miRNA expression during human T-cell development. miR-125b is highly expressed at early immature stages. (A) Schematic representation of human T-cell development. Human thymocytes obtained from healthy donors were sorted according to established cell surface markers: immature DN including pro-T (CD34⁺/CD7⁺CD1a⁻/CD4⁻) and pre-T (CD34⁺/CD7⁺/CD1a⁺/CD4⁻), ISP (CD34⁻/CD1a⁺/CD4⁺/CD8⁻/CD3⁻), IDP (CD4⁺/CD8⁺/CD3⁻), mature DP (CD4⁺/CD8⁺/CD3⁺), and mature SP (SP4: CD4⁺/CD8⁻/CD3⁺ and SP8: CD4⁻/CD8⁺/CD3⁺) thymocytes. (B) Heatmap of the 36 most differentially expressed miRNAs at 7 sequential stages of T-cell development (n = 3). Shown are miRNA expression levels relative to the average of the row. The white color corresponds to the mean expression across all stages (ΔCt = 0). Red and green colors correspond, respectively, to overexpression (ΔCt < 0) and underexpression (ΔCt > 0). (C) Comparative expression of the 20 most abundant miRNAs in T-ALLs and in thymi. miRNA expression is given as percentage of RNU48 control. (D-F) Relative miRNA expression in T-cell subsets (2^−∆∆Ct). Levels in DP cells were used as calibrator (n = 3) (D) miR-125b; (E) miR-99a; (F) miR-100. ***P < .001, Mann-Whitney U test. HSC, hematopoietic stem cell; MLP, multilymphoid progenitor; MPP, multipotent progenitor.