Use of fluorescent latex microspheres (FLMs) to follow the fate of transplanted myoblasts.

A potential treatment for Duchenne muscular dystrophy (DMD) is injection of normal myoblasts into dystrophic muscles to induce formation of muscle fibers. To develop this therapy it is important to identify the injected myoblasts and the muscle fibers that they form in the host muscles. Fluorescent latex microspheres (FLMs) were used for this purpose in this study. Normal myoblasts were labeled with FLMs and injected into dystrophin-deficient (mdx) mice. The FLMs clearly indicated the location of injected myoblasts in the host muscle. Muscle fibers containing dystrophin were localized by immunofluorescence and immunoperoxidase. They were observed in clusters near the myoblasts labeled with FLMs. FLMs were also observed in some of these dystrophin-positive fibers in each cryostat section. These results indicate that: labeling myoblasts with FLMs can be used to trace the injected myoblasts in the muscle and to identify the muscle fibers that they formed; injected myoblasts remain near the injected site and do not migrate very far; most of the dystrophin-positive muscle fibers around the injected myoblasts result from fusion of the injected myoblasts; and the low percentage of dystrophin-positive muscle fibers is likely related to limited diffusion and lack of fusion of many injected myoblasts.

Utilization of an antibody specific for human dystrophin to follow myoblast transplantation in nude mice.

Human myoblasts were transplanted in nude mice. The efficacy of these transplantations was analyzed using a monoclonal antibody (NCLDys3) specific for human dystrophin. This antibody did not reveal any dystrophin in nude mice that did not receive a human myoblast transplantation. However, about 30 days after a human myoblast transplantation, dystrophin-positive muscle fibers were observed. They were not abundant, and were present either in small clusters or isolated. This technique follows the fate of myoblast transplantation in animals that already have dystrophin, and distinguishes between new dystrophin-positive fibers due to the transplantation and the revertant fibers in mdx mice. Moreover, this technique does not require any labelling of the myoblasts before transplantation. It can also be used to detect dystrophin produced following the fusion of myoblasts transfected with the human dystrophin gene.

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.

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.

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.

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.

Proximodistal gradients of the postjunctional folds at the frog neuromuscular junction: a scanning electron microscopic study.

Frog endplates were studied with the scanning electron microscope following the removal of the presynaptic terminal by collagenase and acid treatments. Endplates had 2-14 branches of primary cleft. The longest branches were parallel to the muscle fiber. Short branches oblique or perpendicular to the muscle fiber were also present near the central region of the endplates. The openings of postjunctional folds in the primary cleft were clearly visible at the bottom of the primary cleft and could be counted and measured. The longest primary cleft branches of each endplate were divided into segments of 20 microns (length corrected for shrinkage). The number of postjunctional folds per micrometer of primary cleft, the average postjunctional fold length (i.e. across the primary cleft) and the total postjunctional fold's length per micrometer of primary cleft were evaluated for each 20-microns segment of primary cleft. Negative proximodistal gradients were observed for these three parameters for the long branches of primary cleft, i.e. values were higher in the proximal region (near the motor axon) than in the distal region. These postsynaptic gradients probably reflect similar or smaller proximodistal presynaptic gradients for the active zones along the nerve.

Scanning electron microscopic study of the neuromuscular junction of dystrophic mice.

The structure of the end-plate regions of normal and dystrophic 3-month-old mice were studied by scanning electron microscopy after the presynaptic terminals were removed by hydrochloric acid treatment. Quantitative analysis revealed that the end-plate area correlated positively with the muscle fiber diameter in both the normal and dystrophic animals. However, the motor end-plate area was significantly smaller in the dystrophic mice. The total length of the primary cleft of an end-plate correlated positively with the end-plate area and with the muscle fiber diameter in both normal and dystrophic mice. However, the total length of the primary cleft of an end-plate was significantly shorter in dystrophic mice, especially in large-diameter muscle fibers. Finally, the end-plate of dystrophic mice was characterized by shorter primary clefts with less branching points. These changes of several morphometric characteristics of the postsynaptic membrane suggest that the functional denervation of the mouse dystrophic neuromuscular junction has a postsynaptic origin.