Ectopic expression of miR-125b in HPCs mimics the effects of TLX3 on T-cell differentiation. Sorted human HPCs were transduced with either miR-125b/OE or CTRL mCherry-traceable vectors. T-cell differentiation was followed as described in Figure 4A. (A) Relative miR-125b expression at days 21 (N = 3) and 42 (N = 5) in miR-125b/OE cells compared with CTRL cells. *P < .05, Mann-Whitney U test. (B) miR-125b/OE enhances T-cell growth. Cumulative T-cell numbers at day 42 compared with day 21 of culture for miR-125b/OE and CTRL cells (n = 8). *P < .05, Wilcoxon test. (C) miR-125b/OE induces accumulation of immature DN cells and blocks sCD3 acquisition by DP cells. Shown are the data from the representative experiment at day 42. Proportions of T-cell fractions are relative to gated mCherry⁺/CD7⁺/CD5⁺ T cells. (D-E) Human T-cell development in mouse xenograft model. Sorted human HPCs were transduced with either miR-125b/OE or CTRL mCherry-traceable vectors and IV injected into NSG recipients. Mice were sacrificed at day 90 posttransplantation. (D) Proportion of ISP4, IDP, DP, SP4, and SP8 populations recovered from total thymi was estimated within the mCherry⁺/CD7⁺/CD5⁺ compartment (n = 3). *P < .05, multiple Student t test. (E) Shown are the representative plots; 1 mouse per group. Proportions are given relative to gated CD4⁺/CD8⁻; CD4⁺/CD8⁺ and CD4⁺/CD8⁺ fractions. (F) Flowchart of leukemia competitive transplantation experiments. Sorted T-ALL (M106) infected cells (mCherry⁺) expressing or not miR-125b were mixed at a 1:1 ratio with sorted control (eGFP⁺) or sorted wild-type (WT) cell populations and 500 cells were transplanted into NSG recipients. The hosts were monitored for T-ALL development by BM analysis. (G) Relative miR-125 expression (R-Q-PCR), normalized to RNU44 (2^−∆Ct), in M106 T-ALL blasts yielded from BM of recipient mice at sacrifice (n = 4 for CTRL; n = 3 for 125b/OE). (H) T-ALL cell expansion in periphery assessed by CD45⁺/CD7⁺ cells in BM at 2 time points. The percentage of CD45⁺/CD7⁺ cells was measured gating on live cells. (I) Percentage of (mCherry⁺) CD45⁺/CD7⁺ T-ALL cells in BM was measured at day 55. The dashed line indicates the percentage of mCherry⁺ cells (50%) at the day of injection. (H-I) CTRL (n = 11); 125b/OE (n = 12). (J) Kaplan-Meier survival plot depicting accelerated leukemia onset in the mouse recipients with ectopic expression of miR-125b. Death points of the recipients were recorded when found dead or moribund. The dashed line indicates median survival time. Data: (G-I) ****P < .0001; *P < .05, Mann-Whitney U test; (J) Mantel-Cox test.