Extended Data Figure 2: K-means clustering analysis of quiescent satellite cells and activation capacity.

a, Representative pictures of Pax7 (red) and MyoD (green) immunostaining of FACS isolated satellite cells from adult mice and quantification of activated (Pax7/MyoD double-positive) satellite cells at 14 and 24 h after injury by CTX injection (before the first division) of adult, old and geriatric muscles. Arrows indicate double-positive cells. Scale bar, 50 μm. b, Equal numbers of quiescent satellite cells FACS purified from resting muscle of adult, old and geriatric mice, labelled with a GFP-expressing lentivirus, were transplanted into injured muscle of young wild-type (immunodeficient) mice, and allowed to adapt to the new muscle host for 3 weeks, where they formed new myofibres (see Fig. 1d) and returned to quiescence. After the 3-week adaptation period, the muscle was re-injured to provoke satellite cell reactivation, and GFP⁺ satellite cells were FACS isolated 24 h after injury, and analysed for activation markers (MyoD and Ki67 immunostaining). MyoD quantification is shown. c, Gene expression microarrays were performed in freshly FACS isolated satellite cells from resting muscle of adult, old and geriatric wild-type mice compared to satellite cells of young wild-type mice (young (n = 3), adult (n = 3), old (n = 3) and geriatric (n = 5) satellite cells). Venn diagrams of overlapping significantly upregulated or downregulated genes in the microarrays (FDR <0.05 and fold change ≥ 1.5). d, K-means clustering analysis of the gene expression microarrays in panel c allowed us to isolate five different gene clusters. One of them was composed of genes with increased expression in geriatric satellite cells (cluster 5, hereafter called cluster G1), and that included genes belonging to our senescence gene set (see Fig. 2d). Heat maps depicting expression levels for genes included in these five clusters. Cluster 2 (not shown) is composed of genes with no changes in expression levels. Red, increased expression; black, neutral expression; green, decreased expression. Expression levels of all the probes present in each cluster were represented. e, Heat maps depicting expression levels for genes included in cluster G2 (generated by K-means clustering of gene expression data from young (n = 3), adult (n = 3), old (n = 3), geriatric (n = 5) and SAMR1 (n = 3) and SAMP8 (n = 3) satellite cells). Red, increased expression; black, neutral expression; green, decreased expression. Venn diagram of overlapping genes between cluster G1 and cluster G2. f, Heat map depicting expression levels for genes included in cluster G3 from young (n = 3), adult (n = 3), old (n = 3), geriatric (n = 5), Bmi1^+/+ (n = 3), Bmi1^−/− (n = 3), SAMR1 (n = 3) and SAMP8 (n = 3) satellite cells. Red, increased expression; black, neutral expression; green, decreased expression. Functional annotation analysis of the Gene Ontology (GO) was performed in DAVID to identify biological processes enriched in cluster G3 (including Bmi1 data). The top annotation clusters are shown according to their enrichment score. Names are based on enriched GO annotations. g, Venn diagram of the overlap between significantly upregulated genes in geriatric, progeric and Bmi1-deficient satellite cells, genes composing cluster G3 and genes belonging to our senescence gene set (see Supplementary Table 3). h, i, Scheme of transplantation assay of labelled satellite cells into pre-injured young hosts. Equal numbers of FACS-purified satellite cells from resting muscle of Bmi1^+/+ and Bmi1^−/− mice were transplanted into muscles of young mice (as in Fig. 1d), and activated satellite cells (h) and new regenerating myofibres (i) were analysed at 24 h and 7 days after transplantation, respectively. i, Representative images of transplanted muscles after 7 days are shown. Graph shows numbers of GFP⁺ myofibres scored per muscle. Scale bar, 50 μm. Data are mean ± s.e.m. Two-sided Mann–Whitney U-test was used to assess statistical significance. P values are indicated. a, n = 6 biological replicates; b, h, n = 4 donor mice; i, n = 5 donor mice. h, i, At least three independent engraftments per donor mouse. n.s., not significant.