shRNA-mediated knockdown of ENT1 negatively impacts ex vivo erythroid differentiation. (A) Schematic representation of the schema used for ex vivo human erythropoiesis and the experimental design. Cord blood CD34⁺ cells were differentiated using a 3-phase culture system (containing rEPO) that recapitulates human erythropoiesis up to the enucleated reticulocyte. Cells were transduced at day 4 of the expansion phase with a lentiviral vector harboring an ENT1 shRNA or scramble together with a GFP reporter gene. Representative MGG-stained cells from healthy control are shown for corresponding differentiation stages. (B) The evolution of surface ENT1 expression during rEPO-induced (3 U/mL) erythroid differentiation was monitored by flow cytometry and representative histograms showing nonspecific IgG staining (gray shaded) and specific (black line) staining at days 0, 3, 7, and 10 of erythroid differentiation are presented, differentiating negatively and positively stained cells. The geometric mean fluorescence intensity (GMFI) is indicated in each histogram. GMFIs from 3 independent experiments were quantified and are presented as means ± standard error of the mean (SEM; right). (C) Progenitors transduced with shControl and shENT1 vector were FACS-sorted on the basis of GFP expression 3 days posttransduction and ENT1 mRNA expression levels were analyzed by quantitative real-time polymerase chain reaction and normalized to actin mRNA. mRNA ± SD relative to control cells is presented (n = 3; unpaired Student t test; ****P < .0001). (D) Transduced progenitors were cultured in the presence or absence of rEPO as indicated. The evolution of shRNA-expressing cells was followed and the percentages of GFP⁺ cells relative to day 1 are shown. Results correspond to data obtained from 3 independent experiments. (E) Progenitors transduced with the shControl and shENT1 vectors were FACS-sorted on the basis of GFP expression and then differentiated in the presence of EPO. Cells were counted at the indicated days and growth curves are presented for shENT1-GFP⁺ (gray line) and shControl-GFP⁺ (black line) cells. Data presented are means ± SEM of 4 independent experiments; *P < .05, ***P < .001. (F) Progenitors were transduced with shControl or shENT1 vectors, both harboring a GFP transgene. Gene transfer efficiency was evaluated as a function of GFP expression 3 days following transduction and representative histograms are shown (top). The percentages of cells expressing GPA and CD71 were evaluated by flow cytometry and representative profiles in GFP⁻ and GFP⁺ subsets are presented. The percentages of positively stained cells are indicated. (G) Erythroid differentiation of control-GFP- and shENT1-GFP-transduced progenitors was monitored as a function of GPA and CD71 expression within the GFP⁺ cells. The percentages of GPA⁺ and CD71⁺ cells ± SEM in control (black circles) and shENT1-GFP (gray triangles) transduced progenitors are shown at days 3, 5, and 7 of culture (n = 3; unpaired Student t test; *P < .05, **P < .01, ***P < .001). (H) Enucleation was monitored at day 10 of differentiation as a function of Syto-16 staining and the percentages of Syto-16-negative cells are presented (n = 3 independent experiments; unpaired Student t test; *P = .02). (I) At day 4 of differentiation, cells were FACS-sorted on the basis of GFP expression and differentiation was continued until day 10. Erythroid differentiation was monitored as a function of Band3 expression and myeloid differentiation was monitored by CD11a/CD33 surface expression (black) and nonspecific control IgG staining (gray). Representative histograms and quantification are shown within GFP⁺ cells. (n = 3 independent experiments; *P < .05).