Fig. 4

MuSCs/MPCs in BALB/c mice undergo precocious differentiation after HLI surgery. A UMAP representation displaying quiescent (depicted in yellow), activated/proliferative (in blue), and differentiating (in pink) muscle stem cells (MuSCs) and muscle precursor cells (MPCs). Total MuSC/MPC count stands at 9,217. B MuSCs/MPCs from part A are arranged in a pseudotime sequence, initiating from quiescent cells (left) and advancing to activated/proliferative (middle) and subsequently to differentiating cells (right). The heatmap's color gradient represents the expression intensity of the specified genes in MuSCs/MPCs in accordance with the pseudotime progression. C (Top) Violin plot showing the distribution of quiescent (depicted in yellow), activated/proliferative (in blue), and differentiating (in pink) MuSCs/MPCs in the two strains along the pseudotime trajectory shown in B. (Bottom) Curve plot showing the distribution of MuSC/MPCs at indicated time points, before (sham) and 1, 3, and 7 dpi post-HLI, in the two mouse strains along the pseudotime trajectory shown in B. D Gene set enrichment analysis (GSEA) highlighting the genes related to “skeletal muscle cell proliferation” are significantly enriched in the up-regulated genes in MuSCs/MPCs in C57BL/6 mice compared to those in BALB/c mice. E, F At 7 dpi, TA muscles were collected from both C57BL/6 and BALB/c for immunostaining using antibodies against Pax7 and cell proliferation marker Mki67. E n = 5 mice per strain. Data expressed as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01. F Representative Pax7 (red) and Mki67 (green) immunofluorescence staining images along with DAPI (blue). G Representative RNAscope data showing that Adgre1+ macrophages (F4/80, green) and Myod1+ MuSC/MPCs (red) are spatially proximal to each other in the limb muscle of BALB/c and C57BL/6 mice at day 3 after HLI. Three mice per strain were used for RNAscope analysis. H Inferred ligand-receptor interactions between macrophages and MuSCs in BALB/c and C57BL/6 following HLI at 3 days post HLI surgery. I IGF1 promotes proliferation of primary MPCs purified from BALB/c and C57BL/6 strains. (Left) Representative images of EdU incorporation by C57BL/6 and BALB/c primary MPCs cultured with or without recombinant IGF1 for 72 h. EdU was added to the culture medium 6 h prior to cell fixation and imaging. Nuclei stained with Hoechst. Arrows indicate EdU+ cells. (Right) Quantification of the percentage of EdU+ cells for the indicated strains and treatment conditions. *P < 0.05, **P < 0.005