Comparison of chondrocyte sub-populations as a function of population doublings. Cells were analyzed at the end of the first passage after MACS and subsequent expansion until cells had undergone more than 8 population doublings (pds). A) Flow cytometry analysis with percentage of W5C5 positive cells after W5C5 MACS sorting and W8B2 positive cells after W8B2 MACS sorting. Representative examples are shown. B) Adipogenic differentiation of subpopulations. Adiponectin expression relative to GAPDH. C) Osteogenic differentiation. ALP gene expression relative to GAPDH and ALP enzyme activity were presented. Boxes represent 25% / 75% percentiles, whisker 5% / 95% percentiles, mean (dotted line) and median (solid line) values. (n = 3 donors after MACS, n = 4 donors + > 8pds). *: significant differences (p < 0.05) between cells differentiated directly after MACS and after expansion, and between subpopulations under the same experimental conditions. Significant differences existed in all cases between the control and the induction conditions (not specifically indicated).