β3-Integrin-mediated maintenance of long-term HSC repopulating activity during ex vivo expansion is dependent on TPO, but not SCF. (A) To assess the influence of β3-integrin signaling, 40 CD34⁻KSL cells (Ly5.2) derived from WT (A) or Y747A mice (B) were cultured for 5 days in the presence of 2C9.G2 or IgG in serum-free medium supplemented with 50 ng/mL SCF or 50 ng/mL TPO. After the culture, whole cultured cells were transplanted with 2 × 10⁵ BM competitor cells (Ly5.1) into lethally irradiated Ly5.1 mice. The plots depict the percentage of donor (Ly5.2)–derived cells in the peripheral blood of individual mice 12 weeks or 20 weeks after transplantation. Bars represent mean values. *P < .01. Recipient mice with donor cell chimerism of less than 1.0% for any lineage were considered not to be reconstituted (negative mice). (C) After culture of 1000 sorted WT CD34⁻KSL cells (Ly5.1) for 5 days with 2C9.G2 or hamster IgG (isotype control) in the presence of TPO, the percentages of KSL and CD48⁻KSL cells were determined by flow cytometric analysis. The values in the dot plots are mean ± SD. (D) After culture, the total cell number was counted. The graph shows the fold increase in total cell number after 5 days of culture. Data are mean ± SD.