Engineering synthetic signaling receptors to enable erythropoietin-free erythropoiesis

Blood transfusion plays a vital role in modern medicine. However, frequent shortages pose a significant healthcare challenge. Ex vivo manufacturing of red blood cells (RBCs) derived from universal donor pluripotent stem cells is limited by the high cost of recombinant cytokines required for ex vivo erythroid differentiation. Erythropoietin (EPO) signaling through the EPO receptor is indispensable to RBC development, and EPO is one of the most expensive components in erythroid-promoting media. Here, we use design-build-test cycles to develop highly optimized small molecule-inducible synthetic EPO receptors (synEPORs) which are integrated at a variety of genomic loci using homology-directed repair genome editing. We find that integration of synEPOR at the endogenous EPOR locus in an induced pluripotent stem cell producer line cultured with small molecule yields equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production compared to cells cultured with EPO. Due to the dramatically lower cost of small molecules vs. recombinant cytokines, these efforts eliminate one of the most expensive elements of ex vivo culture media—EPO cytokine. As cytokines dependence is a common barrier to ex vivo cell production, these strategies could improve scalable manufacturing of multiple clinically relevant cell types.