Leukemic engraftment of CD34⁺ cells from NPM1-mutated AML in immunodeficient mice. (A-D) Bone marrow paraffin sections from a NOG mouse, inoculated (12 weeks before) with 2 × 10⁶ CD34⁺ cells (purity 98.3%) isolated from 1 patient (patient 17) with NPM1-mutated AML, showing infiltration by human cells, which are positive at immunostaining with a specific anti–human CD45 antibody (A), display a myeloid phenotype (myeloperoxidase, MPO-positive; B) and aberrant cytoplasmic expression of nucleophosmin (NPM; C arrow). Double arrows in panel C indicate a normal osteoblast with nucleus-restricted NPM on the endosteal surface of bone. As expected, nucleolin/C23 was also restricted to the nucleus (D, arrow). (A-D) APAAP; hematoxylin counterstaining. Images were collected using an Olympus B61 microscope and a UPlanApo 20×/0.70 (A-B) and a UPlan FI 100×/1.3 NA oil objective (C-D) Camedia 4040, Dp_soft Version 3.2; and Adobe Photoshop 7.0. (E) Flow cytometric analysis of murine bone marrow confirmed engraftment of human cells (hCD45, 52%) which are prevalently CD33⁺ (92.7%) and express the monocytic marker CD11b (18.6%). CD34/CD38 staining pattern of engrafted hCD45⁺ cells is also shown (E right panels). CD34⁺/CD38⁻ cells (gate on F4) were also CD123⁺ (E right bottom panel). (F) May-Grünwald-Giemsa staining of a cell cytospin preparation from murine bone marrow of the same case (patient 17) showing large-size leukemic cells with monocytoid appearance (cleaved nucleus, basophilic cytoplasm; arrow) admixed with cells of murine origin (double arrow indicate a normal murine polymorphonucleated cell). (G) WB analysis with anti-NPM mutant specific antibodies (anti-NPMm) of mouse bone marrow cells confirmed expression of NPM1 mutated protein in cells engrafted in mice (lane 2) as well as in the original AML patient sample (lane 3). Positive (OCI/AML3, lane 1) and negative (U937, lane 4) controls are shown.