HSC specification to the erythroid lineage is dependent on the expression and function of the SLC7A1 arginine transporter. (A) Cell-surface expression of SLC7A1 was evaluatedafter rEPO-induced erythroid differentiation of CD34⁺ progenitors (day 4) and representative histograms in the absence or presence of rEPO are presented (left). Relative MFIs of SCL7A1 in rEPO-induced progenitors are compared with levels detected in the absence of EPO (right, n = 5 independent experiments). (B) Arginine uptake in the absence or presence of rEPO (day 4) was monitored using L-[2,3,4-³H)] arginine monohydrochloride (2 μCi) for 10 minutes at room temperature. Uptake in the presence of EPO was arbitrarily set at 1 (means of triplicates in 3 independent experiments). (C) Surface SLC7A1 levels were monitored at days 4, 7, and 10 of rEPO-mediated differentiation and representative histograms are shown (left). Mean fluorescent intensities (MFIs) of SLC7A1 staining relative to day 4 were quantified (right, n = 9). (D) Arginine uptake was evaluated at days 4, 7, and 10 of erythroid differentiation; arginine uptake at day 4 was arbitrarily set at 1 (means of triplicates in 4 independent experiments). (E) CD34⁺ progenitors were transduced 3 days with GFP-tagged shCTRL and shSLC7A1 lentiviral vectors and representative histograms of cell-surface SLC7A1 expression on GFP⁺ cells (left) as well as quantification of SLC7A1 expression relative to control-transduced cells is shown (right, n = 8 independent experiments). (F) Arginine uptake was monitored in fluorescence-activated cell sorter–sorted progenitors transduced with shCTRL and shSLC7A1 vectors at day 4 and uptake levels relative to the shCTRL condition are presented (n = 3). (G) The evolution of shCTRL- and shSLC7A1-transduced progenitors was monitored as a function of GFP expression at days 0, 3, and 7 of rEPO-induced differentiation and representative histograms are shown (left). Quantification of the percentages of GFP⁺ cells relative to day 0 is presented (right, n = 6). (H) Differentiation of rEPO-induced shCTRL- and shSLC7A1-transduced progenitors was monitored as a function of CD34 and CD36 expression on IL3R⁻GlyA⁻ cells; CFU-E are defined by an IL3R⁻GlyA⁻CD34⁻CD36⁺ phenotype (left, day 3). Quantification of CFU-E relative to shCTRL-transduced progenitors is presented (right, n = 9). (I) The differentiation of progenitors to an erythroid (GlyA) vs myeloid (CD11b) fate was evaluated by flow cytometry at day 3 of rEPO-induced differentiation. Representative plots (left) and quantifications (right) are presented. Surface expression of GlyA and CD11b was monitored on shCTRL- and shSLC7A1-transduced progenitors at day 3 of differentiation (left). Quantification of erythroid (n = 14) and myeloid (n = 12) differentiation is shown in independent experiments (right). (J) CD34⁺CD38⁻ progenitors transduced with shCTRL and shCAT1 vectors were evaluated for CFU potential using the StemMACS HSC-CFU Assay Kit and the numbers of burst-forming unit erythroid (BFU-E) and CFU–granulocyte-macrophage (GM) colonies generated at day 14 are presented for 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. IgG, immunoglobulin G; ns, not significant.