In vitro CD146 expression under normoxic and hypoxic conditions. CD146 expression of CD271⁺/CD45⁻/CD146^−/low and CD271⁺/CD45⁻/CD146⁺ cells was monitored over time. Sorted cells from the CD146^−/low (A) and the CD146⁺ (B) fractions were plated on glass chamber slides. Original sorting gates are shown in red on the left. Cells were incubated at 37°C, 5% CO₂, and 21% O₂. On day 6, cells were stained with antibodies against CD271 (green) and CD146 (red) plus the corresponding secondary antibodies. Small inserted photographs on the top right show controls stained with secondary antibodies only. Scale bars indicate 20 μm. (C) CD146 and CD271 expression in cultured cells after the second passage. Stromal cultures were generated from unsorted BM-MNCs (top left), bulk-sorted CD271⁺/CD45⁻/CD146^−/low cells (top middle), or CD271⁺/CD45⁻/CD146⁺ cells (top right). FACS plots in the bottom row show CD146 and CD271 expression of clonal second-passage cultures from a CD271⁺/CD45⁻/CD146^−/low–sorted cell (bottom left) and a CD271⁺/CD45⁻/CD146⁺ sorted cell (bottom right), respectively. (D) Representative FACS histogram plot of cells cultured for 1 week with 100μM DFO (red open histogram). Controls were cultured without DFO (gray tinted histogram). Gray open histogram indicates isotype control. (E) Representative FACS plots for CD146 expression of established stromal cells cultured at 21% O₂ (gray tinted histogram, top left) or at 1% O₂ (red tinted histogram, bottom left). Increased expression and reexpression of CD146, respectively, was observed when both normoxic and hypoxic stromal cells were passaged and both were incubated for an additional 2 weeks at 21% O₂ (gray and red tinted histograms in right top and bottom row, respectively). Open histograms represent isotype controls.