MN1 and NUP98HOXD13 cooperate to induce leukemogenesis from human CB cells in vivo. (A) Outline of the experimental design to evaluate the in vitro leukemic stem cell expansion potential of transduced CD34⁺ CB cells in suspension culture. (B) In vitro proliferation of transduced and sorted cells cultured in myeloid-promoting conditions (Steel factor, FL, IL-3, IL-6, and TPO, each 10 ng/mL) (starting between 33, 38, and 50 days after transduction; mean ± standard error of the mean, CTL n = 4, for MN1, ND13, MN1+ND13 n = 6). *P < .05 for the comparison CTL vs ND13, CTL vs MN1+ND13, MN1 vs ND13, and MN1 vs MN1+ND13 on day 15. (C) Survival of NSG-3GS mice receiving transplants of CB cells cultured for 1 to 3 months in vitro (MN1, n = 6; ND13, n = 16; MN1+ND13, n = 28). **P < .01. (D) Correlation between diagnosis of AML and survival and the number of MN1⁺ND13⁺ CB cells transplanted in NSG-3GS mice (mice from survival curve in Figure 2C). Each dot represents a mouse. Red circle indicates the absence of AML at the time of sacrifice. (E) Number of disease initiating cells per 10⁶ injected cells in ND13 and MN1+ND13-transduced CB cells transplanted in NSG-3GS mice after 1 to 3 months of culture. (F) Morphology of BM cells of ND13 (103 days posttransplant, left panel) and MN1+ND13 (38 days posttransplant, right panel) mice at sacrifice. Wright-Giemsa–stained marrow cytospins from representative mice. Micrographs were acquired using an Axioplan2 Zeiss microscope and images were captured using OpenLab software (Perkin Elmer); original magnification ×400. (G) Immunophenotype of GFP⁺YFP⁺ BM cells from a MN1+ND13 mouse with AML at sacrifice. Representative FACS blots are shown. (H) Gene set enrichment analysis of indicated signatures comparing MN1⁺ND13⁺ and ND13⁺ CB cells cultured for 60 days in myeloid promoting conditions. FDR, false discovery rate; NES, normalized enrichment score.