Pomalidomide partially reverses the fetal-to-adult globin switch through the reciprocal expression of globin genes. (A) Schematic representation of human erythropoiesis and experimental design. Adult CD34⁺ cells from peripheral blood were expanded for 4 days, then differentiated using a three-phase culture system that recapitulates human erythropoiesis up to the enucleated reticulocyte (phases 1 to 3, detailed in the table at the bottom). 1 μM pomalidomide (Pom 1μM) was added at the end of the expansion phase and replaced every 3 to 4 days for the duration of the culture unless otherwise stated. (B) qRT-PCR of γ- and β-globin mRNA levels normalized to β-actin at D11 of differentiation in the presence of Pom 1μM or DMSO control. The data are shown as the mean fold difference of DMSO ± standard error of the mean (SEM) (N = 5): HBB (***P = 8.0785 × 10⁻⁵), HBG1 (**P = .0034), HBG2 (n.s. P = .0803). (C-I) D14 of differentiation: (C) western blot of cell lysates for α-, β-, and γ-globin chains (N = 3), and (D) hemoglobin production was assessed by HPLC using supernatants from dH₂O lysed cells. Representative chromatograms from DMSO and pomalidomide-treated cultures. Gray and white arrows denote fetal hemoglobin (HbF) and adult hemoglobin (HbA), respectively. (E-F) The area under the curve was calculated, and relative hemoglobin composition was expressed as (E) the mean percent HbF/(HbF + HbA) ± SEM (N = 6): ***P = 5.5585 × 10⁻⁷ and (F) the mean percent HbA/(HbA + HbF) ± SEM (N = 6): ***P = 6.2138 × 10⁻⁷. (G) Cells were fixed, permeabilized, and stained with a PE-conjugated anti-HbF antibody. Flow cytometry data displayed as HbF against FSC. Box delineates HbF^pos cells (F-cells), which possess a fluorescent intensity greater than unstained control (N = 3). (H) Western blot of γ-globin chain expression and (I) F-cells in cultures treated with Pom 1μM for 14 days, the first or second 7 days of differentiation (N = 3).