Overexpression of TLX3 induces miR-125b expression and correlates with expansion and differentiation arrest of T-cell progenitors. (A) Design of gain-of-function experiments. Human HPCs isolated from UCB were transduced with lentiviral expression vectors encoding, respectively, TLX3 (TLX3/OE) or eGFP alone (CTRL), miR-125b (125b/OE), or mCherry alone (CTRL) and cocultured with the murine MS5 stromal cells constitutively expressing human NOTCH ligand Δ-like 1 (MS5/DL1) in the presence of a cytokine cocktail. (CD7⁺/eGFP⁺) or (CD7⁺/mCherry⁺) T/NK progenitors were sorted and replated at day 21 of coculture with MS5/DL1 stromal cells. Thereafter, differentiation was weekly monitored and HPC-derived T cells were fractionated at day 42. (B) Left, Comparison of TLX3 expression in transduced T cells and in primary TLX3 T-ALL by R-Q-PCR (n = 8). Shown are absolute copy numbers of TLX3 normalized to housekeeping gene ABL quantified by using external DNA dilution standards. Top right, Western blot analysis illustrating TLX3 protein expression in sorted CD7⁺/eGFP⁺ T cells at day 21. (C) miR-125b expression levels in TLX3/OE vs CTRL CD7⁺/CD5⁺ T cells measured at day 21 (N = 5) and 42 (N = 7) of differentiation. Bar represents medians. ***P < .001, **P < .01, Mann-Whitney U test. (D) TLX3 induces proliferation of T-cell progenitors. Ratios of T-cell numbers at day 42 vs day 21 obtained in 7 independent experiments. *P < .05, Wilcoxon test. (E) Fluorescence-activated cell sorting (FACS) data from a representative experiment at day 42 of culture are shown. Proportions of T-cell fractions are given relative to gated eGFP⁺/CD7⁺/CD5⁺ T cells. IL, interleukin; sCD3, soluble CD3; SCF, stem cell factor.