Fig. 2: MAD-iPSCs derived mesenchymal stem cells (MAD-MSCs) recapitulated accelerated cellular senescence.

a, Human MSCs derived from WT and MAD-iPSCs by a temporal neuralized ectoderm induction method. b Growth curve of MSCs. Bars represent the mean ± SD.; n  =  3 independent biological replicates; ***p <  0.001; n.s., non-significant; p value was calculated using two-way ANOVA test. c Proliferative capability measured by Ki 67 (red) using P13 MSCs. DAPI, blue. Scale bar 10 μm. Data represent the mean ± SD, n  =  5. d SA-β-gal staining of MSCs at passage 13; Scale bar 100 μm. Data are mean ± SD, n =  3. e Representative image of γ-H2A.X (red) immunostaining at passage 13. DAPI, blue. Scale bar 10 μm. Data are mean ± SD, n =  8. The p values were calculated using two-tailed unpaired t-test. Experiments in c-e were repeated three times with similar results. f Representative transmission electron micrographs (TEM) of P13 WT- and MAD-MSCs. Scale bar 250 nm. The percentage of damaged mitochondria was  quantified and calculated in MSCs. Data are mean ± SD, n = 462 WT, n = 368 MAD. The p value was calculated using two-tailed unpaired t-test. Three independent replicates were performed with similar results. g Representative images of co-staining of lamin B1 (LMNB1, green) and HP1a (red), lamin A/C (LMNA, green) and LAP2 (red), lamin A/C (LMNA, green) and H3K9me3 (red), lamin A/C (LMNA, green) and H3K27me3 (red), lamin A/C (LMNA, green) and H3K27ac (red), and lamin A/C (LMNA, green) and H3K4me3 (red) in passage 9 MSCs. DAPI, blue. Scale bars 10 µm. h Fluorescence intensity of g were quantified, including Lamin B1 and HP1a (WT n = 189, MAD n = 79), lamin A/C and LAP2 (WT n = 84, MAD n = 51), H3K9me3 (WT n = 88, MAD n = 64), H3K27me3 (WT n = 71, MAD n = 31), H3K27ac (WT n = 163, MAD n = 55), H3K4me3 (WT n = 145, MAD n = 79) and nuclei size (WT n = 293, MAD n = 143). Data are mean ± SD; the lines in scatter dot plot indicate the averaged intensity and the p values were calculated using two-tailed unpaired t-test. Three independent biological experiments were performed with similar results. i GO and KEGG enriched signaling pathways in MAD-MSCs. All p-values were determined by two-sided modified Fisher’s exact test using DAVID. j Comparison of aging-associated gene profilings between MAD-MSCs and other human MSCs aging models. Color depth indicates the level of transcriptional similarity. Source data are provided as a Source Data file.