Studies with AML patient-derived xenograft cells

Primary MLL-AF9-expressing cells were obtained from BM samples of diagnosed AML pediatric patients stored in the BioBank of the laboratory of Pediatric Hematology of the University Hospital of Padua, (Italy) according to the guidelines of the local ethics committee. Initial AML xenografts were established by tail vein injection with 8 × 10⁶ primary cells suspended in 300 μl of PBS in 6- to 8-week-old NSG mice, which were purchased from Charles River (Wilmington, MA, USA). All animal experiments were performed in accordance with institutional guidelines and established protocols [53]. Engraftment was monitored by weekly blood collections and flow cytometry analysis with antihuman CD45 (BD Biosciences). The engraftment rate was defined by the number of days required for the transplanted human CD45+ cells to reach at least 20% in the peripheral blood. Human leukemic cells from the spleens of engrafted mice were collected and cultivated in RPMI supplemented with 10% Human serum (Euroclone), antibiotics, and cytokines SCF, FLT-3L and TPO (40 ng/ml for each), IL-3 and IL-6 (20 ng/ml for each). (All cytokines were obtained from Inalco, Milan, Italy). For ex vivo experiments, two independent biological replicates were performed.