from 15 to 300 days. In 30 further mdx mice, one leg was irradiated 1 h after 3H-TdR injection to block DNA synthesis. Post-mitotic labelling was measured in both legs 10-15 days later. Between 20 and 60 days of age a very high proportion (up to 2%) of muscle (satellite cell) nuclei were replicating pre-mitotically; from 80-300 days cell replication was detectable but at much lower levels. Centrally placed nuclei within muscle fibres appeared at 24 days, increased rapidly to 50% by 50-100 days, declining thereafter to 25% at 300 days. In post-mitotic samples, labelled myotubes and labelled peripheral muscle nuclei (satellite cell nuclei and myonuclei) appeared at 28 days and were present in the mdx muscles through to 310 days, indicating continued cell replication and muscle regeneration. Myogenic cell replication was both retarded and inhibited by irradiation. These data demonstrate that muscle cell replication in mdx mice commences at about 3 weeks of age, is maximal at 4-8 weeks, but continues at lower levels until at least 44 weeks.

Long-term persistence and migration of myogenic cells injected into pre-irradiated muscles of mdx mice

Experiments were conducted to study the fate(s) of normal muscle precursor cells (mpc) which had been injected into the muscles of mdx mice. Right legs of mdx nu/nu mice were X-irradiated (18 Gray), to inhibit the proliferation of host mpc. Normal mpc were injected into the tibialis anterior (TA) muscles of these legs and the non-irradiated, contralateral legs. In pre-irradiated legs injected with normal mpc, the number of dystrophin-positive fibres was similar at 35, 49 and at 250 days after injection, but the number of dystrophin-negative fibres was much less at the latter time point, indicating prolonged survival of dystrophin-positive muscle fibres. Non-injected muscles neighbouring the injected TA muscle rarely contained muscle of donor origin 49 days after injection, but frequently did so 250 days after injection. This indicates that some of the injected mpc must have retained the ability to proliferate, to migrate into a neighbouring muscle and to differentiate into new muscle for a considerable period after the original cell implant. In non-irradiated legs, the implanted normal mpc formed markedly fewer dystrophin-positive fibres than in the contralateral, irradiated muscle, and undertook little or no migration to adjacent muscles.

Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse

Normal muscle precursor cells, prepared by the enzymatic disaggregation of neonatal mouse muscle, were implanted into an area of regenerating muscle in a genetically different inbred strain. This was done in an attempt to determine, first, whether donor muscle precursor cells prepared in this way would fuse with the developing muscle fibres of the host; and second, whether in the mosaic muscle fibres thus formed donor as well as host genes were expressed. As markers of the host and donor genes we used the allelic isoenzyme variants of glucose-6-phosphate isomerase (GPI). In 43 out of 60 grafts we detected the presence of a hybrid isoenzyme intermediate between host and donor types. This hybrid indicated that donor muscle precursor cells had fused with regenerating host muscle cells, and had expressed their GPI genes within the resulting mosaic muscle fibres. We have developed this technique with a view to inserting normal genes into genetically abnormal myopathic muscle.

Muscle precursor cells injected into irradiated mdx mouse muscle persist after serial injury

Muscle of donor origin was formed after implantation of H‐2Kb‐tsA58 muscle precursor cells (mpc) into irradiated mdx nu/nu mouse muscles. A series of injections of the myotoxin, notexin, which destroys mature muscle fibers but spares muscle precursor cells and other tissues, was made into the mpc‐injected muscles, leaving time for regeneration to occur between each injection. New muscle fibers of donor origin were formed after up to four notexin treatments, providing evidence that some of the implanted mpc reentered an undifferentiated, quiescent, stem cell‐like state and were capable of myogenesis after further injuries to the muscle. A similar model could be used to assay whether preparations of human mpc contain long‐lasting precursor cells, prior to their implantation into patients. In control mdx muscles, which had been irradiated, injected with tissue culture medium, and given three notexin injections, regeneration also occurred, indicating that radiation‐resistant mpc were present, presumably within the treated muscle.

Are Human and Mouse Satellite Cells Really the Same

Satellite cells are quiescent cells located under the basal lamina of skeletal muscle fibers that contribute to muscle growth, maintenance, repair, and regeneration. Mouse satellite cells have been shown to be muscle stem cells that are able to regenerate muscle fibers and self-renew. As human skeletal muscle is also able to regenerate following injury, we assume that the human satellite cell is, like its murine equivalent, a muscle stem cell. In this review, we compare human and mouse satellite cells and highlight their similarities and differences. We discuss gaps in our knowledge of human satellite cells, compared with that of mouse satellite cells, and suggest ways in which we may advance studies on human satellite cells, particularly by finding new markers and attempting to re-create the human satellite cell niche in vitro.

A novel morpholino oligomer targeting ISS-N1 improves rescue of severe spinal muscular atrophy transgenic mice.

In the search for the most efficacious antisense oligonucleotides (AOs) aimed at inducing SMN2 exon 7 inclusion, we systematically assessed three AOs, PMO25 (-10, -34), PMO18 (-10, -27), and PMO20 (-10, -29), complementary to the SMN2 intron 7 splicing silencer (ISS-N1). PMO25 was the most efficacious in augmenting exon 7 inclusion in vitro in spinal muscular atrophy (SMA) patient fibroblasts and in vitro splicing assays. PMO25 and PMO18 were compared further in a mouse model of severe SMA. After a single intracerebroventricular (ICV) injection in neonatal mice, PMO25 increased the life span of severe SMA mice up to 30-fold, with average survival greater by 3-fold compared with PMO18 at a dose of 20u2009μg/g and 2-fold at 40u2009μg/g. Exon 7 inclusion was increased in the CNS but not in peripheral tissues. Systemic delivery of PMO25 at birth achieved a similar outcome and produced increased exon 7 inclusion both in the CNS and peripherally. Systemic administration of a 10-μg/g concentration of PMO25 conjugated to an octaguanidine dendrimer (VMO25) increased the life span only 2-fold in neonatal type I SMA mice, although it prevented tail necrosis in mild SMA mice. Higher doses and ICV injection of VMO25 were associated with toxicity. We conclude that (1) the 25-mer AO is more efficient than the 18-mer and 20-mer in modifying SMN2 splicing in vitro; (2) it is more efficient in prolonging survival in SMA mice; and (3) naked Morpholino oligomers are more efficient and safer than the Vivo-Morpholino and have potential for future SMA clinical applications.