Purpose To examine whether adipogenic cells may be prospectively isolated from skeletal muscle and to perform preliminary characterization of these cells.
Introduction Whether due to aging, diabetes, obesity, or other pathologies, the disregulation of adipocyte growth and maturation presents a major obstacle for human health. The fat content of skeletal muscle can increase with age or with muscle deterioration, as in Duchenne's muscular dystrophy. Efforts to isolate muscle stem cells (satellite cells) often rely on retrospective analysis, but there have been several reports of adipogenesis arising from these satellite cell populations, the reasons for which remain unclear.
Methods and Results To determine whether adipogenic and myogenic activities may be resolved at the cellular level, we performed several experiments using freshly dissociated hind limb skeletal muscles from 8- to 12-week-old mice. Using flow cytometry and fluorescence-activating cell sorting (FACS), we have prospectively isolated adipogenic progenitors (AP) as a population of small, Hoechstmid, propidium iodide (PI)low, CD45−, CD31−, CD34+, and Sca1+ cells. The AP population is distinct from the myogenic progentors (MP), which comprise a population of Hoechstmid, propidium iodide (PI)low, CD45−, CD31−, CD34+, and Sca1− cells. After 8 to 12 days in culture, the AP and MP populations spontaneously differentiated into lipid-laden adipocytes and contracting myotubes respectively. qRT-PCR analysis reveals that AP cells express higher levels of adipocyte-specific transcripts (including PPAR-γ, C/EBP-β), compared with MP cells, which were observed to express myogenic transcripts (including Pax7 and Myf5). Colony-forming assays reveal adipogenesis and myogenesis at the clonal level, with a CFU-adipocyte occurring every 1/45 AP cells and a CFU-myotube every 1/54 MP cells.
Conclusion and Future Work These results are consistent with the notion that adipogenesis and myogenesis arise from two distinct cell types in adult skeletal muscle. We aim to further dissect the AP population using FACS to resolve adipose-forming cells at different developmental stages.
We wish to acknowledge the Canadian Institutes of Health Research and the Michael Smith Foundation for Health Research for their support.
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