Expanded CM Pre-Meg/E progenitors are capable of leukemia-initiation. (A) Schematic of leukemia initiation experiment design. Cbfb^+/56M/Mx1-Cre mice were induced by poly (I:C). LSKs, Pre-Meg/Es, Pre-GMs, and GMPs were sorted 2 weeks later and subjected to (B) apoptosis analysis, (C) CFC and replating, or (D) transduced with MIG-Mpl retrovirus followed by transplantation and monitored for survival, (E-F) phenotype, and (G) engraftment in serial transplants. (B) The frequency (%) of apoptotic cells defined by Annexin-V⁺ in CM or Ctrl LSK, Pre-GM, GMP, or Pre-Meg/E cells 24 hours after IR (2 Gy). Bars and lines indicate mean ± SD. (C) Number of CFCs derived from Pre-GM (left) or Pre-Meg/Es (right) in first plating (1000 cells) and in serial replating (2 × 10⁴ cells) using MethoCult M3434. Shown is mean ± SEM of ≥3 independent experiments performed in duplicate. (D) Kaplan-Meier survival curves of recipients transplanted with CM/Mpl LSKs (7-8 × 10³ cells), Pre-Meg/Es, Pre-GMs, or GMPs (2.5 × 10⁴). (E) Peripheral white blood cell counts (10³/μL; left) and spleen weights (g; right) in CM-Mpl LSK– or Pre-Meg/E–derived leukemic mice. The gray area indicates the normal range detected in normal Ctrl mice (n = 8). (F) The frequency (%) of various immunophenotypic populations in GFP⁺ BM of leukemic mice transplanted with CM-Mpl LSK (n = 4) or CM-Mpl Pre-Meg/E (n = 6). (G) Kaplan-Meier survival curves of secondary (n = 11) and tertiary (n = 6) recipients transplanted with 1 × 10⁶ BM cells from moribund mice transplanted with CM-Mpl Pre-Meg/E. *P < .05; **P < .01; ***P < .001.