American Society of Hematology

Figure 1

$Figure 1. All CFU-Fs in human bone marrow are contained in the CD271+/CD45−/CD146−/low and the CD271+/CD45−/CD146+ fraction. Freshly isolated human BM-MNCs were stained with antibodies against CD271, CD146, and CD45, and analyzed by flow cytometry. Representative contour plots of CD271- and CD146-expressing human bone marrow cells after forward/side scatter and 7-AAD gating are shown. (A) CD271 and CD146 expression in whole viable BM-MNCs. (B) CD271 and CD146 expression after exclusion of CD45+ cells. (C) Percentage of CD271+/CD45−/CD146+, CD271+/CD45−/CD146−, and CD271−/CD45−/CD146+ cells in viable, nondepleted human BM-MNCs (n = 9). Horizontal lines indicate mean values. Error bars show SEM. (D) Gates for the CD271/CD45/CD146 sorts from lineage-depleted bone marrow were set according to the appropriate FMO controls for CD45 (top row left plot). The top right plot illustrates the CD45-sorting gate, with P10 and P5 indicating the sorting gates for CD45+ cells and CD45− cells, respectively. Red dots indicate all gated events shown in the CD271/CD146 plot (E) after back-gating on CD45. Sorting gate definition based on the FMO controls for CD271 and CD146 are illustrated in the bottom plots. (E) Sorting gates from a representative bone marrow sample for CD271−/CD146− (P8), CD271−/CD146+ (P9), CD271+/CD146−/low (P7), and CD271+/CD146+ (P6) after lineage depletion and CD45 exclusion. As shown in this analysis, CD146 expression increased gradually with increasing CD271 expression, and therefore it is impossible to identify a clear-cut CD271+/CD146− cell population within the CD271+ cells. We therefore chose instead to use the term CD271+/CD146−/low for cells sorted on gate P7 to indicate this fact. (F) CFU-F frequencies per 100 freshly isolated BM-MNCs of total viable cells sorted only based on 7-AAD exclusion (viable, n = 4), sorted total CD45+ cells (n = 4), and the different sorted CD271/CD146 populations after CD45 exclusion (n = 5). Each dot represents the mean frequency of CFU-Fs from one bone marrow sample. Horizontal lines indicate mean values of at least 4-5 independent samples. (G) CFU-F frequencies per 96-well plate of single-sorted CD271+/CD45−/CD146−/low and CD271+/CD45−/CD146+ primary cells. Data are shown as mean ± SEM (n = 8).$

All CFU-Fs in human bone marrow are contained in the CD271⁺/CD45⁻/CD146^−/low and the CD271⁺/CD45⁻/CD146⁺ fraction. Freshly isolated human BM-MNCs were stained with antibodies against CD271, CD146, and CD45, and analyzed by flow cytometry. Representative contour plots of CD271- and CD146-expressing human bone marrow cells after forward/side scatter and 7-AAD gating are shown. (A) CD271 and CD146 expression in whole viable BM-MNCs. (B) CD271 and CD146 expression after exclusion of CD45⁺ cells. (C) Percentage of CD271⁺/CD45⁻/CD146⁺, CD271⁺/CD45⁻/CD146⁻, and CD271⁻/CD45⁻/CD146⁺ cells in viable, nondepleted human BM-MNCs (n = 9). Horizontal lines indicate mean values. Error bars show SEM. (D) Gates for the CD271/CD45/CD146 sorts from lineage-depleted bone marrow were set according to the appropriate FMO controls for CD45 (top row left plot). The top right plot illustrates the CD45-sorting gate, with P10 and P5 indicating the sorting gates for CD45⁺ cells and CD45⁻ cells, respectively. Red dots indicate all gated events shown in the CD271/CD146 plot (E) after back-gating on CD45. Sorting gate definition based on the FMO controls for CD271 and CD146 are illustrated in the bottom plots. (E) Sorting gates from a representative bone marrow sample for CD271⁻/CD146⁻ (P8), CD271⁻/CD146⁺ (P9), CD271⁺/CD146^−/low (P7), and CD271⁺/CD146⁺ (P6) after lineage depletion and CD45 exclusion. As shown in this analysis, CD146 expression increased gradually with increasing CD271 expression, and therefore it is impossible to identify a clear-cut CD271⁺/CD146⁻ cell population within the CD271⁺ cells. We therefore chose instead to use the term CD271⁺/CD146^−/low for cells sorted on gate P7 to indicate this fact. (F) CFU-F frequencies per 100 freshly isolated BM-MNCs of total viable cells sorted only based on 7-AAD exclusion (viable, n = 4), sorted total CD45⁺ cells (n = 4), and the different sorted CD271/CD146 populations after CD45 exclusion (n = 5). Each dot represents the mean frequency of CFU-Fs from one bone marrow sample. Horizontal lines indicate mean values of at least 4-5 independent samples. (G) CFU-F frequencies per 96-well plate of single-sorted CD271⁺/CD45⁻/CD146^−/low and CD271⁺/CD45⁻/CD146⁺ primary cells. Data are shown as mean ± SEM (n = 8).

This Feature Is Available To Subscribers Only