Ex vivo erythroid differentiation of CD34⁺ from ENT1_null patients. (A) Erythroid differentiation of CD34⁺ progenitors was evaluated following rEPO-induced differentiation as a function of expression of the erythroid surface markers GPA and Band3. Representative histograms showing the differentiation of CD34⁺ progenitors from P1 (blue), P2 (green), P3 (red), controls (black), and nonspecific immunoglobulin G (IgG) staining (gray shaded) are presented at days 5 and 8 of differentiation. Crossed boxes are present when insufficient cells were available for analyses. (B) Enucleation of erythroblasts from P1 and P2 ENT1_null patients and respective controls (Ctrl1 and Ctrl2) was monitored at day 12 of erythroid differentiation as a function of DRAQ5 staining. Representative fluorescence-activated cell sorting plots and the percentages of enucleated cells (DRAQ5⁻) are shown. (C) The percentages of GPA⁺ cells and Band3⁺ cells were quantified at day 5 of differentiation. Mean percentages ± standard error of the mean for controls (black dots, n = 7 independent experiments) and P3 ENT1_null patient progenitors (red triangles, n = 2 independent experiments in duplicate) are presented. ****P < .0001; ***P = .0002 (unpaired Student t test). (D) Growth curves of progenitors from the 3 ENT1_null patients (P1, n = 1; P2, n = 1; P3, n = 4) and healthy donors (n = 9) were evaluated at indicated days of erythroid differentiation. **P < .01 (unpaired Student t test). (E) Representative images of MGG-stained Cytospins at day 5 of rEPO differentiation of control and P3 ENT1_null progenitors are shown (top). The bar represents 10 µm. Quantification of the different cell types is presented (n = 3 independent experiments, bottom). (F) Characterization of P3 and control progenitors were evaluated at day 7 of the expansion phase (before addition of rEPO) as a function of IL-3R, CD34, CD36, CD38, and CD33. Representative histograms showing nonspecific IgG staining (gray shaded), control (black line) and P3 (red line) staining are presented. The expression of CD34 and CD36 surface markers was analyzed on the IL-3R^neg cells to identify burst-forming unit erythroid (IL-3R⁻CD34⁺CD36⁻, green square) and CFU-E (IL-3R⁻CD34⁻CD36⁺, red square) and representative percentages are indicated. (G) CD34⁺ progenitor cells from a healthy donor and the P3 ENT1_null patient were cultured in a methylcellulose semisolid culture assay and representative images of the generated colonies are shown. Quantification of the generated colonies is presented for 2 technical replicates.