Patterns of engraftment of CD34− cells from NPM1-mutated AML in immunodeficient mice. (A-F) Example of engraftment (pattern 3) in NOG mice inoculated with 10 × 106 CD34− cells (purity 99.9%) from patient 22. (A) Flow cytometric analysis of bone marrow (6 weeks after inoculum) showing engraftment of human myeloid (93% hCD45+/CD33+) cells, which appeared CD34− (0.3% CD34+ cells) and CD11b+ (70.8% hCD45+/CD11b+). A small percentage of T cells (5.9% hCD45+/CD3+) was also detected. (B-D) Tibia paraffin sections showing massive marrow infiltration by a double population: (1) mononuclear cells close to bone trabeculae (B double arrows; inset, from a different section), which are MPO+ and PGM1(CD68)+ (C-D double arrows); (2) mature histiocytes located in the central area of bone marrow (B single arrow), which are MPO− and PGM1+ (C-D single arrows). Asterisk in panel B inset and in panel C indicates bone area. (E-F) Leukemic origin of these cells was confirmed by WB with anti-NPM mutant (anti-NPMm) antibody (E) and genomic DNA fragment analysis showing double peaks (F). (G-I) Example of engraftment (pattern 4) in NOG mice inoculated 6 weeks before with 2 × 106 CD34− cells (purity 99.99%) isolated from patient 21. Tibia paraffin sections showing marrow infiltration by a mixed population of mature histiocytes (single arrow) and lymphocytes (double arrows). Immunostaining for hCD45 confirmed the human origin of these cells. (B,G) Hematoxylin and eosin. (C-D,H-I) APAAP; hematoxylin counterstaining. Images were collected using an Olympus B61 microscope and a UPlanApo 40×/0.85 (B-D,G-H) and a UPlan FI 100×/1.3 NA oil objective (B inset, I); Olympus E330-ADU1.2× camera; and Adobe Photoshop 7.0.
