Figure 1. CD47 expression levels distinguish functionally and molecularly distinct aged muscle stem cell subsets.

(A) Cell surface marker analysis of MuSCs from Pax7-ZsGreen reporter mice²⁰. Histogram overlay shows CD47 expression in ZsGreen⁺ cells (blue) and the CD47 isotype control (gray). (B) CyTOF analysis of the MuSC population in TA and GA muscle isolated from young mice. Gated Live/Lineage⁻/α₇integrin⁺/CD34⁺ MuSCs were analyzed with the X-shift algorithm (K=30) yielding 3 clusters that were visualized using single-cell force-directed layout²⁰. Expression levels of Pax7 and CD47 distinguish unique MuSC subsets (representative experiment, n= 3 mice; 4 independent experiments). (C) Scatter plot shows CD47 expression in MuSCs from young and aged mice, measured by single-cell RNA-seq analysis (mean ± SEM). Two-tailed unpaired t-test analysis with Welch’s correction. (D) Histogram overlay shows CD47 expression in MuSCs from young (blue) and aged (red) mice and the FMO + CD47 isotype control (gray) (representative experiment, n= 3 young and 3 aged mice). (E) Stacked columns indicate the relative proportion of CD47^lo and CD47^hi MuSC subsets in young and aged mice (mean ± SEM from n=9 mice, 3 independent experiments). Two-way ANOVA analysis with Sidak correction for multiple comparisons. (F) Scheme depicting the in vivo assay of regenerative capacity. Overlaid blue dots in the biaxial plot indicate the FMO + CD47 isotype control. Representative BLI images at 4 weeks post-transplant are shown (lower right panel). (G) Scatter plot shows the percentage of transplants from each condition that engrafted above threshold (dashed line) into recipient tissue and the BLI signal intensity (y axis). Line represents the median BLI signal (n= 26 mice, 3 independent experiments). Kruskal Wallis test with significance determined by Dunn’s multiple comparisons test.