Fig. 6
In vitro characterisation of Stro-1 immuno-selected populations. Isolated cell populations were seeded into CFU-F assay (1 × 102 cells/cm2) over serial passage (P1 to P10) and cultured for 14 days in T25 cm2 flasks before fixation in 85 % ethanol and staining for ALP expression a (n = 4; 55, 55, 55 and 56 days of age foetal samples). ALP+ colonies are presented as a percentage of the total number of colonies counted b. Population doubling rate (c, foetal samples, n = 6; 55, 55, 55, 55, 56 and 56 days post conception), a measurement of proliferation, and specific ALP activity (d, foetal samples, n = 4; 55, 55, 56 and 56 days of age post conception) were also assessed over serial passage in monolayer cultures seeded at 1 × 103 cells/cm2 (P2). Specific ALP activity was also assessed in cell populations (P2) following 14 days culture in basal or osteogenic medium (e, foetal samples, n = 3; 55, 56, and 59 days (Stro-1); 55, 55 and 61 days (unsorted) of age post conception). Additional monolayer cultures (1 × 103 cells/cm2, P2) were treated with osteogenic and adipogenic (rosiglitazone) medium for 14 days, and stained with Alizarin red f and Oil Red O g, respectively. Micromass cultures (2.5 × 105 cells per pellet, P2) were treated with chondrogenic medium for 14 days and stained with Alcian blue h. Control monolayers were cultured in basal medium. Representative images were of Stro-1 immuno-selected cultures in differentiation media. Error bars are SD. *** P ≤ 0.001 (foetal samples, n = 3; 55, 56 and 59 days (Stro-1); 55, 55 and 61 days (unsorted) of age post conception). ALP alkaline phosphatase