The mechanism of action of pomalidomide is conserved in CD34⁺ cells derived from sickle cell patients. CD34⁺ cells derived from the peripheral blood of SCD patients were expanded for 4 days and differentiated toward reticulocytes using the three-phase culture system. (A) Growth curves of DMSO and pomalidomide-treated cultures. The data are shown as the mean cells/mL ± SEM (N = 3). (B-C) Flow cytometric analyses of (B) BFU/CFU-E and (C) terminal erythroid differentiation as described in Figure 2 (N = 3). (D) May-Grunwald Giemsa–stained cytopreps at indicated days. Bar represents 5 μm. (E) Western blot for IKZF1 after treatment with DMSO, 1 μM pomalidomide, 5 μM MG132, or the combination of Pom 1μM and MG132 5μM for indicated times (N = 3). (F) Western blot for the expression levels of SOX6, BCL11A, KLF1, and CoREST at day 4 and day 11 (N = 3). (G) Quantification of immunoreactive bands normalized to GAPDH. (H) Immunoblot analyses of α-, β-, and γ-globin chains (N = 3). (I-J) Hemoglobin production was assessed by HPLC using supernatants from dH₂O lysed cells. (I) Representative chromatograms from DMSO and pomalidomide-treated cultures. Gray and white arrows indicate peaks corresponding to HbF and HbA, respectively. (J) Quantification of hemoglobin HPLC displayed as the mean percent HbF/(HbF + HbS) ± SEM (**P = .0063; N = 3) and the mean percent HbS/(HbS + HbF) ± SEM (**P = .0127; N = 3). (K) Cellular distribution of HbF by flow cytometry at day 14. The box indicates HbF^pos cells (F-cells) (N = 3).