Opn⁺CD45⁻Ter119⁻ cells isolated from bone exhibit osteoblast function. (A) Flow cytometric analysis of ALP activity, determined using a modified version of the fluorogenic ELF-97 assay (Invitrogen). Histograms show kinetics (1 minute, 15 minutes, and 30 minutes) of detection of ALP activity in purified Opn⁺ cells (black line) compared with the ALP activity in a control cell population of total BM cells (gray filled, n = 4). Numbers in the upper right indicate frequency of ALP⁺ cells among Opn⁺ cells/frequency of ALP⁺ among BM control cells. This is representative of 3 independent experiments (*P < .05 at each time point indicated). (B) Sorted osteoblasts form bone nodules in culture. Ten OPN⁺ cells were cultured in α-MEM/15% serum (d1 of culture, 10 times) and at day 15 (d15, 4 times) osteogenesis was induced by the addition of 20 mg/mL ascorbic acid plus 50 μM β-glyceraldehyde. Cell clusters form after 7 days (d22 of culture, 4 times) and distinct bone nodules are visible by 15 days (d30 of culture, 4 times). Mineralization was confirmed at d30 by the von Kossa method. Micrographs shown are representative of 3 independent experiments (n = 20 wells/experiment). Plates were viewed using a UPLanF1 lens at 10×/0.30 Ph1 (first micrograph) or a UPLan FLN lens at 4×/0.13 (all other micrographs). The efficiency of bone nodule formation was 89% plus or minus 3% of seeded wells over the 3 experiments performed. (C) Differentiating osteoblasts in bone nodule cultures showed increasing expression of ALP mRNA (graph, middle panel, representative of 3 to 5 different wells sampled individually at each time point indicated; *P < .05) and increasing ALP activity (histogram, right panel, representative of 3 individual wells sampled at each time point, day 15, day 20, or day 30, after culture initiation). Data are representative of 3 independent experiments; error bars represent SD.