Transplantation of myogenic cells has been discussed as a treatment for genetic disease, in muscle and other tissues (75,76), and also holds promise as a tool for investigating the regulation of expression of muscle genes in vivo. However, there are two major obstacles to full exploitation of these techniques: the finite mitotic capacity of primary myoblasts limits the cell numbers available from clones, whereas the tumorigenic tendency of established myogenic lines makes them difficult to use in vivo (77).
To generate cell lines with the above characteristics, J. Morgan and T. Partridge and their colleagues isolated conditionally immortal myoblast cell lines from the limb muscles of 19-d-old embryonic, and 10-d-old and 4-wk-old H-2KbtsA58 transgenic mice (78). Cell growth was sufficiently robust to allow the generation of clones directly by growth at low density. Clones exhibiting a typical myogenic morphology grew readily in the permissive conditions, but not in semipermissive conditions (of 33°C, IFN-y-negative). Of the clones chosen, 14 of 15 readily formed myotubes when grown at high density in nonpermissive conditions. Confluent cultures switched from permissive to nonpermissive conditions showed greatly reduced DNA synthesis and began to form myotubes within 24 h. The number of myotubes continued to increase for the next several days. Some fusion also occurred in permissive conditions if cultures were allowed to become very dense. In both cases, the resultant myotubes expressed dystrophin and muscle-specific myosin. Nonfused cells did not express muscle-specific myosin or dystrophin.
Cells from eight different clones were injected into the leg muscles of dystrophin-negative MDX nu/nu mice of the Gpi Isa isotype in which either the right or both legs had been subjected to 18G X-rays at 15-17 d of age. Irradiation inhibits the regeneration of mdx muscle, which eventually atrophies, thus presenting a good environment for the formation of new muscle from implanted myogenic cells (79,80).
Seven of eight clones injected in vivo formed new muscle histologically indistinguishable from that formed by injection of primary myoblasts (81), but did not form tumors in both irradiated and nonirradiated mdx muscles. No systematic difference in the ability to form muscle was seen between different clones, or between irradiated vs. nonirradiated legs.
Even after 19 passages (approx 60 doublings) in culture, cells remained dip-loid and formed normal muscle in the absence of tumor formation on injection into irrradiated mdx nu/nu muscle, in contrast to the neoplastic behavior of established muscle cell lines (77,82,83), no tumors were found up to 120 d postimplantation (78).
One striking aspect of the above experiments was that rare clones of H-2KbtsA58-derived cells could be reisolated from injected muscle after several weeks of in vivo growth, injected into a second generation of hosts (where the injected cells formed new muscle), reisolated again, and injected to form normal muscle in still a third generation of hosts. Clones were reisolated successfully from both irradiated and nonirradiated muscle. Only rare clones were obtained in these reisolation experiments, suggesting that myoblast cell lines derived from H-2KbtsA58 mice are able both to form myotubes and also to enter the pool of slowly dividing, or quiescent, myogenic cells known to exist in normal muscle.
In contrast with the problems of senesence and neoplasia associated with the use of primary cells or spontaneously arising cell lines, respectively, clonal myoblast lines derived from H-2KbtsA58 mice can be grown for extended periods in vitro without exhibiting any loss of conditionality or capability of undergoing normal differentiation in vitro or in vivo. Moreover, the ease of isolation of cell lines from H-2KbtsA58 mice of different ages raises the possibility of comparing the properties of myoblast lines representing different developmental stages.
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