Claude Labrecque

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.

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.

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.