Kras ^G12D -carrying MPP show increased proliferation but lack self-renewal. (A–G) Pdzk1ip1-CreER R26^Tom/Tom reporter mice with WT or conditional mutant (Kras^G12D) Kras alleles were administered tamoxifen to induce simultaneous Tom expression and Kras mutation in HSC. At the indicated time points after tamoxifen treatment, mice were injected i.p. with EdU, and EdU incorporation in the stem/progenitor populations in the BM was measured by flow cytometry. (A–C) Mice at 5 wk after tamoxifen treatment were injected with EdU and analyzed 2 h later. (A) Representative staining profiles of gated LSK cells from Kras^G12D mice, showing EdU staining within gated Tom⁻ (blue) and Tom⁺ (red) HSC and MPP. (B) Fraction of EdU⁺ cells in the Tom⁺ (red) or Tom⁻ (blue) subset within the indicated populations from Kras^G12D reporter mice. Total stem/progenitor populations from WT mice were used as controls (gray) due to minimal Tom labeling of progenitors at this time point. Symbols represent individual mice; bars represent the mean ± SD of data pooled from two independent experiments. (C) Fraction of EdU⁺ cells in MPP3 and MPP4 subsets determined as above (data from a single experiment). (D) Mice at 3 wk after tamoxifen treatment were injected with EdU and analyzed 2 h later. The fraction of EdU⁺ cells in HSC and MPP is shown as above (data pooled from two independent experiments). (E–G) Mice at 3 wk after tamoxifen treatment were injected with EdU twice daily and analyzed 48 h after the first injection. (F) Representative staining profiles of gated HSC or MPP from WT (total cells) or Kras^G12D (Tom⁺ or Tom⁻ cells) mice, showing staining for EdU and DNA content. Total EdU⁺ cells or EdU⁻ cells with 2n or 4n DNA content are indicated. (G) Fractions of EdU⁺, EdU⁻ 2n, or EdU⁻ 4n populations among gated HSC or MPP shown as in panels B–E (data from a single experiment). (H) Reconstitution capacity of Kras^G12D MPP. Kras^G12D reporter mice were administered tamoxifen, and their BM was isolated 5 wk later. Tom⁺ HSC (50 cells) or Tom⁺ MPP (1,000 cells) were mixed with 2 × 10⁵ total BM from CD45.1 congenic mice and transferred into lethally irradiated CD45.1 recipients. Two donor mice were induced, and donor cells from each mouse were transferred separately. The accrual of Tom⁺ donor-derived cells was measured in the PB at the indicated time points. Symbols represent the mean ± SD of the recipients of HSC (n = 7) or of MPP (n = 13). Statistical significance was estimated by the Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.