Overexpression of N5A fusion in CB-CD34⁺cells induces acute megakaryoblastic leukemia and multilineage leukemia subtypes in xenograft models. (A) Representation of the experimental procedures used to establish in vivo models of N5A-driven leukemia. Human CD34⁺ cells were isolated from CB and transduced as described in Figure 1A. After GT evaluation, 70% of the cells from each well were injected into a primary recipient mouse. Leukemia xenograft cells were collected and characterized phenotypically, molecularly, and functionally. (B) Distribution of generated xenograft models classified by leukemia subtypes based on molecular markers and cytology analyses (supplemental Table 3). Models originated from 6 experimental groups initiated from 7 independent CB samples (supplemental Table 2). (C) Detection of N5A fusion transcript expression by RT-PCR with RNA isolated from BM or spleen (Sp) cells of leukemic mice, as indicated. Normal nontransduced CB-CD34⁺ sample was used as negative control (CTL). (D-J) AMKL xenograft models. (D) Brittle white bones and mild splenomegaly were observed in AMKL xenograft models (xAMKL-3, N5A vector) compared with control xenograft-recipient mice (CTL, empty vector). (E) Representative hematoxylin-phloxine-saffron–stained longitudinal sections of tibia BM harvested from xCTL and xAMKL-3 recipient mice. (F) Blasts infiltration percentage (hCD45^lowCD41⁺/CD61⁺ cells) in BM and spleen of primary AMKL (n = 6). (G) Giemsa-stained peripheral blood (PBL) smear and BM cytospin from an AMKL primary (1^ary) recipient mouse (xAMKL-3) highlighting the presence of leukemic blasts (48 weeks after transplantation). (H) Fluorescence-activated cell sorting (FACS) profiles revealing typical hCD45^lowCD34⁻CD41⁺ megakaryoblasts in the BM of a primary recipient mouse (xAMKL-3), along with CD45^hiCD3⁺-activated T cells. The spleen is infiltrated by CD45^hiCD3⁺ activated T cells with hCD45^lowCD34⁻CD41⁺ barely detectable. Characterization of an additional mouse xenograft model (xAMKL-1) with detailed T-cell immunophenotype is shown in supplemental Figure 2. (I) Giemsa-stained BM cytospin and spleen touch preparations showing leukemic blasts derived from a secondary (2^ary) recipient mouse with a 2.2 × 10⁶ xAMKL-3 BM–cell transplant (33 weeks after transplantation). (J) FACS profiles revealing hCD45^lowCD34⁻CD41⁺GFP⁺ megakaryoblasts in the BM and spleen of the secondary recipient mouse. *P < .05. HSC, hemopoietic stem cell; LSC, leukemic stem cell.