Figure 5
Figure 5. Integrin-β3–mediated signaling leads to the suppression of expansion and cell division on HSCs during ex vivo culture. Forty sorted WT CD34−KSL cells (Ly5.1) were cultured for 5 days with 2C9.G2 or hamster IgG (isotype control) in S-Clone SF-03 serum-free medium supplemented with 50 ng/mL SCF plus 50 ng/mL TPO. (A) After the culture, total cell number was counted. Graph represents fold increase of total cell number after 5 days of culture. Data are mean ± SD. (B) To confirm 2C9.G2 binding to cells during culture, cells were stained with fluorescently labeled hamster IgG and analyzed by flow cytometry: white represents IgG; and gray, 2C9.G2. (C) The percentages of KSL and CD48−KSL cells were also determined by flow cytometry after culture. **P < .05. (D) HSC frequency among the cultured cells was determined using limiting dilution assays. After groups of 10, 30, 50, 100, or 500 whole cultured cells were counted exactly and sorted, the groups were individually transplanted into lethally irradiated Ly5.2 mice along with 2 × 105 BM cells from Ly5.2 mice. This was followed by analysis for chimerism 20 weeks after the transplantation. The table shows the rate of positive mice (multilineage reconstituted mice); the numbers in parentheses are the positive mice/tested mice. In the case of fresh CD34−KSL cells, a single cell was transplanted. After determining the percentage of reconstructed mice (table), the percentage of unreconstructed mice (percentage of negative mice on y-axis) was plotted versus the number of input cells, leading to a theoretical HSC frequency based on a Poisson distribution. Inputting 500 cells resulted in 0% negative mice, and these data are not plotted. (E) Fresh or whole cultured cells (Ly5.1) were transplanted into lethally irradiated mice (Ly5.2) along with 5 × 105 BM cells (Ly5.2). Twenty weeks later, peripheral blood from the recipient mice was analyzed by flow cytometry. The plots represent the percentage of donor-derived cells (percentage of Ly5.1+ cells) in the peripheral blood of individual recipients. Bars represent the mean values. **P < .05. (F) In addition, the MAS reflects the repopulating ability of single HSCs, as estimated from the RU value (supplemental Table 2) and HSC number (supplemental Table 2). Data are mean ± SD. *P < .01. **P < .05.

Integrin-β3–mediated signaling leads to the suppression of expansion and cell division on HSCs during ex vivo culture. Forty sorted WT CD34−KSL cells (Ly5.1) were cultured for 5 days with 2C9.G2 or hamster IgG (isotype control) in S-Clone SF-03 serum-free medium supplemented with 50 ng/mL SCF plus 50 ng/mL TPO. (A) After the culture, total cell number was counted. Graph represents fold increase of total cell number after 5 days of culture. Data are mean ± SD. (B) To confirm 2C9.G2 binding to cells during culture, cells were stained with fluorescently labeled hamster IgG and analyzed by flow cytometry: white represents IgG; and gray, 2C9.G2. (C) The percentages of KSL and CD48−KSL cells were also determined by flow cytometry after culture. **P < .05. (D) HSC frequency among the cultured cells was determined using limiting dilution assays. After groups of 10, 30, 50, 100, or 500 whole cultured cells were counted exactly and sorted, the groups were individually transplanted into lethally irradiated Ly5.2 mice along with 2 × 105 BM cells from Ly5.2 mice. This was followed by analysis for chimerism 20 weeks after the transplantation. The table shows the rate of positive mice (multilineage reconstituted mice); the numbers in parentheses are the positive mice/tested mice. In the case of fresh CD34−KSL cells, a single cell was transplanted. After determining the percentage of reconstructed mice (table), the percentage of unreconstructed mice (percentage of negative mice on y-axis) was plotted versus the number of input cells, leading to a theoretical HSC frequency based on a Poisson distribution. Inputting 500 cells resulted in 0% negative mice, and these data are not plotted. (E) Fresh or whole cultured cells (Ly5.1) were transplanted into lethally irradiated mice (Ly5.2) along with 5 × 105 BM cells (Ly5.2). Twenty weeks later, peripheral blood from the recipient mice was analyzed by flow cytometry. The plots represent the percentage of donor-derived cells (percentage of Ly5.1+ cells) in the peripheral blood of individual recipients. Bars represent the mean values. **P < .05. (F) In addition, the MAS reflects the repopulating ability of single HSCs, as estimated from the RU value (supplemental Table 2) and HSC number (supplemental Table 2). Data are mean ± SD. *P < .01. **P < .05.

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