Effects of overexpression of Ezh2 in HSCs in tertiary recipients. (A) Representative fluorescence-activated cell sorting (FACS) plot showing donor contribution in a recipient transplanted with 2.5 × 10⁶ twice serially transplanted Ezh2-overexpressing cells in competition with 5 × 10⁵ freshly isolated CD45.2⁺ BM cells. Left panel shows contribution when gated on myeloid cells; right panel shows donor contribution for lymphoid cells. (B) The percentage of CD45.1⁺GFP⁺ cells in the peripheral blood in all recipients (n = 28) 3 months after tertiary transplantation. Cells were cotransplanted in different ratios (5:1, ▵; 2:1, ⋄; 1:1, ○) with freshly isolated BM cells. (C) Survival curve of tertiary recipients that received transplantations of serially transplanted BM cells without cotransplantation of freshly isolated BM cells (control, ▪; Ezh2, ). (D) CRI comparing transduced CD45.1⁺GFP⁺ stem cells versus nontransduced CD45.1⁺GFP^- stem cells in primary, secondary, and tertiary recipients (control, ▪; Ezh2, ). (E) CRI comparing transduced CD45.1⁺GFP⁺ stem cells with freshly isolated BM cells after 1, 2, and 3 serial transplantations (control, ▪; Ezh2, ).