FIG. 4.

Passaging effects on OACs. Pooled P2 OAC cultures from five single cell-derived colonies were inoculated at 100 cells/cm² into T-150 flasks containing 20 mL growth medium and serial passaged at 50% confluence. MFI ratio of NG2 (A) and CD146 (B) surface expression was determined by flow cytometry as in Figure 1. Growth properties were evaluated by monitoring cumulative doublings (C) and doubling time (D). Senescence-associated β-galactosidase activity (E) in 30 randomly selected images of subconfluent cultures. Cell potency to exhibit osteo- (F), adipo-, (G) and chondrogenesis (H) was quantified after 21 days of differentiation as in Supplementary Figure S1. Threshold values of positive differentiation are given in Supplementary Figure S1. Data are presented for n=3 independent cultures. Each symbol represents a different pooled group of five single cell-derived colonies from the same donor.