By David N. Leff

Muscles don¿t last forever.

Whether from injury, disease ¿ such as muscular dystrophy ¿ or simply aging, the body¿s muscle mass is heir to weakening and wasting. But there¿s an artifactual factor as well, a consequence of research itself, which can turn muscle into grease.

Cell and developmental biologist Marie Csete, at the University of Michigan, Ann Arbor, explains: ¿Oxygen (O₂) is an obvious environmental factor that has received little attention in culturing skeletal muscle progenitor cells. Too much oxygen can kill stem cells, slow their growth, even trigger an alternate developmental pathway that converts pre-muscle stem cells into adipose cells.¿

Csete, who teaches anesthesiology at the university medical school, is senior author of a paper in the November 2001 issue of the Journal of Cellular Physiology. Its title: ¿Oxygen-mediated regulation of skeletal muscle satellite cell proliferation and adipogenesis in culture.¿

¿Our overall finding is that these satellite stem cells, and other stem cells, are exquisitely sensitive to the oxygen environment around them,¿ Csete told BioWorld Today. ¿One unexpected experimental result,¿ she went on, ¿was that survival of mature muscle fibers increased in the physiologic 6 percent oxygen vs. non-physiologic 20 percent oxygen_, which is used in virtually all traditional cell-culture laboratory incubators.¿

To reach their conclusions, Csete and her co-authors performed an experiment in mice. ¿We derived the stem cells from the muscles in their hind legs,¿ she recounted. ¿This preparation was interesting, because we incubated single muscle fibers in culture, then allowed the stem cells to emerge from those fibers. So this process recapitulates what the stem cells do in muscle regeneration.

¿First, we affirmed their cellular characteristics, by looking through the microscope to see what kind of cell they made ¿ morphologically. Did it look like a muscle cell or a fat cell? Then we examined the characteristics of the daughter cells: What kinds of genes they expressed, what shape they assumed in culture. Our results,¿ Csete continued, ¿were that satellite stem cells are quite likely to not generate muscle, but under the influence of the local lab environment, make fat cells instead. That¿s the finding of our paper.¿

Taking On Gas Physiology Skeptics

¿I think that normal gas physiology,¿ she observed, ¿is just not part of what biologists are taught any more. So they assume that by handling their stem cells as they do in their labs the gases are taken care of. Very often, in response to our concerns, their reaction is: Boom! I¿ve been doing it this way for 30 years, and it¿s just fine.¿ But some people who looked at our data were concerned that since we can get some stem cells to make muscle, for example, in high or low oxygen, the pattern of gene expression is different, depending on the oxygen conditions.

¿Our initial hypothesis,¿ Csete observed, ¿was that if I lowered oxygen down to levels that were more reflective of the normal tissue environment in which stem cells are found ¿ 2 percent to 6 percent instead of 20 percent ¿ I would enrich the stem-cell pool. The fact that we can activate that pool specifically with gases was a nice surprise. Its significance,¿ Csete added, ¿is twofold: First, that traditional methods for culturing stem cells may actually be toxic. And second, that we can use gases to program stem cells to produce specific outcomes, without having to change anything else in the way they¿re cultured.

¿We are continuing this work,¿ Csete said, ¿to show that high oxygen is indeed associated with high oxidative stress. So our follow-on work, which is not published yet, raises all sorts of clinical flags. For one, high oxidative stress may be exactly the condition in muscle of very old people. So looking at stem cells in aging muscle, we¿re finding that they are reacting like high-oxygen cultures. They don¿t proliferate as well and are more likely to die. We¿re working under the hypothesis that they are also more likely to make fat.

¿What people know is that muscle mass is dramatically reduced as we age, and people do accumulate fat in their muscle. The assumption has been that this is a kind of nonspecific degenerative process. But I think that part of it at least may be an abnormal regeneration from these cells, under the influence of abnormal oxidative stress.

¿The presumption in President Bush¿s edict,¿ Csete pointed out, ¿is that the 72 human embryonic stem cell lines he released for research last week might make every kind of cell. That if we put them into a mother they would make a human.¿

Download Lab Oxygen ¿ Or Else

¿Our work really raises the caution that the way the embryonic stem cells [ESCs] are handled in the lab ¿ not only by oxygen but also exposure to serum, and all sorts of artificial things that we do to cultured cells ¿ may damage their abilities to efficiently generate certain kinds of daughter cells. As we understand how better to capture the normal environment of an ESC, we should preserve its function better.

¿The function of our adult myogenic stem cells,¿ she continued, ¿is specifically to replace skeletal muscle throughout life ¿ as we damage our muscle through the normal stress of wear and tear that we put it through. We suggest that they may also contribute to fat formation. In this way the satellite cell, which was thought to be directed only to muscle, is now starting to look more and more like what we call mesenchymal stem cells. Those are adult ¿ not embryonic ¿ stem cells, and I think they¿re probably the most abundant solid-organ stem cells late in life.

¿These cells are a potential source of replacement therapy in any situation where you lose muscle mass,¿ Csete pointed out. ¿People have tried to transplant these cells in various muscular dystrophies, and those cells have not been successful ¿ mostly for immunologic reasons. But if we understand the biology of the cells more, which I¿m hoping this work will do, then we can have a population of cells programmed to be a bit more efficient at replacing muscle.

¿What we¿re uncovering in our work,¿ Csete concluded, ¿is that this is a useful tool for identifying new pharmacologic targets for treating disease processes ¿ from obesity and diabetes to aging.¿