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D J Watt

Publications and source records attributed to D J Watt.

34 records · Page 2Linked to original sources
PubMed1991

X-irradiation improves mdx mouse muscle as a model of myofiber loss in DMD.

The mdx mouse, although a genetic and biochemical homologue of human Duchenne muscular dystrophy (DMD), presents a comparatively mild histopathological and clinical phenotype. These differences are partially attributable to the greater efficacy of regeneration in the mdx mouse than in DMD muscle. To lessen this disparity, we have used a single dose of X-irradiation (16 Gy) to inhibit regeneration in one leg of mdx mice. The result is an almost complete block of muscle fiber regeneration leading to progressive loss of muscle fibers and their replacement by loose connective tissue. Surviving fibers are mainly peripherally nucleated and, surprisingly, of large diameter. Thus, X-irradiation converts mdx muscle to a model system in which the degenerative process can be studied in isolation from the complicating effect of myofiber regeneration. This system should be of use for testing methods of alleviating the myofiber degeneration which is common to mdx and DMD.

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PubMed1991

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.

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PubMed1990

Segregation of the myogenic cell lineage in mouse muscle development.

With increasing interest in the idea of therapeutic implantation of normal muscle precursor cells into muscle lacking the protein product of the dystrophin gene, it has become important to obtain enriched populations of myogenic cells from biopsied muscle sources. Myogenic cells for implantation are highly favoured as they are the only cells that will fuse readily with host muscle fibres into which they are implanted, thus carrying the introduced gene into the target fibre with the maximum of efficiency. Second, myogenic cells appear less immunogenic than those of a non-myogenic nature; and third, the use of mononuclear myogenic cells may permit the introduction of multiple copies of a deficient gene into the patient's own cells. From a mixed population of cells obtained by the enzymic disaggregation of neonatal murine muscle we have selected, utilising a modification of the panning technique, for a cell population rich in myogenic cells. Segregation was accomplished using Mab H28, an antibody to the mouse neuronal cell adhesion molecule (N-CAM), derived from mouse/rat hybridoma cells. Following incubation with Mab H28, disaggregated muscle was applied to the surface of a bacteriological grade dish previously coated with anti-rat immunoglobulin. Cells segregated into two populations; those bearing N-CAM, and hence labelled with Mab H28, were adherent to the dish, whereas those not expressing N-CAM remained in suspension. Use of this technique, which involves minimal cell loss, resulted in the segregation of prefusion myogenic cells together with fibroblasts in the 'non-adherent' fraction, whereas cells in the adherent fraction consisted of a highly enriched population of actively dividing myogenic cells.

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PubMed1988

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.

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PubMed1987

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

Regeneration of mature skeletal muscle fibres 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 consisting 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.

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PubMed1984

Long term survival of allografted muscle precursor cells following a limited period of treatment with cyclosporin A.

Previous work (Watt et al., 1982) has shown that, in the mouse, skeletal muscle can be transplanted successfully in the form of a suspension of it's mononucleate precursors. Eventual therapeutic application of this technique by the implantation of precursor cells derived from normal muscle into myopathic individuals would require a means of preventing allograft rejection applicable to man. We have therefore investigated the use of the drug cyclosporin A (CyA) as a means of prolonging the survival in mice of allografts of mononucleate muscle cells made into a region of regenerating host muscle. We have administered CyA to the hosts at doses of either 75 or 150 mg/kg body weight/day for 42 days from the day of grafting. By using isoenzyme allotypes as markers of host and donor tissues, we have shown that allografted mononucleate cells become incorporated in host muscle fibres and that the mosaic host/donor muscle fibres so formed survive for as long as we continued the experiment, a maximum of 107 days after grafting, or 65 days after the end of CyA treatment.

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PubMed1982

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.

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PubMed1981

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.

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PubMedDate not supplied

Use of mononuclear precursor cells to insert allogeneic genes into growing mouse muscles.

Our long-term aim is to use allografts of normal muscle precursor cells to insert donor myonuclei, containing a normal genome, into the growing defective muscle fibers of young animals with inherited myopathies. In the present article, we have used isoenzyme variants of glucose-6-phosphate isomerase (GPI) as genetic markers of host and donor genomes in the mouse. We show that mononucleated cells, prepared by enzymatic disaggregation of neonatal donor muscle, 39 can be used to introduce donor GPI genes into muscle fibers of growing muscles of young hosts. The expression of both donor and host GPI genes in the resulting mosaic muscle fibers leads to the formation of a "hybrid" GPI isoenzyme dimer. Such hybrid GPI was found in 10 of 102 host muscles examined. Our results indicate that grafts of muscle precursor cells can be used to alter the genetic constitution of both normal and mildly myopathic muscle.

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PubMedDate not supplied

Direct enzyme transfer from lymphocytes is specific.

Lymphocytes are known to interact directly with other cells in vivo and in vitro, and have recently been shown to transfer the lysosomal enzyme, beta-glucuronidase, to fibroblasts from patients with an inherited deficiency of the enzyme. This process requires cell-cell contact, is unaffected by inhibitors of 'classical' receptor-mediated endocytosis and is abolished by inhibitors of protein synthesis. Although it is not yet known to what extent the transfer of enzymes by direct cellular interaction is a general phenomenon, a similar mechanism could possibly be involved in the transfer of other lysosomal enzymes in vivo and in the exchange of protein in vitro. We show here that the direct transfer of enzymes from lymphocytes to fibroblasts is restricted to only certain lysosomal enzymes.

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PubMedDate not supplied

Factors which affect the fusion of allogeneic muscle precursor cells in vivo.

Skeletal muscle allografts in mice have been used to study the factors which promote the fusion in vivo of muscle precursor cells derived from different strains of mice. Tolerance of host mice to allogeneic donor tissues has been induced either by the injection of donor spleen cells into newborn recipients, or by the irradiation and reconstitution, with donor bone marrow cells, of the haemopoietic system of the thymectomized recipient. The isoenzymes of glucose-6-phosphate isomerase (GPI) have been used as markers to distinguish the contribution of host and donor tissues to these allografts. In 174 of 204 such grafts evidence of both host and donor tissues was found. In 57 grafts which yielded a "hybrid' isoenzyme of GPI, it was inferred that host and donor muscle cells had fused to form mosaic fibres. The following factors appear to influence the fusion of muscle precursor cells: a the presence within grafts of adequate numbers of precursor cells of both the host and the donor strains; b the particular strain-combinations used; c the immunological state of the host.

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PubMedDate not supplied

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 beta-galactoside binding protein (betaGBP), 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 betaGBP 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.

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