Ferric citrate and apo-transferrin enable erythroblast maturation with beta-globin from hemogenic endothelium

Red blood cell (RBC) generation from human pluripotent stem cells (PSCs) offers potential for innovative cell therapy in regenerative medicine as well as developmental studies. Ex vivo erythropoiesis from PSCs is currently limited by the low efficiency of functional RBCs with beta-globin expression in culture systems. During induction of beta-globin expression, the absence of a physiological microenvironment, such as a bone marrow niche, may impair cell maturation and lineage specification. Here, we describe a simple and reproducible culture system that can be used to generate erythroblasts with beta-globin expression. We prepared a two-dimensional defined culture with ferric citrate treatment based on definitive hemogenic endothelium (HE). Floating erythroblasts derived from HE cells were primarily CD45(+)CD71(+)CD235a(+) cells, and their number increased remarkably upon Fe treatment. Upon maturation, the erythroblasts cultured in the presence of ferric citrate showed high transcriptional levels of beta-globin and enrichment of genes associated with heme synthesis and cell cycle regulation, indicating functionality. The rapid maturation of these erythroblasts into RBCs was observed when injected in vivo, suggesting the development of RBCs that were ready to grow. Hence, induction of beta-globin expression may be explained by the effects of ferric citrate that promote cell maturation by binding with soluble transferrin and entering the cells.