Pomalidomide regulates HbF synthesis and erythropoiesis in transgenic sickle cell mice. Homozygous sickle cell mice were treated with vehicle, pomalidomide, or hydroxyurea for 8 weeks by intraperitoneal injection and killed for analysis. (A) HbF protein levels as a percentage of total hemoglobin were determined by high performance liquid chromatography at the end of the study to avoid artifactual increases of HbF from repeated blood draws. F cells were analyzed by flow cytometry after immunolabeling formalin-fixed RBCs with anti–human fluorescein isothiocyanate-conjugated HbF antibody. (B) Representative images depicting the M:E ratio in hematoxylin and eosin-stained bone marrow sections of the proximal femur. Erythroid cells are recognized by their round, dense, and deeply basophilic nuclei. All images were collected using a Zeiss Axioplan 2 microscope and a Plan-Apochromat 63×/1.4 oil objective. (C) Analysis of bone marrow cellularity, M:E ratio, and megakaryocyte counts. Cellularity in the active treatment groups was determined in reference to the 100% cellular marrow in vehicle-treated sickle cell mice. Megakaryocytes were counted in 5 low magnification optical fields per animal and converted to megakaryocytes/mm2. All sections were analyzed by 2 blinded investigators. Veh indicates vehicle; PL, pomalidomide; HU, hydroxyurea; C/HD, combination high-dose treatment (10 mg/kg pomalidomide, 100 mg/kg hydroxyurea); and C/LD, combination low-dose treatment (10 mg/kg pomalidomide, 10 mg/kg hydroxyurea). *Significantly different from Veh and HU (P < .05). **Significantly different from Veh and PL (P < .01).
