Abstract
A new conditionally immortal satellite cell-derived cell-line, H2K 2B4, was generated from the H2Kb-tsA58 immortomouse. Under permissive conditions H2K 2B4 cells terminally differentiate in vitro to form uniform myotubes with a myogenic protein profile comparable with freshly isolated satellite cells. Following engraftment into immunodeficient dystrophin-deficient mice, H2K 2B4 cells regenerated host muscle with donor derived myofibres that persisted for at least 24 weeks, without forming tumours. These cells were readily transfectable using both retrovirus and the non-viral transfection methods and importantly upon transplantation, were able to reconstitute the satellite cell niche with functional donor derived satellite cells. Finally using the Class II DNA transposon, Sleeping Beauty, we successfully integrated a reporter plasmid into the genome of H2K 2B4 cells without hindering the myogenic differentiation. Overall, these data suggest that H2K 2B4 cells represent a readily transfectable stable cell-line in which to investigate future stem cell based therapies for muscle disease.
Highlights
Primary myoblasts are routinely used as a cell model for muscle research
As previous conditionally immortal cell-lines have been generated from proliferating myogenic cells and not individual satellite cells, it is possible that the commonly used cell models do not exhibit all of the muscle stem cell qualities [8]
Within the first twenty-four hours myogenin expression was markedly upregulated and the number of nuclei expressing myogenin increased over the 96 hours, indicating that the majority of myoblasts had committed to terminal differentiation
Summary
Primary myoblasts are routinely used as a cell model for muscle research. their limited proliferation capacity in vitro hinders their ability to be used in repeated experiments and numerous isolations of primary cells are required. An alternative approach is the use of established muscle cell-lines which have extensive mitotic capacity whilst retaining their ability to terminally differentiate in vitro and in vivo. Studies conducting 2- and 3 dimensional cell culture experiments have shown differences in myotube maturation as well as myogenic and adhesion protein expression in C2C12 cells compared to primary myoblasts, highlighting the importance of using a suitable cell-line when investigating myogenic differentiation in vitro. Since 1994, conditionally immortal myoblasts have been routinely used as cell models for muscle research; extensive in vitro and in vivo characterisation on individual cell-lines has yet to be conducted. Whilst several studies have used conditionally immortal satellite cell derived cell-lines to address particular hypothesis, the clones were not extensively characterised in vitro prior to their used in vivo [11,12]
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