Identification of the APL-initiating cells. (A) Kaplan-Meier survival curve of sublethally irradiated mice transplanted with different sorted cell populations isolated from leukemic bone marrow. Injections of sorted cells from subpopulations 3 and 4 (3000 cells) and subpopulation 9 (3000 and 15 000 cells) and 2000 KSL (2000 cells, c-kit⁺, Sca1⁺, lineage⁻) isolated from leukemic bone marrow were injected into sublethally irradiated mice. Cells isolated from subpopulations 3 and 4 were able to generate leukemia with a latency of 38 days (○, n = 8). Mice injected with a high concentration of subpopulation 9 (15 000 cells, ■, n = 4) developed leukemia with incomplete penetrance, whereas none of the mice injected with 3000 cells from subpopulation 9 (□, n = 6) or KSL (crosshatched line, n = 6) developed leukemia. (B) Cells accumulating in leukemic bone marrow and their counterparts isolated from wild-type bone marrow were sorted (subpopulations 3 and 4; Bi; red bold rectangle in CD34⁺/c-kit⁺ fraction) and stained (Bii) with Wright-Giemsa (original magnification ×100). (Biii) As few as 30 cells accumulating from populations 3 and 4 initiate leukemia in sublethally irradiated recipient mice. Decreasing amounts of cells were injected into sublethally irradiated recipient mice (3000 cells, ○, n = 6; 300 cells, ●, n = 8: and 30 cells, ▲, n = 10). (Biv) Injection of cells accumulating in leukemic bone marrow (subpopulations 3 and 4) results in a 100-fold enrichment of LICs compared with injection of unsorted cells (○, injection of 3000 sorted cells, n = 6, compare with ●, injection of 300 000 unsorted leukemic bone marrow cells, n = 3). (C) Kaplan-Meier survival curve of mice after secondary transplantation. Thirty sorted LICs from subpopulations 3 and 4 (CD34⁺, c-kit⁺, FcγRIII/II⁺, Gr1^int) were used in the primary transplantation. Subsequently, 2.5 × 10⁵ to 5 × 10⁶ unfractionated leukemic spleen cells were isolated from a moribund primary leukemic mouse and transplanted into sublethally irradiated secondary recipients (n = 6), which subsequently developed a secondary leukemia.