Diana J. Watt

Muscle stem cells

Since its discovery four decades ago, the satellite cell of skeletal muscle has been implicated as the major source of myogenic cells involved in growth and repair of muscle fibres. This review not only looks at the role of the satellite cell in these processes but discusses how cells derived from other sources and tissues have recently been implicated in muscle formation and regeneration. Muscle itself also yields cells that contribute to other cell lineages although it is currently debated as to whether these cells originate within muscle or have migrated there from other tissues. The reality of using cells from muscle or other tissues to repair diseased muscle fibres is also addressed.

The movement of muscle precursor cells between adjacent regenerating muscles in the mouse

SummaryRegeneration of mature skeletal muscle fibers involves the formation of new multinucleate muscle fibres by the fusion together of mononucleate muscle precursor cells. Such precursor cells appear to be largely or entirely derived from satellite cells, located between the basement membrane and the sarcolemma of the muscle fibre. We have previously presented evidence that precursor cells which contribute to regenerating muscle in a region of muscle damage are not all locally derived but that some migrate in from exogenous sources. The present study examines the possibility that a regenerating muscle might receive muscle precursor cells from neighbouring muscles. To do this we have made whole muscle allografts in the mouse and used the two murine isoenzyme allotypes of the dimeric enzyme Glucose-6-Phosphate Isomerase (GPI) as markers to demonstrate whether there is movement of muscle precursor cells between these allografts and adjacent host muscles. In host muscles adjacent to some allografts, a “hybrid” form of GPI was detected, each molecule consiting of one donor and one host GPI subunit. Such heterodimers can form only where host and donor nuclei share a common cytoplasm: in muscles this means that mosaic host/donor muscle fibres are present. The presence of such fibres implies that muscle precursor cells must have migrated into the host muscle from the neighbouring allograft.

Partial correction of an inherited biochemical defect of skeletal muscle by grafts of normal muscle precursor cells

We have attempted to use allografts of normal muscle precursor cells (mpc) to insert donor nuclei, containing a normal genome, into growing or regenerating skeletal muscle fibres of mice with an inherited deficiency of the enzyme phosphorylase kinase (PhK). Analysis of the glucose-6-phosphate isomerase (GPI) isoenzymes of treated muscles showed that myonuclei of donor origin became incorporated into host muscle fibres in 8 of 9 regenerating autografts, but PhK activity was found only in the 3 grafts into which the largest numbers (1-3 x 10(6)) of mpc had been implanted. Following injection of normal mpc into growing PhK-deficient skeletal muscle, mosaic fibres containing myonuclei of donor origin were detected in only 11 of 192 muscles examined from 64 mice, but, of these 11 muscles, 5 contained PhK activity detectable by two separate assays in a further 4 muscles activity was detected by one or other assay.

Cyclosporin A as a means of preventing rejection of skeletal muscle allografts in mice.

Isografts and allografts of skeletal muscle inserted into the limbs of mice initially degenerate. After some 5 to 8 days newly formed myotubes appear in the graft which develop into mature muscle fibers. In nontolerant hosts allografts are rejected between the 10th and 12th days. In mice treated with cyclosporin A, this effect persists for some 12 days after the end of treatment. Isoenzyme marker studies indicate that the regenerated graft is composed of both host and donor tissue. Donor isoenzyme does not persist when grafts are rejected.

FACTORS WHICH AFFECT THE FUSION OF ALLOGENEIC MUSCLE PRECURSOR CELLS IN VIVO

Watt D.J. (1981) Neuropathology and Applied Neurobiology 8, 135–147

The muscle-specific marker desmin is expressed in a proportion of human dermal fibroblasts after their exposure to galectin-1

We have previously shown that galectin-1 is a factor capable of converting mouse dermal fibroblasts to the myogenic lineage [Cell Transplant 2000;9:519]. Here, we report that human dermal fibroblasts are also capable of expressing the myogenic marker, desmin, when grown in muscle-cell-conditioned media. Furthermore, the human foetal skin cells also express this marker when grown in the presence of galectin-1. These results highlight the importance of galectin-1 in the conversion of both human and murine skin cells to a myogenic lineage. Thus galectin-1 could be an important tool for use in autologous cell therapies for the treatment of human muscular dystrophies.

Dermal fibroblasts participate in the formation of new muscle fibres when implanted into regenerating normal mouse muscle.

Both in vitro and in vivo studies have described the conversion of fibroblasts to myogenesis when in the presence of dysfunctional myogenic cells. Myogenic conversion of fibroblasts subjected to a normal, as opposed to a diseased muscle environment has only been reported in vitro. The primary aim of this work was to determine if fibroblasts can convert to a myogenic lineage and contribute to new fibre formation when implanted into the regenerating muscle of a normal mouse. Dermal fibroblasts were prepared from neonatal mouse skin and labelled prior to implantation with the fluorescent nuclear marker 4′,6‐diamidino‐2‐phenylindole (DAPI). Cells were implanted into muscles of host mice that had been subjected to either cold/crush or minced muscle injury. Some host muscles were x‐irradiated to deplete the muscle of endogenous muscle precursor cells. Muscles were removed at 3 wk postimplantation and analysed both histologically and for the presence of DAPI labelled nuclei. Fibres containing DAPI labelled central nuclei indicated that the implanted cells had participated in the regenerative process. Mouse dermal fibroblasts therefore do contribute to muscle fibre formation in regenerating normal mouse muscle but the extent of their contribution is dependent on the nature of the trauma induced in the host muscle. The study also showed that regeneration was more successful in muscles which had not been irradiated, which is contrary to the previous studies where dermal fibroblasts were introduced into myopathic mouse muscle.

A factor implicated in the myogenic conversion of nonmuscle cells derived from the mouse dermis

Using the mdx mouse model for human Duchenne muscular dystrophy we have shown that a cell population residing in the dermis of C57B1/10ScSn mouse skin is capable of converting to a myogenic lineage when implanted into the mdx muscle environment. It was important to determine the characteristics of the converting cell. A previous in vitro study indicated that 10% of cells underwent conversion but only when the cells were grown in medium previously harvested from a myogenic culture. In the present study we cloned cells derived from the dermis to identify the converting cells. Clones grown in normal growth medium showed no conversion, but when grown in medium conditioned by muscle cells around 40% conversion was achieved in several individual clones. We investigated whether the protein β-galactoside binding protein (βGBP), which is secreted by myoblasts and acts as a cell growth regulator of fibroblasts, could be a candidate factor responsible for conversion. Medium harvested from COS-1 cells infected with a construct containing βGBP has been used for this investigation. Growth of dermal fibroblasts in medium enriched with this factor showed a high rate of conversion to cells expressing muscle-specific factors.

Allografts of muscle precursor cells persist in the non-tolerized host

Implantation of normal muscle precursor cells into myopathic fibres to alleviate recessively inherited diseases of skeletal muscle has received much attention since the discovery of a defective or deficient gene coding for the protein dystrophin in the Duchenne and Becker forms of muscular dystrophy. Therapeutic allografting of cells would require some means of preventing their immune rejection. Here we have allografted muscle into the non-tolerant and non-immunosuppressed murine host. Precursor cells introduced in the form of a single cell suspension survive for prolonged periods post-implantation. Allografts of minced muscle often failed to survive, even though host and donor were compatible at the major histocompatibility locus. Differences at minor loci may well have contributed to such rejection. Where allografted tissue was rejected, there was a decrease in the amount of surviving host muscle at the graft site, an important observation in terms of the therapeutic implantation of cells.

Myoblast transplantation in inherited myopathies

Amongst the different forms of disease of human skeletal muscle are those where the lesion and subsequent pathological state is manifest within the muscle tissue itself: these are termed primary myopathies. Within this group are the muscular dystrophies, in which there is progressive degenerative wasting of striated musculature. The most debilitating of the muscular dystrophies is the X-linked Duchenne muscular dystrophy, which has an incidence of 1 in 3000 males at birth. In this disease, atrophy and loss of fibres from both locomotory and respiratory muscles results in their replacement by fibrous connective tissue and fat. There is regeneration of skeletal muscle, but this is insufficient to compensate for the degeneration. Affected boys become progressively weaker, are usually wheelchair-bound by their early teens and die in their late teens or early twenties (Walton and Gardner-Medwin, 1974).