Progressive loss of competitive repopulating activity is accompanied by an increase in SA-β-gal expression in hematopoietic stem progenitor cells. (A) BM cells derived from Ews^−/− and WT littermates were stained as described in Methods for Lin⁻, LSK, and CD150⁺CD48⁻ LSK populations and subsequently analyzed for apoptotic cell death. The percentages of Annexin V-positive cells are indicated. (B) Top panel: BM cells from Ews^−/− mice (KO, CD45.2; right panel) and their Ews^+/+ littermates (WT, CD45.2; left panel) were mixed with an equal number of competitor cells (CD45.1) and transplanted into lethally irradiated recipient (CD45.1.2) mice. Peripheral blood from the transplanted recipients was analyzed for CD45.1/CD45.2 expression by flow cytometry every 4 weeks after transplantation. Bottom panel: representative FACS profile showing donor (Ews^−/−, CD45.2) and competitor (CD45.1) contributions at 20 weeks after transplantation in the peripheral blood of recipients. (C) Top panel: recipient mice were killed 20 weeks after transplant. Ews^+/+- and Ews^−/−-derived LSK cells were stained for SA-β-gal activity. At least 30 cells from 3 random fields were counted in each experiment and the percentage of SA-β-gal–positive cells is shown on the y-axis. *Statistically significant difference (2-tailed Student t test, n = 2, P = .01). Bottom panel: flow cytometric analysis of Ews^−/− and WT littermate-derived LSK cells for the expression of SA-β-gal before and after transplantation. The cells were stained with a fluorescent β-galactosidase substrate (C₁₂FDG) and analyzed by flow cytometry.