KLF1-GATA1 fusion protein reduces hypoxia-related sickling in erythroid cells cultured from CD34+ cells isolated from blood samples of patients with SCD. (A) CD34+ cells isolated from patients with SCD were lentivirus-transduced with vector only, KLF1, or KLF1-GATA1 fusion protein, then grown in a 3-phase erythroid culture system.39 (A) At day 19, cultured erythroid cells were stained with TO for sorting enucleated erythroid cells. Unsorted or TO− sorted cells (enucleated erythroid cells) were cytospun, followed by Giemsa staining. (B) Representative bright-field images of enucleated erythroid cells that were incubated for another 16 hours in a 2% oxygen environment in culture medium before image processing. All images were taken within 3 minutes after removal from 2% oxygen without further manipulating the cells. Original magnification ×35. (C) Mean percentage of nonsickled (round) erythroid cells after a 16-hour incubation in a 2% oxygen environment. ∗P < .05 vs vector control-transduced cells. Error bars indicate the mean ± SD results of 3 independent experiments. (D) Flow cytometry analysis of cultured erythroid cells for erythroid differentiation markers glycophorin A and transferrin receptor (CD71). At least 3 independent experiments were performed. (E) Bromodeoxyuridine incorporation assay of cultured erythroid cells for cell proliferation. R2, R3, and R4 represent the G1, S, and G2 phases, respectively. At least 3 independent experiments were performed. KG-M, KLF1-GATA1 medium form; KG-L, KLF1-GATA1 long form.
