PT birth accelerates cellular senescence and proliferative arrest of ECFCs. SA-β-gal activity and cell-cycle analyses were assessed as markers of cellular senescence. (A) Representative images from CT- and PT-ECFCs at passage 3 (P3), 6 (P6), and 8 (P8) are shown in left panel (magnification ×20; scale bar, 100μm). The percentage of senescent cells in PT-ECFCs was determined as the number of cells expressing SA-β-gal (blue cells) relative to the total number of cells in each field in PT neonates (n = 16) and CT neonates (n = 16). Graph (right panel) represents means ± SEM of 16 independent samples in continuous passage assay. Experiments were done in triplicate, *P < .05; **P < .01; ***P < .001. (B) SA-β-gal activity negatively correlated with gestational age in samples from all groups (n = 32, P3); r2, correlation coefficient of determination; r, Pearson correlation coefficient. (C) Cell-cycle analysis by flow cytometry. The DNA content in each cell-cycle phase in CT- and PT-ECFCs were analyzed by flow cytometry after propidium iodide staining. The representative histograms are from 2 samples per groups (left panel). Graph (right panel) represents a mean percentage of cells at different phases of the cell cycle determined by the DNA content ± SEM for 5 to 8 independent samples. *P < .05.