Immortalization of human myoblasts

The muscle stem cells known as myoblasts have therapeutic potential in the treatment of neuromuscular diseases such as Duchenne muscular dystrophy. Progress has been slow, however, because these cells don’t fare well in long-term culture, making it difficult to make stable genetic modifications to cells from patients. (Generally, genetic manipulation of culture cells relies on rare events, followed by expansion of a clone of cells that has acquired a modification of interest. Finite division potential limits clonal expansion, and by the time the cells are ready to transplant, they’re often close to replicative exhaustion).

To make matters worse, telomerase — the silver bullet of benign immortalization — isn’t sufficient to prevent myoblasts from lapsing into cellular senescence.

Happy news, then, from Zhu et al., who — in addition to having sneaked an impressively long title past the editors of Aging Cell — report that addition of just a single gene (along with telomerase) allows myoblasts to persist and divide in culture indefinitely:

Cellular senescence in human myoblasts is overcome by human telomerase reverse transcriptase and cyclin-dependent kinase 4: consequences in aging muscle and therapeutic strategies for muscular dystrophies

Cultured human myoblasts fail to immortalize following the introduction of telomerase. The availability of an immortalization protocol for normal human myoblasts would allow one to isolate cellular models from various neuromuscular diseases, thus opening the possibility to develop and test novel therapeutic strategies. The parameters limiting the efficacy of myoblast transfer therapy (MTT) could be assessed in such models. Finally, the presence of an unlimited number of cell divisions, and thus the ability to clone cells after experimental manipulations, reduces the risks of insertional mutagenesis by many orders of magnitude. This opportunity for genetic modification provides an approach for creating a universal donor that has been altered to be more therapeutically useful than its normal counterpart. It can be engineered to function under conditions of chronic damage (which are very different than the massive regeneration conditions that recapitulate normal development), and to overcome the biological problems such as cell death and failure to proliferate and migrate that limit current MTT strategies. We describe here the production and characterization of a human myogenic cell line, LHCN-M2, that has overcome replicative aging due to the expression of telomerase and cyclin-dependent kinase 4. We demonstrate that it functions as well as young myoblasts in xenotransplant experiments in immunocompromized mice under conditions of regeneration following muscle damage.

Why might myoblasts require the CDK in addition to telomerase? To biologists of senescence, this presents no great mystery: Replicative exhaustion, which runs out the telomere “clock” at the ends of chromosomes, is just one way to get to senescence. Other paths include detection of supraphysiological levels of growth signals (implying that the cell itself or a neighbor has dysregulated an oncogene) and the presence of persistent (i.e., irreparable) DNA damage. These stimuli have in common that they both identify cells that are at risk for neoplastic growth.

Thus, cells with perfectly functional telomeres can senesce as a result of genotoxic damage or other risk factors. If myoblasts are more prone to accumulating certain types of damage, or a lower threshold for oncogene-induced senescence, then they might well senesce with long telomeres. Since all senescence pathways converge at downregulation of cell-cycle progression factors, and in particular the suppression of the cyclin-dependent kinases, providing the CDKs ectopically allows myoblasts to overcome the senescence barrier.

Why the telomerase, then? I would speculate that the CDK merely alleviates a limiting factor in myoblast replicative potential. Once that limit is abolished, the cells are only free to divide until arrested by the telomere checkpoint, eventually triggered when telomeres drop below some critical length. Like most other cells, then, myoblasts need telomerase to divide indefinitely.

As the authors point out, indefinite culture of myoblasts will allow more thorough characterization of muscle stem cells from neuromuscular disease patients [Editorial interpolation: unless the problem in vivo happens to be premature senescence, in which case immortalizing myoblasts will hopelessly confuse some unlucky researcher.]. Furthermore, immortalized myoblasts could be much more readily genetically manipulated, raising the possibility that those suffering from genetic diseases might someday benefit from gene therapy on their own muscle stem cells.

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2 comments

  1. I’m skeptical of the therapeutic potential of myoblasts. Myoblasts don’t exist as a stable population in vivo and are therefor completely incapbable of replenishing the true muscle stem cell population (the satellite cell). The max potential of myoblasts is therefor limited to repair of acute muscle damage. It isn’t clear how long muscle can maintain itself w/o endogenous stem cell support (days? years? no one knows).

    As far as overcoming senescence by messing with CDK4… I’m not so sure about this being a great idea for therapies either. I’m pretty sure that cultured myoblasts undergo spontaneous genomic mutation as all kinds of wacky things happen to myoblasts in long term culture (at least the mouse myoblasts that I culture at 20% oxygen).

    I don’t think we’ll see much progress on therapies involving the ex-vivo manipulation of muscle stem cells until someone figures out how to maintain satellite cells in culture.

  2. okee —

    Thanks for the comment.

    I hear what you’re saying about mouse culture artifacts. In our lab, we don’t grow mouse cells at 20% oxygen anymore, but only at 3% — this prevents a weird kind of oxygen-specific arrest that is unrelated to telomere length, and (to judge from the secretory profile of the cells) is distinct from cellular senescence. A lot of the long-term culture problems with mouse cells of all kinds has to do with the increased sensitivity of this species to oxygen. I suspect that the problems that have arisen in your long-term culture of myoblasts is a special case of this general phenomenon.

    For more information on mouse cell culture and oxygen concentration, see Parrinello et al. The paper focuses on fibroblasts but our subsequent experience is that all types of mouse cells fare badly (and appear to either exhibit genomic instability or premature arrest) in atmospheric oxygen.

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