Expression of homing-associated cell adhesion molecule (H-CAM/CD44) on human CD34+ hematopoietic progenitor cells  Takao Deguchi, Yoshihiro Komada, Kenji Sugiyama, Xao-Li Zhang, Eiichi Azuma, Hatsumi Yamamoto, Minoru Sakurai  Experimental Hematology  Volume 27, Issue 3, Pages 542-552 (March 1999) DOI: 10.1016/S0301-472X(98)00047-2

Figure 1 Cell surface expression of CD44 on bone marrow (BM), cord blood (CB), and peripheral blood (PB) CD34+ cells. Cells were stained with PE-conjugated anti-CD34 and FITC-conjugated anti-CD44 MoAb. Histograms of CD34+ cells stained with control MoAb (dotted line) or anti-CD44 MoAb (solid line) are shown. Data are from a representative experiment. Nine independent experiments gave similar results Experimental Hematology 1999 27, 542-552DOI: (10.1016/S0301-472X(98)00047-2)

Figure 2 Cell surface expression of B-lymphocyte–associated CD19 antigen on CD34+CD44+ and CD34+CD44− cell populations. (A) BM cells were stained with FITC-conjugated anti-CD44, PE-conjugated anti-CD19, and PC5-conjugated anti-CD34 MoAbs. (B, C) After gating CD34+CD44+ (R1) and CD34+CD44− (R2) cells, cells were analyzed for the expression of CD19. The data were shown in histograms of sorted cells stained with control MoAb (dotted line) or anti-CD19 MoAb (solid line). Triple-color analysis showed that CD19 was expressed in 26.9% of CD34+CD44+ cells (B) and in 99.5% of CD34+CD44− cells (C). This experiment was repeated five times and yielded similar results Experimental Hematology 1999 27, 542-552DOI: (10.1016/S0301-472X(98)00047-2)

Figure 3 Expression of CD34 and CD44 on sorted BM cells in short-term suspension culture. Sorted CD34+CD44+ and CD34+CD44− BM cells (1 × 104/mL) were cultured as described in Materials and methods. Cells were harvested on days 0, 2, 3, and 5 and stained with FITC-conjugated anti-CD44 MoAb and PE-conjugated anti-CD34 MoAb. The data of green (FITC/CD44) and orange (PE/CD34) fluorescence are depicted in dot plot graphs. The percentages of stained cells in each region are shown. This experiment was repeated four times and yielded similar results Experimental Hematology 1999 27, 542-552DOI: (10.1016/S0301-472X(98)00047-2)

Figure 4 Expression of lineage-specific markers on sorted BM cells in short-term suspension culture. Sorted CD34+CD44+ (A–C) and CD34+CD44− (D–F) BM cells were cultured for 5 days as described in Materials and methods. Cells were stained with a combination of MoAbs (CD3/CD19, CD45/CD14, and GlyA/CD33). The data of green (FITC) and orange (PE) fluorescence are depicted in dot plot graphs. This experiment was repeated four times and yielded similar results Experimental Hematology 1999 27, 542-552DOI: (10.1016/S0301-472X(98)00047-2)

Figure 5 Expression of surface antigen on viable cells in 14 day-BMSC culture initiated with CD34+CD44− cells. Sorted CD34+CD44− cells were cultured on human BMSC monolayers in the presence of Flt3L and IL-7. After 14 days of culture, cells were harvested and stained with a combination of MoAbs (CD10/19, CD44/CD19, CD19/CD34, and kappa/lambda). The data of fluorescence on cultured BM progenitor cells are depicted in dot plot graphs. This experiment was repeated four times and yielded similar results Experimental Hematology 1999 27, 542-552DOI: (10.1016/S0301-472X(98)00047-2)

Figure 6 Flow cytometric analysis of propidium iodide PI-stained nuclei of sorted BM cells. Sorted CD34+CD44+ (A,C,E) and CD34+CD44− (B,D,F) BM cells were cultured in cytokine-supplemented medium (A–D) or on BMSC monolayers (E,F). Colorimetric DNA fragmentation assay was performed on day 0 (A,B) and day 2 (C–F). The percentages of cells in sub-G1, G0/G1, S, and G2/M fraction are indicated. These experiments were repeated three times and yielded similar results Experimental Hematology 1999 27, 542-552DOI: (10.1016/S0301-472X(98)00047-2)

Figure 7 DNA fragmentation of sorted BM CD34+CD44+ (A,C) and CD34+CD44− (B,D) cells. Sorted BM cells were cultured for 2 days in cytokine-supplemented medium (A,B) and on normal BMSC monolayers (C,D). Harvested cells were labeled with biotinylated dUTP using TUNEL assay. The data of green fluorescence on BM progenitor cells incubated with (solid line) of without (dotted line) biotinylated dUTP are presented in histograms. The percentage of dUTP-labeled cells is indicated Experimental Hematology 1999 27, 542-552DOI: (10.1016/S0301-472X(98)00047-2)