Marrow Derived Muscle Colonies Are a Clonal Phenomenon.

We have previously established a model of plasticity of marrow to skeletal muscle that is based on transplantation of GFP positive marrow and cardiotoxin injury to the tibialis muscle. In these experiments, we have observed colonies of GFP positive muscle fibers, defined as 3 or more myocytes. We noted that individual GFP positive fibers showed marked heterogeneity of fluorescence. In sharp contrast, colonies of GFP+ cells, showed homogeneous fluorescence. This was confirmed by using confocal microscopy and by measuring the mean fluorescent intensity of the individual cells in the colonies. When the intensity of the green fluorescence in individual fibers was graded to dull, moderate or bright, in 95% of the colonies counted, showed homogenous intensity of green fluorescence while for single GFP fibers dispersed throughout the specimen the probability of 3 cells being the same color was only 14.12%. Therefore the probability of 95% of colonies to have a minimum of 3 homogenous fibers by random association is less than 0.0001. To show that colonies are not from clumps of cells injected into the muscle, we compared the number of the colonies in two different experimental setting where in both GFP positive marrow cells were injected intravenously after 900cGy of radiation followed by cardiotoxin injury to the muscle. Animals who received two cycle of G-CSF mobilization were compared with those who had lineage negative marrow cells directly injected into their muscle, one day after injury. The incidence of muscle colonies remained the same between the groups. The incidence increased in experiments with multiple cycles of mobilization and injury and also in mdx mouse that has spontaneous ongoing regeneration of its muscles. Cotransplantation of marrow cells from yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) transgenics into C57BL/6 mice followed by cardiotoxin injury showed colonies that were either from YFP or CFP cells. No mixed colonies were found. Transplantation of YFP marrow cells before injury followed by CFP cells after injury and vice versa showed colonies only from cells that were infused at the time of transplantation. Colonies were seen only in samples that received CD45, C-Kit and/or Sca positive marrow cells and not in those negative populations. These cells resulted in a GFP+ CD45 negative population 4 weeks after transplantation and produced Myf5+ and MyoD+ myoblasts three days after injury. These data suggests that marrow derived muscle colonies are a clonal phenomenon similar to CFU-S cells in recipient spleens after transplantation.