a, Left, representative immunofluorescence images of young and old fibroblasts at passage 3 stained for αSMA (which is encoded by the Acta2 gene) . Right, quantification of the percentage of αSMA+ cells in young (3 months, n = 8) and old (29 months, n = 8) fibroblasts at passage 3 (1 experiment). Based on reprogramming efficiency, old cultures are shown as good old, bad old or old. Each dot represents cells from one mouse. Lines depict median. P value, two-tailed Wilcoxon rank-sum test. b, Left, representative immunofluorescence images of young and old fibroblasts at passage 3 incubated with EdU for 4 h, then stained for αSMA (red), EdU (green) and DAPI (blue). White arrows indicate an EdU-positive activated and a non-activated cell. Right, FACS quantification of the percentage of EdU-positive THY1−PDGFRα+ (THY1−) and THY1+PDGFRα+ (THY1+) cells in young (3 months, n = 5; 3 independent experiments) and old (29 months, n = 5; 1 experiment) cultures at passage 3. Dots and lines as in a. P values, two-tailed Wilcoxon rank-sum test. c, PAGODA of single-cell RNA-seq from young and old fibroblasts described in Extended Data Fig. 4c. Top heat map, PAGODA clustering of cells. Maroon and blue colours indicate increased and decreased expression of the associated gene sets, respectively. Middle heat map, expression of genes in the GO cellular senescence gene set, for which expression is shown as VST-transformed read counts, scaled row-wise. Bottom heat map, cells that originate from good and bad old cultures. The scale for expression fold changes is indicated on the right. d, RT–qPCR of p16Ink4a expression in cultures of THY1−PDGFRα+ (THY1−) and THY1+PDGFRα+ (THY1+) young and old cells at passages 4–6. Results are shown as fold change in expression over THY1−PDGFRα+ cells. Data are from young (3 months, n = 5) and old (29 months, n = 5) cultures (6 independent experiments). One young and three old cultures were used in 2–3 independent experiments. In this case, an average of the measurements was determined. Dots and lines as in a. P values, two-tailed Wilcoxon signed-rank test. e, Percentage of SA-β-galactosidase-positive cells in young (3 months, n = 11) and old (28–29 months, n = 22) fibroblast cultures at passage 3 (3 independent experiments). log 2 -transformed fold change in SA-β-galactosidase-positive cells over median of young fibroblasts. Line indicates median. P values, two-tailed Wilcoxon ranked sum test. f, g, RT–qPCR of old THY1−PDGFRα+ (THY1−) and THY1+PDGFRα+ (THY1+) cells at passage 4–6 (3 independent experiments) untreated (f) (n = 6, 3 independent experiments) or treated with the indicated shRNA constructs for 72h (g) (n = 5, 4 independent experiments). Box-and-whisker plot of fold change in expression over THY1−PDGFRα+ populations originating from the same culture (f) or over shLuciferase (shLuc) treated cells (g). Box plots depict the median and interquartile range, with whiskers indicating minimum and maximum values. *P < 0.06, one-tailed Wilcoxon signed-rank test with Benjamini–Hochberg correction. h, RT–qPCR on young (3 months, n = 5) fibroblasts after overexpression of Ebf2 for 48 h (2 independent experiments). Box-and-whisker plot of fold change in expression over cells treated with empty vector. Box plots as in f. *P < 0.06, one-tailed Wilcoxon signed-rank test with Benjamini–Hochberg correction. i, Heat map of significantly differentially expressed genes (determined by DESeq2) between freshly FACS-sorted THY1−PDGFRα+Lin− (THY1−) and THY1+PDGFRα+Lin− (THY1+) cells described in Fig. 3b and enriched KEGG pathways. Expression is shown as VST-transformed read counts, scaled row-wise. The scale for expression fold changes is indicated on the left. All depicted KEGG pathways were significantly enriched (one-sided Fisher’s exact test with Benjamini–Hochberg correction, FDR-adjusted P < 0.05). For a complete list of KEGG terms, see Supplementary Table 4e. j, Pathway enrichment analysis of KEGG pathways associated with ageing in dataset described in Fig. 3b. For a complete list of KEGG terms, see Supplementary Table 4g. **P = 0.01, ***P = 0.001; two-sided nominal P value with Benjamini–Hochberg correction. k, Top, ranked fold change (old/young) in levels of the indicated cytokines in plasma (see Extended Data Fig. 1a). Bottom, ranked fold change (old/young) in expression for the indicated cytokines in freshly FACS-sorted THY1−PDGFRα+Lin− (THY1−) and THY1+PDGFRα+Lin− (THY1+) cells from young and old ears. See ‘Cytokine profiling analysis on plasma and conditioned medium using Luminex multi-analyte’ for calculation of ranked fold changes. Gene expression related to wounded fibroblasts is from datasets described in Extended Data Fig. 7e–g. l–n, Correlation between the proportion of THY1+PDGFRα+ (THY1+) fibroblasts in old cultures (29 months, n = 23) (l), young (3 months, n = 21) and bad old (29 months, n = 6) cultures (m), and young (3 months, n = 21) and good old (29 months, n = 6) cultures (n), and the reprogramming efficiency of the culture (3 independent experiments). Dots as in a. P values, two-sided algorithm AS 89 in R. The y axis denotes the fold change in the proportion of THY1+PDGFRα+ fibroblasts relative to the median of young mice, and x axis denotes the fold change in reprogramming efficiency of the culture relative to the median of young mice. o, p, Correlation between the proliferation rate (o) or the percentage of SA-β-galactosidase-positive cells (p) of a given fibroblast culture and reprogramming efficiency of the culture. Proliferation rate was determined by calculating the growth slope of young (3 months, n = 15), middle-aged (12 months, n = 10) and old (28–29 months, n = 27) ear fibroblast cultures at passage 3 (4 independent experiments). Senescence was assessed by SA-β-galactosidase staining of young (3 months, n = 11), middle-aged (12 months, n = 11) and old (28–29 months, n = 22) ear fibroblast cultures at passage 3 (3 independent experiments). Dots as in a. P values, two-sided algorithm AS 89 in R. The y axis denotes the fold change in the proliferation rate or percentage of SA-β-galactosidase-positive cells relative to the median of young mice, and x axis denotes the fold change in reprogramming efficiency of the culture relative to the median of young mice. q, Reprogramming efficiency of FACS-sorted young (3 months, n = 8) and old (29 months, n = 7) THY1−PDGFRα+ (THY1−) and THY1+PDGFRα+ (THY1+) fibroblasts at passages 4–6, assessed using AP staining (3 independent experiments). log 2 -transformed fold change in reprogramming efficiency of the cells relative to the median of young THY1−PDGFRα+ fibroblasts. One old culture was used in two independent experiments. In this case, an average of the measurements was determined. Dots and lines as in a. P values, two-tailed Wilcoxon signed-rank test. r, Reprogramming efficiency of FACS-sorted young (3 months, n = 5) THY1−PDGFRα+ (THY1−) and THY1+PDGFRα+ (THY1+) fibroblasts at passages 4–6, assessed as in q (3 independent experiments). Reprogramming was induced using a non-lentiviral piggyBac transposon system. Results are shown as number of AP+ colonies. Dots and lines as in a. P values, one-tailed Wilcoxon rank-sum test. s, t, Cytokine profiles of conditioned medium collected from cultures of old (s) (29 months, n = 6, 3 independent experiments) and young (t) (3 months, n = 6, 2 independent experiments) THY1−PDGFRα+ (THY1−) and THY1+PDGFRα+ (THY1+) fibroblasts at passages 4–6 . Comparisons were made between THY1−PDGFRα+ and THY1+PDGFRα+ from the same original culture. Based on cytokines that are significantly different in conditioned medium from fibroblasts (Fig. 1b). Box-and-whisker plot of log 2 -transformed fold change in mean fluorescence intensity (MFI) over THY1−PDGFRα+ fibroblasts. Box plots as in f. *P < 0.05, one-tailed Wilcoxon rank-sum test with Benjamini–Hochberg correction. Exact P values are in Supplementary Table 4h. u, Reprogramming efficiency, assessed as in q, of FACS-sorted young (3 months, n = 6) THY1−PDGFRα+ (THY1−) and THY1+PDGFRα+ (THY1+) fibroblasts at passages 4–6 treated with fresh conditioned medium daily starting from day 1 after infection (3 independent experiments). Conditioned medium was collected daily from the THY1−PDGFRα+ or THY1+PDGFRα+ fibroblasts from the same original culture. log 2 -transformed fold change in reprogramming efficiency relative to the reprogramming efficiency of THY1−PDGFRα+ fibroblasts treated with conditioned medium from THY1−PDGFRα+ fibroblasts. One young culture was used in two independent experiments. In this case, an average of the measurements was determined. Dots and lines as in a. P values, two-tailed Wilcoxon signed-rank test. For individual experiments in b, d–h, l - u and exact P values, see Supplementary Table 7.