Deficiency in the Matk/CHK gene alters early hematopoietic stem cell populations and leads to the hyperproliferation of pre-B cells in the presence of IL-7. (A) Frequency of SPKLS cells in the Matk/CHK^–/– mice. BM cells from Matk/CHK^+/+ (WT) and Matk/CHK^–/– (KO) mice were stained as described.¹⁵  Lineage-positive cells were depleted using magnetic columns and a cocktail of biotinylated lineage-specific antibodies. The BM cells were stained with Hoechst 33342 followed by antibody staining (anti–Sca-1 and anti–c-Kit). The boxed regions define the SPKLS cells. The results shown are representative of 3 independent experiments. The lack of Matk/CHK protein in the homozygous mice was confirmed by Western blot analysis of adult brain samples (not shown), which normally express high levels of Matk/CHK. (B) Frequency of CMP and CLP in the Matk/CHK^–/– mice. BM cells from the Matk/CHK^+/+ (WT) and Matk/CHK^–/– (KO) mice were stained as described.¹¹  Lineage-positive cells were depleted using magnetic columns and a cocktail of biotinylated lineage-specific antibodies. CMP was defined by the Lin^– IL-7Rα^–Thy-1^–Sca1^–c-Kit⁺ phenotype. CLP was defined by the Lin^– IL-7Rα⁺ Thy-1^– Sca1_low c-Kit_low phenotype. The results shown are representative of 2 independent experiments. (C) Matk/CHK^–/– mice produce a higher number of pre-B colonies, but not of the CFU-Mix. Total BM cells from Matk/CHK^+/+ (WT) and Matk/CHK^–/– (KO) mice were placed in methylcellulose cultures with a cocktail of cytokines either for the CFU-Mix or pre–B cell colony formation. The cells were plated in triplicate cultures. After 10 to 12 days of culture, pre–B cell colonies were counted. CFU-Mix colonies were counted after 10 to 14 days of culture. Analysis of colony formation was conducted using an inverted microscope. A colony was defined as consisting of at least 200 cells for the CFU-Mix and 50 cells for the pre-B colony. There were no significant differences between genotypes in the CFU-Mix assay (mean 27.4 versus 29.8, WT versus Matk/CHK^–/– cells, based on 4 independent experiments). The number of pre-B colonies from the Matk/CHK-deficient BM cells was approximately 4-fold higher than that from the WT BM cells (mean 7.5 versus 29.7, WT versus Matk/CHK^–/–, based on 3 independent experiments).