FACS Analysis and Immunohistochemical Analysis of Human Myogenic Stem Cell Number and Cell‐cycle Kinetics in Response to Acute Myotrauma

In humans, myogenic stem cell (SC) enumeration is an important measurement used to determine the SC response in vivo to various physiological stimuli. The current standard for enumeration is immunohistochemistry (IHC) with antibodies against common SC markers (i.e. Pax7, NCAM). Fluorescent activated cell sorting (FACS) analysis may provide a more accurate determination of changes in the SC pool and provide additional analysis unachievable with IHC. FACS analysis revealed Pax7+ cells/mg isolated from 50mg fresh tissue increased 36% 24h after injury. The number of Pax7+ cells/mg in G2/M phase of the cell cycle increased 202% after 24h and cells/mg in G1/G0 and S‐phase increased 32% and 59% respectively. IHC data illustrated, in relation to N‐CAM or C‐Met alone, Pax7 alone was expressed on a greater number of cells. Furthermore, all 3 markers appear to sufficiently and similarly report SC expansion after injury (26–36%). Here we illustrate the use of FACS as a precise method of enumerating SC number on a per milligram tissue basis, providing a more easily understandable relation to muscle mass as opposed to number of myonuclei or fiber number. Although IHC is a powerful tool for SC analysis, FACS is an objective, reliable and effective method for SC quantification and can provide additional information such as cell‐cycle kinetics more accurately than IHC alone.