Complement is locally produced and required for efficient OC differentiation in humans. (A) Zymosan-based C3b uptake assays using factor B^−/− sera plus 1:5 diluted control media (solid line) or human BM cell–conditioned media (dotted line), showing that BM cell-conditioned media compensated the absence of factor B. (B) Zymosan-based C3b uptake assays using factor D^−/− sera plus 1:5 diluted control media (solid line) or human BM cell-conditioned media (dotted line), showing that BM cell-conditioned media compensated the absence of factor D. (C) E^shA-hemolytic assays using C5-depleted sera plus 1:5 diluted control media or BM cell-conditioned media, showing that BM cell-conditioned media compensated the absence of C5, therefore inducing C5b-9–mediated hemolysis. (D) Human BM cells were incubated with 1 × 10⁻⁸M 1,25(OH)₂ vitamin D3 in the presence of placebo (control), C3aRA, C5aRA, or C3aRA·C5aRA, showing that efficient OC differentiation in humans also requires C3aR/C5aR as in mice. Representative results of 2 independent experiments. Data are mean ± SD: *P < .05, #P < .05. (E) Both mesenchymal cells and OC progenitors are involved in the complement-regulated OC differentiation. Samples of 2 × 10⁴ primary WT or C3^−/− calvarial OBs were cultured with 2 × 10⁶ WT and C3^−/− splenocytes (as source of OC progenitors) together with 1 × 10⁻⁸M 1,25(OH)₂ vitamin D3. The resultant TRAP-positive cells were counted after 12 days of coculture.