Figure 2. Isolation and stress resistance capacity of ALDH^hi myoblasts.

(a, b) ALDH^hi murine myoblasts were isolated via fluorescence activated cell sorting (FACS) from a heterogeneous population of skeletal muscle derived cells using DEAB, a potent inhibitor of ALDH, as a gating control. (c, d) A significantly increased rate of proliferation of ALDH^hi myoblasts compared to ALDH^lo myoblasts was observed in conditions of oxidative stress (H₂O₂, 250 µM) (* indicates p<0.05) and inflammatory stress conditions (TNF-α, 2.5 ng/ml) (n = 9). (e) ALDH^lo and ALDH^hi myoblasts underwent myogenic differentiation by fusing into MHC+ myotubes (red) under oxidative stress conditions (H₂O₂, 250 µM). Nuclei were stained with DAPI (blue). (f) Significantly increased myogenic differentiation indices (MDI) were observed in ALDH^hi cells at several concentrations of oxidative stress (0, 250 and 500 µM) when compared to ALDH^lo (n = 3). (g) Similarly increased MDIs of ALDH^hi cells were observed under all inflammatory stress conditions when compared to ALDH^lo myoblasts. (h) An increased number and density of dystrophin positive myofibers (stained in red) were observed in mdx mice injected intramuscularly with ALDH^hi cells compared to those injected with ALDH^lo myoblasts. Nuclei are DAPI (blue) stained. Green FluoSphere beads are observed to localize at the area of initial injection. (i) A significantly increased regeneration index was observed in ALDH^hi transplanted mice compared to those injected with ALDH^lo and unsorted cells. (n = 3) (j, k) Measurements of intracellular antioxidants glutathione (GSH) and superoxide dismutase (SOD) levels demonstrated statistically significant elevation of both antioxidants in ALDH^hi myoblasts compared to ALDH^lo myoblasts (n = 3).