Fig. 2.

Senescence and replicative stress in UCSCs and infant BMSCs

RT-qPCR analysis of senescence-related genes, including p16 (A), p21 (B), p53 (C), and TERT (D) in UCSCs and infant ADSCs at passages 3–5 (early passages) and passage 7–9 (late passages). Each value was normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the relative fold changes of each value were compared to UCSCs at early passages. Using the data from UCSCs in the early passages as the baseline, we calculated the relative fold change of UCSCs in the late passages and infant BMSCs in both early and late passages. Statistical comparisons of differences were individually analyzed using Mann-Whitney U analysis for the following groups: differences between UCSCs in the early and late passages, differences between infant BMSCs in the early and late passages, differences between UCSCs and infant BMSCs in the early passage, and differences between UCSCs and infant BMSCs in the late passages. (E) Senescence-associated β-galactosidase (SA-β-gal) staining of UCSCs and infant BMSCs at late passages was detected using a Senescence Detection Kit, and images were captured using a Nikon Eclipse TS100 microscope. Blue-stained cells indicate positive SA-β-gal staining. The cell images were captured at 200 × magnification (the scale bar = 100 μm). (F) Immunostaining of γH2AX (green) and DAPI (blue) in UCSCs and infant BMSCs at late passages, detected using an Olympus BX43 microscope at a magnification of × 400, with the scale bar representing 100 μm. Quantification of γH2AX immunofluorescence intensity in the nuclei of UCSCs and infant BMSCs. Values, quantified using Image-Pro Plus v4.5.0.29, were normalized to the cell numbers and compared to show the relative fold changes. Mean ± SEM of quantified values with three replicates are expressed. Statistical significance of comparing UCSCs and infant BMSCs was determined by the Mann-Whitney U test. The star symbol (∗) will indicate significant differences between the compared groups. “∗" represents p < 0.05, "∗∗" represents p < 0.01, and "∗∗∗" represents p < 0.001.