Rapid selection of donor myoblast clones for muscular dystrophy therapy using cell surface expression of NCAM.

This study describes an easy 3 step-procedure to prepare rapidly and at low cost, pure myoblast cell cultures from a normal muscle biopsy. Following collagenase and trypsin treatment of the tissue (step 1), dissociated cells were cloned at a density of 10 cells/ml in MCDB 120 medium (0.2 ml/well). Clones that grew were then tested for NCAM cell surface expression by cytofluorometric analysis (CFA) using Coulter CD56-PE monoclonal antibodies (step 2). Only those clones with more than 98% strongly labelled positive cells were expanded (step 3) for further trials in cell transfer therapy for dystrophic patients. Visualization of the pattern of NCAM expression was performed by immunoperoxidase assay, while the potential ability to form myotubes was confirmed by the observation of their formation within a period of 1 to 2 weeks. The 65% of the CD56+ clones in CFA were the same clones that proved to be myogenic with positive immunoperoxidase assay and myotube formation. This method avoids the fastidious and costly approach of cell sorting (whenever available), avoids contamination hazards due to many manipulations of the clones. Moreover this approach leads to a pure myoblast population free of any contaminating fibroblast which could contribute to connective tissue implement already deleterious in dystrophic patients.

Human myoblast transplantation: preliminary results of 4 cases.

Myoblasts from immunocompatible donors have been transplanted into the muscles (tibialis anterior, biceps brachii, and/or extensor carpi radialis longus) of 4 Duchenne patients in the advanced stages of the disease. Although no immunosuppressive treatment was used, none of the patients showed any clinical signs of rejection such as fever, redness, and inflammation. One patient transiently produced antibodies against the donor myoblasts as determined by cytofluorometric analysis. This patient and 2 others were shown to form antibodies against their donor's myotubes. Muscle biopsies of the injected tibialis anterior of 4 patients revealed that 80%, 75%, 25%, and 0% of the muscle fibers, respectively, showed some degree of dystrophin immunostaining. The contralateral noninjected muscles of the latter 3 patients did not contain any dystrophin positive fibers, while that of the first patient showed dystrophin expression in 16% of the fibers examined. Myoblasts were also injected into the extensor carpi radialis longus or the biceps brachii of these patients. A few months subsequent to injection, one patient was shown to have a 143% increase of strength during static wrist extension. This result must be interpreted with caution because a double-blind strength-measuring protocol was not used. Furthermore, we have noted that this change slowly decayed over time. The strength of 2 other patients was increased less remarkably (41% and 51%), while the strength of the fourth patient was unchanged.

A light and electron microscopic study of dystrophin localization at the mouse neuromuscular junction.

Duchenne muscular dystrophy (DMD) is characterized by a lack of dystrophin expression. Dystrophin is a 420 Kd protein localized in the muscle sarcolemma that most likely provides stability to the muscle plasma membrane. Neuromuscular junctions (NMJs) were localized by revealing either the acetylcholine receptors (AChRs) with alpha-bungarotoxin coupled with cascade blue or by revealing desmin, a protein found in higher concentration at the NMJs using immunochemistry. An accumulation of dystrophin was observed in normal mice by immunoperoxidase labelling at NMJs identified with these markers. Dystrophin was pinpointed on the postjunctional folds of NMJs by electron microscopy and was more abundant on the postjunctional membrane than on the remaining muscle membrane. Our observations are similar to previous observations suggesting that dystrophin may interact with the AChRs.

In vitro bromodeoxyuridine labeling of nuclei: application to myotube hybridization.

Rat myoblast nuclei were labeled with various concentrations of bromodeoxyuridine (BrdU), an analogue of thymidine, for 24 or 48 hr. Almost every myoblast was labeled with BrdU at concentrations between 10(-7) M and 10(-5) M. When the cells were labeled with 0.5 microM or more, the percentage of labeled cells remained over 90% and 80% at 2 and 5 days, respectively. However, when the cells were labeled with BrdU concentration lower than 10(-7) M the percentage of labeled nuclei decreased more rapidly with time. The BrdU-labeled cells were mixed with an unlabeled population to determine whether their capacity to fuse was reduced. At a BrdU concentration of 0.5 x 10(-6) M, labeled myoblasts fused to a similar extent as unlabeled myoblasts, and a high percentage of marked cells were still perceptively labeled after 5 days. In contrast, the fusion capacity of myoblasts incubated with more than 10(-6) M BrdU was inhibited after only few rounds of DNA synthesis. These myoblasts were eventually able to fuse, however, when the BrdU diminished in the DNA due to cell division. These results indicate that labeling with BrdU at a concentration of 0.5 x 10(-6) M and an incorporation time of 48 hr is optimal to obtain perceptible immunocytochemical staining without affecting myoblast fusion. Such BrdU immunolabeling could be used as a nuclear marker for hybridization studies.

Dystrophin expression in myotubes formed by the fusion of normal and dystrophic myoblasts.

Mdx mouse dystrophy is characterized by the absence in the muscle cytoplasmic membrane of a high molecular weight protein called dystrophin. A possible avenue for treatment of muscular dystrophies is to inject normal myoblasts in a dystrophic muscle to form hybrid muscle fibers. Hybrid myotubes were formed in vitro by the fusion of normal rat and dystrophic mouse (mdx) myoblasts. Staining with Hoechst dye 33258 permitted the clear distinction of mouse and rat nuclei. Immunostaining demonstrated that dystrophin was present over the entire membrane of all hybrid myotubes even when nuclei ratio normal/dystrophic was low.

Is dystrophin present in the nerve terminal at the neuromuscular junction? An immunohistochemical study of the heterozygote dystrophic (mdx) mouse.

Neuromuscular junctions (NMJs) were identified by revealing the presence of cholinergic receptors (AChR) with alpha-bungarotoxin coupled to the fluorescent dye cascade blue in 9- and 60-day-old normal and heterozygote mdx mice. Dystrophin was detected by an immunoperoxidase technique. All the muscle fibers of the normal animals observed in cross sections were immunoreactive for dystrophin and an accumulation of dystrophin was observed at all NMJs identified by alpha-bungarotoxin. In the 9-day-old mdx heterozygote animals, dystrophin positive, negative, and partially positive muscle cross sections were observed. Four different observations were made in these heterozygote animals on the coexistence of AChR and dystrophin. First, alpha-bungarotoxin sites (i.e., NMJs) were observed on dystrophin positive muscle fiber cross sections with an accumulation of dystrophin at these sites. Second, alpha-bungarotoxin sites were observed on dystrophin positive fibers without a dystrophin accumulation at NMJs. Third, there was a coexistence of alpha-bungarotoxin and dystrophin labelling at NMJs of muscle fibers with perimeters labelling negative for dystrophin. Fourth, NMJs, identified by alpha-bungarotoxin, were observed on muscle fibers negative for dystrophin even at the NMJ. These observations suggest that dystrophin is present not only in the muscle membrane but also in the presynaptic nerve terminals.

A new technique to identify hybrid myotubes in vitro without culture fixation.

Fluorescent latex microspheres (FLMs) were used to label myoblasts and to permit the observation of hybrid myotubes before culture fixation. This type of labeling did not affect survival, development, or fusion of these cells. The FLMs were retained for several weeks. Labeled mouse myoblasts were co-cultured with unlabeled rat myoblasts to verify whether the marker was released and spread from labeled to unlabeled cells. The nuclear stain Hoechst 33258 was used to distinguish the myoblasts from both species and permitted the demonstration that there was virtually no re-uptake. Hybrid myotubes were also obtained by co-culturing mouse myoblasts containing rhodamine FLMs and rat myoblasts containing green FLMs. These mixed cultures were observed repeatedly with a fluorescent microscope without any cytotoxic effect. Several myotubes were observed before fixation of the cultures to contain both types of fluorescent labels. Subsequent fixation and staining with Hoechst dye confirmed that these myotubes were hybrids.