A cell cycle G1 checkpoint likely cooperates with SDF-1–mediated adhesion in EPC differentiation of c-kit⁺ cells. (A) Stem cells were expanded for 7 days in cytokine containing serum-free medium, after which they were plated onto FN without cytokines ± SDF-1. The results showed that cytokine washing partially restored EPC differentiation of ex vivo–expanded cells. *P < .05; n = 5. Error bars indicate standard errors. (B) Overexpression of p16^INK4 was performed by incubating U2OS cells with retrovirus supernatants (null, lane 1 or increasing amounts of p16^INK4 retrovirus; Δp16^INK4, lanes 2-3) to check transgene expression by Western analysis. (C) C-kit⁺ cells were transduced with retro null or retro p16^INK4 and thereafter analyzed for cell number increase in the presence of mitogens. Both at 3 and 7 days after infection, cell proliferation was blocked in p16^INK4-transduced cells. Data are reported as fold increase in cell number compared with number of cells at the beginning of the time course. *P < .05; n = 4, for the comparison between growth of retro null–infected (squares) and retro p16^INK4–infected (circles) cells. (D) Differentiation test of retro null– and retro p16^INK4–infected stem cells under mitogen stimulation. Although retro null–infected c-kit⁺ did not significantly differentiate into EPCs (filled bars), cells overexpressing p16^INK4 were restored in their ability to differentiate in response to SDF-1 stimulation (open bars). *P < .05; n = 5. Error bars indicate standard errors. C indicates control-untreated cells (A,D).