Overexpression of NUP98-KDM5A efficiently induces maturation block and sustains the proliferative and progenitor capacities of CB-CD34⁺cells. (A) Experimental procedures used to establish in vitro models of N5A-driven leukemia. CD34⁺ cells isolated from single-donor CB were seeded in 96-well plates and infected with lentiviral particles carrying the chimeric NUP98-KDM5A oncogene. The lentiviral vector encodes FLAG-tagged NUP98-KDM5A and a GFP reporter gene, driven by MNDU3 and PGK promoters, respectively. Independent cell lines derived from each well were grown for 3 to 5 days in optimized culture conditions before GT evaluation and further in vitro expansion (20% of the cells from each well). (B) CD34⁺GFP⁺ enrichment in long-term cultures of CB-CD34⁺ cells transduced with a control (CTL, n = 4) or NUP98-KDM5A (N5A, n = 12) vector. (C) Short-term proliferation kinetic of transduced cells in independent cultures of CB-CD34⁺ cells transduced with N5A or control lentiviral vector. Cultures were initiated from 2 independent CBs (eg, CB1 and CB2) transduced with control (n = 6 per CB) or N5A (n = 14 per CB) lentiviral vector, as indicated. (D) Fluorescence-activated cell sorting profiles showing the time course of GFP and CD34 expression in 2 independent samples transduced with control (eg, CTL_C) or N5A lentiviral vector (eg, N5A_A). Transduced CB-CD34⁺ cells were derived from a single donor. (E) Giemsa-stained cytospins showing immature cellular morphology of an N5A-expressing cell line (N5A_C, bottom) at day 80 and differentiation of matched-CTL cells at day 59. Original magnification ×1000. (F) Acquisition by flow cytometry showing differentiation of control cells (GFP⁺CD34⁻ C-KIT^hi) and a maturation arrest of N5A-transduced cells (GFP⁺CD34⁺ C-KIT^low). (G) Graph showing the percentage of GFP⁺KIT^low immature cells in each indicated culture, defined as median fluorescence intensity <1.5 × 10⁴ for KIT^low cells; n = 3 independent experiments, n = 4 CB units, n = 43 cultures of N5A cells, and n = 19 cultures of CTL-cells. (H) Clonogenic progenitor frequency for freshly isolated (day 0, n = 2) and CTL or N5A-transduced CB-CD34⁺ cells, plated at days 8 and 88 of culture (n = 2 for CTL; n = 4 for N5A; mean ± standard error of the mean [SEM]). Phenotypic classification of clonogenic progenitors is presented in supplemental Figure 1. (I) Representative image of a typical long-term colony generated from the N5A_C cell line at day 60 of culture. Top: bright field microscopy; bottom: epifluorescence microscopy. Original magnification ×10. **** P < .0001.