Figure 2: Measuring the number of MDSPCs isolated from murine muscle and their myogenic potential.

(a) A schematic diagram showing the early passage cells analysed in this figure and the method used to isolate various populations of MDSPCs (pp1–pp6). (b) Representative images of pp1–pp2 cells induced to undergo myogenic differentiation. Cells directly isolated from skeletal muscle of mice, using preplate technique (pp1–pp2), were incubated in fusion media to induce myogenic differentiation. Twenty-four hours post-isolation, adhering cells were trypsinized and plated at equal density and induced to undergo myogenic differentiation over 2–3 days. Cells from 2–3 independent populations of each genotype were immunostained for the terminal myogenic differentiation marker, f-MyHC (red). Scale bar=100 μm. (c) rtPCR to measure the expression of the myogenic differentiation marker, myogenin in pp1–pp2 cells, induced to undergo myogenic differentiation. (d) Quantification of Sca-1⁺/CD34⁺/CD45⁻ cells in young WT-, old WT-, and progeroid- (Ercc1^−/−, Ercc1^−/Δ) murine skeletal muscle. Twenty-four hours after isolation from muscle, the cells that did not adhere in preplate 1 and 2 (that is, pp3) were analysed for stem/progenitor cell markers by FACS. The number in the upper right quadrants indicates the percent of Sca-1⁺/CD34⁺/CD45⁻ cells isolated from 3–5 mice of each genotype/age. (e) Graph indicating the average fraction of Sca-1⁺/CD34⁺/CD45⁻ cells normalized to the weight of the cell pellet of pp3 cell populations. Error bars indicate ±s.d. *P<0.05, Tukey's test, relative to young WT cells. (f) Representative images of Sca-1⁺/CD34⁺/CD45⁻ -sorted cells plated at equal density and induced to undergo myogenic differentiation. The cells were stained for the terminal myogenic marker, f-MyHC (red). Scale bar=100 μm. (g) Quantification of myogenic differentiation of Sca-1⁺/CD34⁺/CD45⁻ cells isolated from the skeletal muscle of mice of various genotypes/ages measured as the percent of cells (DAPI, blue) expressing f-MyHC (red). Error bars indicate ±s.d. for cell populations isolated from 2–3 animals per genotype *P<0.001, Kruskal–Wallis ANOVA on ranks relative to young WT cells.