Morphology, surface-marker profile, and gene expression of primary CD271⁺/CD146^−/low and CD271⁺/CD146⁺ CFU-Fs. Freshly sorted cells expressing CD271⁺/CD45⁻/CD146^−/low (A) and CD271⁺/CD45⁻/CD146⁺ (B) were allowed to sediment (0 hours) or to adhere for 24 hours to slides. Photographs were taken on a differential interference contrast microscope (Axiovert 200M; Zeiss). Scale bars indicate 10 μm. (C) Representative multicolor FACS analysis of primary, lineage-depleted BM-MNCs after CD45 exclusion. Events are plotted for CD146 (x-axis) against CD271 (y-axis). Red events in the plots indicate cells that coexpress the marker listed on top of the plot: CD105, CD90, STRO-1, PDGFR-β, SSEA-4, GD2, CD31, and CD34. Gray events represent cells that did not coexpress the listed marker. Each plot represents an individually stained sample tube from the same bone marrow sample. The mean percentage of CD271⁺ cells was of 0.14% ± 0.01% in all plots. (D-E) Single-cell multiplex PCR of sorted, uncultured CD271⁺/CD45⁻/CD146^−/low (D) and CD271⁺/CD45⁻/CD146⁺ (E) cells. Representative results from one donor are shown. Each vertical column represents a single cell and each horizontal row represents a certain analyzed gene. Circles indicate expression of the gene, whereas blanks indicate that no PCR band was detected. In total, sorted cells from 3-4 different donors were analyzed. Numbers indicate the number (#) and percentage (%) of positive cells per plate of the donor sample presented in the figure. Data in the right column represent mean percentages of positive cells of all analyzed samples. The number of CD271⁺ wells is set as 100%. For the mean percentages, cells were only included when they showed a positive band for CD271.