Extended Data Fig. 3: Reprogramming erases features of inflammageing and variability between mice. | Nature

Extended Data Fig. 3: Reprogramming erases features of inflammageing and variability between mice.

From: Heterogeneity in old fibroblasts is linked to variability in reprogramming and wound healing

Extended Data Fig. 3

a–l, To test whether transcriptomic and metabolomics features of inflammageing could be erased by reprogramming, iPS cell lines from young and old fibroblasts at passage 23 were profiled for their transcriptome (RNA-seq) and metabolome. a, All iPS cell lines (n = 11) cluster with previously established bona fide iPS cell lines (n = 3) and ES cells (n = 4)61. Unsupervised hierarchical clustering based on overall transcriptomes of the indicated cell types. The hierarchical clustering was performed using correlation-based dissimilarity (Pearson’s) as distance measure and average for linkage analysis. The y axis indicates the similarity between samples. b, PCA of whole transcriptomes from RNA-seq data of iPS cell lines derived from young (3 months, n = 5) and old (29 months, n = 6) mice at passage 23. Principal components (PCs) 1 and 2 are shown. c, Unsupervised hierarchical clustering of transcriptomes described in b. Hierarchical clustering was performed as in a. d, Strip plot illustrating the log2-transformed fold expression changes of all genes with age for fibroblasts (left; described in Fig. 2b) and iPS cells (right; described in b). Genes detected as significantly upregulated or downregulated (DESeq2, FDR-adjusted P < 0.05, absolute fold change >1.5) with age, are shown in blue and yellow, respectively (see Supplementary Table 2q). e, PCA of metabolomes of iPS cell lines derived from young (n = 5) and old (n = 8) mice at passage 23. Ages as in b. Untargeted metabolomics profiles were generated using ultra-high performance liquid chromatography–mass spectrometry. Principal components 1 and 2 are shown. f, Unsupervised hierarchical clustering of metabolomics profiles described in e. Hierarchical clustering was performed as in a. g, Strip plot illustrating the log2-transformed fold change in signal intensity of all metabolic features with age for fibroblasts (left; described in Extended Data Fig. 2c) and iPS cells (right; described in e). Metabolic features detected as significantly up or down (using a two-tailed Wilcoxon rank-sum test with q value correction) with age are shown in blue and yellow, respectively. h, i, PCA (h) and unsupervised clustering (i) of iPS cell lines derived from young (n = 5) and old (n = 6) mice at passage 23, based on solely the genes that were significantly differentially expressed between young and old at fibroblast level. Ages as in b. Principal components 1 and 2 are shown. Hierarchical clustering was performed as in a. j–l, RT–qPCR of the indicated genes in fibroblasts cultures at passage 3 (j) and 33 (k), and iPS cell cultures at passage 23 (l). The genes shown represent the three major groups of features associated with fibroblast activation that change with age in fibroblasts. Box-and-whisker plot of log2-transformed fold change in MFI over median of young fibroblasts. Box plots depict the median and interquartile range, with whiskers indicating minimum and maximum values. Data are from young (n = 6) and old (n = 6) fibroblast cultures at passage 3, young (n = 5) and old (n = 6) fibroblast cultures at passage 33, young (n = 6) and old (n = 7) iPS cell cultures at passage 23. Ages as in b. *P < 0.05, **P < 0.01; one-tailed Wilcoxon rank-sum test with Benjamini–Hochberg correction. For j: Ccl7 (also known as Mcp3), P = 0.004; Ccl2 (also known as Mcp1), P = 0.004; Acvr2a (which encodes ACVR2α), P = 0.006; Ccl11 (also known as Eotaxin), P = 0.004; Pak6, P = 0.004; Thsb2, P = 0.004; Actn3, P = 0.006; Col1a1 (which encodes COL1α1), P = 0.004; Acta2 (which encodes αSMA), P = 0.004; Lama2, P = 0.004; Dmd, P = 0.008; F2r, P = 0.008. For k: Ccl7, P = 0.027; Ccl2, P = 0.027; Acvr2a, P = 0.027; Ccl11, P = 0.049; Pak6, P = 0.027; Thsb2, P = 0.026; Actn3, P = 0.026; Col1a1, P = 0.035; Acta2, P = 0.027; Lama2, P = 0.027; Dmd, P = 0.027; F2r, P = 0.229. For l: Ccl7, P = 0.800; Ccl2, P = 0.800; Acvr2a, P = 0.800; Ccl11, P = 1.000; Pak6, P = 0.800; Thsb2, P = 1.000; Actn3, P = 0.800; Col1a1, P = 1.000; Acta2, P = 1.000; Lama2, P = 1.000; Dmd, P = 1.000; F2r, P = 1.000. Note that the experiments were conducted independently in fibroblasts at passage 3, 33 and iPS cells, and therefore the statistical comparisons indicated were restricted to each independent experiment. However, a comparison between the expression of secreted factors at passage 3 to 33 shows that expression of Ccl11, but not Ccl2 and Ccl7, significantly decreases upon passaging.

Back to article page