Figure 2: Characterization of muscle stem cells in post mortem tissue.
From: Skeletal muscle stem cells adopt a dormant cell state post mortem and retain regenerative capacity
(a) Enumeration by flow cytometry based on GFP expression of the number of viable satellite cells in one TA muscle from Tg:Pax7-nGFP mice (control, 2720±270 satellite cells; 1980±210 cells at 4, and 1040±230 at 8 days post mortem; n=5 mice, each time point). (b) Evaluation of viable cells in post mortem tissue by calcein incorporation (marker for all viable cells; n=7 mice), and satellite cells from Tg:Pax7-nGFP mice based on GFP expression (n=5 mice). (c) Enrichment of Pax7-expressing cells between day 0 and 4 post mortem indicated as percentage of X-gal+ cells from Pax7nlacZ/+ animals, stained immediately in culture after isolation (n=10 mice). (d) Comparison of loss of satellite cells (Pax7-nGFP+ cells—black line) versus CD34+/Pax7-nGFP-/CD45- cells (containing fibroblasts, endothelial cells and non-satellite stem cells—grey line) (271,000±500 GFP+ cells versus 853,000±158,000 CD34+ cells at day 0 and 92,000±15,000 GFP+ cells versus 237,000±43,000 CD34+ cells 4 days post mortem) (n=3 mice). (e, f) Vimentin-positive fibroblasts cultured from CD34+/Pax7-nGFP-/CD45- cell fraction after sorting (n=3 mice per condition) (e) day 0 post mortem (f) 4 days post mortem; Bar, 50 μm. (g) Percentage of clonogenicity (number of cells forming colonies) after FACS and plating in 96-well dishes at day 0, 4 and 8 post mortem (n=5 mice per condition). (h) Evaluation by videomicroscopy (3% O2, 5% CO2) of time required to perform the first mitosis in cells from day 0, and 4 and 8 days post mortem (n=3 mice per condition). (i) Survival of quiescent or proliferating satellite cells post mortem. Satellite cells were enumerated by FACS from TA muscles of animals exposed to LPS (control, 6,100±1,700 satellite cells; 310±90 cells at 4 days post mortem; n=5 mice, each time point), or from Mdx4cv::Tg:Pax7-nGFP mice (7 months old) (control, 19,900±3,300 satellite cells; 2,400±390 cells at 4 days post mortem; n=9 mice, each time point), or following acute injury (5 days post-notexin injury) of Tg:Pax7-nGFP mice at day 0 or 4 days post mortem (pm) (control 5 days post-notexin, 103,000±8,500 satellite cells; 460±80 cells at 4 days post mortem; n=5 mice, each time point). Graphs indicate the percentage of viable satellite cells between day 0 and 4 days post mortem in the different cohorts. Statistical analysis was performed using paired or unpaired Student's t-tests, a minimum of 95% confidence interval for significance; P-values indicated on figures are <0.01 (**) and <0.001 (***). Figures display average values of all tests ± s.e.m.
