Developmental potential of CD45^–CD123⁺ cells. (A) Representative flow cytometric dot plots demonstrating the relative increase in the percentage of CD45^–CD123⁺ cells throughout the 21-day culture period. CD45^– cells cultured in suspension were dual-labeled for CD45 and CD123 to track CD45^–CD123⁺-expressing cells. The CD45^–CD123⁺ cells, represented by R1, were sorted on day 0 (i), 7 (ii), 14 (iii), and 21 (iv) and the developmental potential of these isolated populations were determined in CFU-F and CFU-O assays. Representative quad plots of day 0, 7, 14, and 21 suspension-derived cells (of a suspension culture initiated with CD45^– cells) stained with CD45 and CD123 are shown here. Positive expression was determined as more than 99% of isotype control and about 15 000 gated events were collected. (B) Total number of CD45^–CD123⁺ cells generated in SF suspension culture conditions supplemented with 100 ng/mL SCF and 20 ng/mL IL-3. Dual fluorescence labeling for CD45 and CD123 was used to track the percentage of CD45^–CD123⁺cells in suspension cultures initiated with CD45^– cells. These values were used to calculate the total number of CD45^–CD123⁺cells generated throughout suspension culture. Statistical significance (P < .05) is denoted by an asterisk. Each data point represents the mean ± SD (n = 3). (C) CFU-F and CFU-O development from CD45^–CD123⁺ suspension-derived cells. CD45^–CD123⁺-sorted cells (grown in CD45^– SSCs) were plated in CFU-F and CFU-O assays following 7 days of suspension culture. (i) CFU-F assays were stained with Giemsa to visualize discrete colonies of fibroblastic cells; (ii) CFU-O assays were incubated with tetracycline (TC), to visualize newly formed bone nodules (seen as white areas) under UV-fluorescence microscopy. Representative scanning electron micrographs of revealing (iii) cement line matrix deposition (arrow) and (iv) collagen mineralization (arrow) derived from CD45^–CD123⁺ sorted cells grown in CFU-O assay conditions (FW for iii and iv = 53 μm and 21 μm, respectively). Note: CFU-F and CFU-O assays were initiated with 1 × 103 cells/cm2.