Tyr747 of β3-integrin is essential for the long-term in vivo repopulating and self-renewal activities of mouse HSCs, independent of ex vivo expansion. (A) HSCs from WT or mutant mice were used for serial competitive repopulation assays. Forty sorted CD34⁻KSL cells (Ly5.2) were transplanted into lethally irradiated mice (Ly5.1) along with 2 × 10⁵ BM competitor cells (Ly5.1). Twelve weeks later, the percentage of donor cells (Ly5.2) was determined in peripheral blood (B). A total of 10⁶ BM cells from primary recipient mice were then transplanted into other irradiated mice, followed by secondary analysis of peripheral blood (C). The plot indicates donor-derived cells (percentage of Ly5.2⁺ cells) in the peripheral blood. In addition, recipient mice with donor cell chimerism of < 1.0% for any lineage were considered not to be reconstituted (negative mice). Bars represent mean values. *P < .01. (D) The table shows the total cell number and frequency of HSC subsets among BMCs from both femurs and tibias. (E) Also shown are the frequencies or relative mean fluorescent intensity (MFI) in CD150⁺ and integrin-β3⁺ cells among the CD34⁻KSL population. The value of the MFI obtained in the presence of the isotype control IgG was used as the control. Data are mean ± SD (n = 6-8). The histograms represent the expression of β3-integrin in murine HSCs (CD34⁻KSL) or hematopoietic progenitors (CD34⁺KSL) derived from WT, integrin-β3^−/−, and Y747A or L746A mutants, all of which are shown in white. The isotype control is in gray.