Fig. 1

Long-term ex vivo culture causes senescence in human PDLSCs. Human PDLSCs were cultured and passaged in vitro; across the ex vivo expansion stage, passage 2 (P2), passage 7 (P7), and passage 15 (P15) cells were collected for subsequent studies. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 represent significant differences between the indicated columns. a The colony-forming ability of PDLSCs changed during long-term ex vivo expansion (top: general views of the cell colonies formed by P2, P7, and P15 cells and a representative single colony from the top panel (scale bar = 100 μm); bottom: quantitative analysis of cell colonies formed by P2, P7, and P15 cells). b Proliferative activity of P2, P7, and P15 cells in terms of CCK-8 assay (P2 vs. P15: *p < 0.05 and **p < 0.01; P2 vs. P7: ^#p < 0.05; P7 vs. P15: ^$p < 0.05). c SA-β-gal in P2, P7, and P15 cells (top: representative images of SA-β-gal expression in P2, P7, and P15 cells (immunocytochemical staining, positively stained cells were dyed in blue; scale bar = 100 μm); bottom: percentage of SA-β-gal-positive cells among all cells and quantitative analysis). d Cell senescence-related proteins in P2, P7, and P15 cells (top: protein levels of p53, p21, p16, and γ-H2AX as determined by Western blot assay; bottom: quantitative analysis of p53, p21, p16, and γ-H2AX expression in P2, P7, and P15 cells)