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.
Poor survival of grafted cells is a major factor hindering the therapeutic effect of cell transplantation; however, the causes of cell death remain unclear. We hypothesized that interleukin-1beta ...(IL-1beta) might play a role in the acute inflammatory response and graft death after cell transplantation and that inhibition of IL-1beta might improve graft survival.
14C-labeled male skeletal muscle precursor cells were implanted into female mouse hearts by direct intramuscular injection. The amount of 14C-label provides an estimate of the surviving cell number, whereas the amount of male-specific Smcy gene measured by polymerase chain reaction indicates the total (surviving+proliferated) number of donor-derived cells. At 10 minutes after implantation, 44.8+/-2.4% of the grafted cells survived and this steadily decreased to 14.6+/-1.1% by 24 hours, and to 7.9+/-0.6% by 72 hours (n=6 in each point). Proliferation of the surviving cells, which began after 24 hours, resulted in an increase in the total cell number from 15.5+/-0.8% at 24 hours to 24.4+/-1.6% at 72 hours. Acute inflammation was prominent at 24 hours and was reduced by 72 hours, in parallel with IL-1beta expression. Administration of anti-IL-1beta antibody improved graft survival at both 24 (25.6+/-1.6%) and 72 hours (14.8+/-1.1%) and resulted in a 2-fold increase in the total cell number at 72 hours (45.8+/-2.4%). The effects of IL-1beta inhibition corresponded with a reduced inflammatory response.
IL-1beta is involved in acute inflammation and graft death after direct intramyocardial cell transplantation. Targeted inhibition of IL-1beta may be a useful strategy to improve graft survival.
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.
Whole muscle grafts were made between mdx and normal mice to investigate whether the mdx myopathic lesion is intrinsic to mdx muscle or is a property of its environment. Grafts were examined between ...20 and 101 days. Unequivocal necrotic muscle fibers and/or newly formed basophilic myotubes were noted in 8 of 16 grafts of mdx muscle made in normal hosts but in none of 16 grafts of normal muscle made in mdx hosts. In older grafts, the proportion of centrally nucleated fibers and variability of fiber diameter were both higher in mdx muscle grafted into normal hosts than in normal muscle grafted into either mdx or normal hosts. Analysis of the glucose-6-phosphate isomerase (GPI) isoenzyme content of the grafts indicated that the muscle formed was predominantly of donor origin. These findings provide evidence that the mdx lesion is a primary myopathy rather than secondary to an extramuscular primary lesion.
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.
Skeletal muscle myoblasts from different sources acquired high levels of the lysosomal enzyme beta-glucuronidase, when they were cultured together with mitogen-activated lymphocytes. ...Immunofluorescent staining, thermal stability, and electrophoretic mobility showed that the increase in enzyme activity in the myoblasts was due to the presence of the lymphocyte form of the enzyme. Although myoblasts were able to take up exogenous beta-glucuronidase from the culture medium by mannose 6-phosphate receptor-mediated endocytosis, enzyme acquisition during co-culture with lymphocytes was independent of this pathway. Enzyme transfer from the lymphocytes was found to require direct cell-cell contact with the muscle cells, and was accompanied by an increase in beta-glucuronidase activity in the lymphocytes themselves. Since this additional activity was also due to the presence of the lymphocyte form of the enzyme, these results indicate that interaction with the muscle cells induced the de novo synthesis of beta-glucuronidase in the lymphocytes.
Inheritance of the Blotchy allele at the X-chromosomal Mottled locus of mice results in changes in the lung which resemble emphysema. Previous studies using measurements of mean linear intercept have ...recognized emphysema in the hemizygous, Blo/Y, male and homozygous, Blo/Blo, female, but not in the heterozygous, Blo/+, female. The aim of this study was to develop a rapid, accurate method for analysis of emphysema and to establish whether it would identify emphysema in the heterozygote. Lungs from wild type and outbred Blo mice were inflated with fixative at a pressure of 25 cm and then sectioned and stained. Transect lengths across air spaces were measured using computerized image analysis and the results were plotted as histograms. Data were also expressed as cumulative frequencies (ogives) and subjected to statistical analysis by the non-parametric Kolmogorov-Smirnov test. Emphysema was shown by significantly increased (P less than 0.001) transect lengths in the Blo/Blo female and Blo/Y male compared with the wild type controls. The heterozygous Blo/+ mice form an intermediate group significantly different (P less than 0.001) from the wild and blotchy mice. The sensitivity of the method is illustrated by the fact that the Blo/+ female formed an intermediate group between wild type and the Blo/Y male and Blo/Blo female.
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