CD34⁺ cells from NPM1-mutated AML generate CD34⁻ NPMc⁺ AML in mice. (A-F) Flow cytometry and immunohistochemistry analysis of CD34 antigen expression in leukemia developed in immunodeficient mice (NOD/SCID) inoculated with CD34⁺ cells (purity 98.5%) isolated from 1 patient (patient 10) with NPM1-mutated AML (27% CD34⁺ cells in the original sample; A). Flow cytometric analysis of mice bone marrow (BM;16 weeks after the inoculum) showing engraftment of myeloid (CD33⁺ and CD117⁺) human cells, which appeared mainly CD34⁻ (1.9% CD34⁺ cells; B). The leukemic nature of these cells was confirmed by WB with anti-NPM mutant antibody (C), as well as morphologic analysis (D hematoxylin and eosin) and expression of nucleophosmin (NPM) in the cytoplasm of human leukemic cells on bone marrow paraffin sections (E, arrow). As staining control, NPM was characteristically nucleus-restricted in normal murine cells (E, double arrows). The asterisk in (E) indicates an empty space originally filled by bone. CD34 immunostaining was negative in leukemic cells (F arrow). (E-F: APAAP; hematoxylin counterstaining). All images were collected using an Olympus B61 microscope and a UPlan FI 100×/1.3 NA oil objective; Camedia 4040, Dp_soft Version 3.2; and Adobe Photoshop 7.0. A vertical line has been inserted in panel C to indicate a repositioned gel lane. (G-I) Serial flow cytometric evaluation of CD34 antigen expression in human leukemic cells grown in immunodeficient mice (NOD/SCID/IL2Rγ^null) killed at 9 (H) and 12 (I) weeks after injection of the same number of CD34⁺ cells (2 × 10⁶, purity 98.3%) isolated from 1 patient (patient 17) with NPM1-mutated AML (27% CD34⁺ cells in the original sample; G).