Myelomonocytic and monocytic cell expansion in the BM of p15Ink4b^fl/fl-LysMcre mice. (A) Representative flow cytometric analysis of BM single-cell suspensions for Gr-1 and Mac-1 highlights expanded mature myeloid (Gr-1⁺/Mac-1⁺) and monocytic (Gr-1^−/lo/Mac-1⁺) populations as well as immature myeloid cells (Mac-1^+/lo/c-Kit⁺) in p15Ink4b^fl/fl-LysMcre mice. Increased population of Mac-1⁺/CD56⁺ cells detected in targeted mice confirms a nonreactive myeloproliferative disease in targeted mice. (B) Histopathologic sections of BM from representative p15Ink4b^wt/wt-LysMcre and p15Ink4b^fl/fl-LysMcre animals. Hematoxylin and eosin-stained and immunohistochemistry using anti F4/80 and MPO antibodies of paraffin-embedded bone sections. Scale bars represent 20 μm. (C) Frequencies of myeloid progenitors in BM cells isolated from targeted and wild-type mice. Lineage-depleted BM cells were purified by SpinSep (StemCell Technologies) and stained with fluorochrome-conjugated monoclonal antibodies to CD34, c-Kit, interleukin-7 receptor (IL-7R), Sca-1, and FcγRII as described previously.¹¹  Myeloid progenitors (CMPs, CD34⁺FγcRII^lo, GMPs, CD34⁺FcγRII^hi, and MEP CD34⁻FcγRII^lo) were identified by flow cytometry within the Lin⁻IL-7R⁻Sca-1⁻c-Kit⁺ BM cell population. Data are mean ± SD (error bars) from 3 independent analyses. (D) In vitro differentiation of FACS-purified CMP progenitors. Lineage-depleted BM cells were purified by SpinSep (StemCell Technologies) and stained with fluorochrome-conjugated monoclonal antibodies to CD34, c-Kit, interleukin-7 receptor (IL-7R), Sca-1, and FcγRII as described previously.¹¹  CMP (CD34⁺FγcRII^lo) purified by FACS from the Lin⁻IL-7R⁻Sca-1⁻c-Kit⁺ BM cell population using FACSAria cell sorter (BD Biosciences) were expanded for 3 days in MyeloCult medium supplemented with SCF (50 ng/mL) and IL-11 (100 ng/mL). Cells were plated in triplicate in methylcellulose media (MethoCult M03434, StemCell Technologies). Burst-forming units–erythroid and granulocyte-macrophage colony-forming cells colonies were counted on days 7 and 12, respectively. The averages of duplicate platings performed in triplicate for each genotype, and SD values (error bars) are shown. (E) Flow cytometric analysis of BrdU incorporation into Mac-1⁺ BM cells indicates an increased proliferation of this population in p15Ink4b^fl/fl-LysMcre mice.