Immortalization of Erythroblasts by c-MYC and BCL-XL Enables Large-Scale Erythrocyte Production from Human Pluripotent Stem Cells

• Immortalized-type erythroblasts are established by overexpression of c-MYC and BCL-XL
• After genes are turned off, imERYPCs differentiate into mature erythroblasts in vitro
• ImERYPC-derived mature cells show oxygen delivery capability similar to normal RBCs
• Enucleated cells reveal circulation potential in immune-deficient mice

SummaryThe lack of knowledge about the mechanism of erythrocyte biogenesis through self-replication makes the in vitro generation of large quantities of cells difficult. We show that transduction of c-MYC and BCL-XL into multipotent hematopoietic progenitor cells derived from pluripotent stem cells and gene overexpression enable sustained exponential self-replication of glycophorin A+ erythroblasts, which we term immortalized erythrocyte progenitor cells (imERYPCs). In an inducible expression system, turning off the overexpression of c-MYC and BCL-XL enabled imERYPCs to mature with chromatin condensation and reduced cell size, hemoglobin synthesis, downregulation of GCN5, upregulation of GATA1, and endogenous BCL-XL and RAF1, all of which appeared to recapitulate normal erythropoiesis. imERYPCs mostly displayed fetal-type hemoglobin and normal oxygen dissociation in vitro and circulation in immunodeficient mice following transfusion. Using critical factors to induce imERYPCs provides a model of erythrocyte biogenesis that could potentially contribute to a stable supply of erythrocytes for donor-independent transfusion.