Shp2 ablation results in significant decrease in BM HSCs/progenitors. (A, B) In vitro CFU assays for whole BM cells or purified LSK cells. 50 000 BM (A) or 250 sorted LSK (B) cells were seeded on methylcellulose medium Methocult 3434 (StemCell Technologies Inc) with cytokines for 14 days before colony numeration. (n = 3-6). (C) Representative spleen colonies from CFU-S day 12 assay. Quantification of CFU-S₁₂ suggests a decrease of HSCs and multipotent progenitors (n = 5-6). (D) Competitive reconstitution strategy 1: injection first/transplantation later. Donor mice were treated with poly-I:C before BM transplantation. (E) Representative FACS plots illustrate BM chimerism in 1:1 donor versus competitor reconstitution assay. (F) Percentage of CD45.2 donor derived cells using 1:1 donor to competitor ratio (n = 6-10). (G) Percentage of CD45.2 donor-derived cells within different lineages in 1:1 ratio competitive reconstitution (n = 4-5). (H) Shp2^Δ/Δ BM contributes to less than 1% of LSK chimerism in 1:1 ratio competitive reconstitution (n = 4). (I) 500 sorted LSK cells were transplanted into lethally irradiated recipients in competition with 5 × 10⁵ CD45.1 cells in the injection first/transplantation later setting. Peripheral chimerism was analyzed at indicated time points. fl/fl:cre⁻ LSK cells barely gave rise to any peripheral blood cells (n = 4-5). ***P < .001, **P < .01,*P < .05, error bars are SD; see also supplemental Figure 2.