Engineering recombinant human erythropoietin (rhEPO) for efficient in vitro erythropoiesis

Abstract

Recombinant human erythropoietin (rhEPO) is one of the critical cytokines for studying erythropoiesis. However, since endogenous EPO is hard to extract, scientists use commercial rhEPO. However, reliance on commercial rhEPO is often hampered by the high cost and biochemical rigidity of commercial reagents. These limitations, compounded by potential contamination risks and a lack of transparency in production, restrict experimental flexibility and compromise research autonomy. Here, I established a robust, two-track in-house production platform to bypass dependency on commercial sources and secure a consistent supply of bioactive rhEPO. Our dual strategy integrates a stable HEK293T cell line for the continuous secretion of rhEPO into conditioned media (CM) for large- scale applications, alongside a transient transfection system utilizing a Flag-int-hEPO construct for high-purity applications via immunoprecipitation (IP). I validated the biological efficacy of these systems using the EPO-dependent HUDEP-2 erythroid progenitor cell line. Both CM-derived and IP-purified rhEPO effectively activated the canonical JAK2/STAT5 signaling pathway, supporting robust cell proliferation and driving terminal erythroid differentiation which is evidenced by precise surface marker (CD71/CD235a) shifts and hemoglobin synthesis. By offering a scalable and customizable framework for cytokine engineering, this system paves the way for advanced, cost-effective, and efficient models of human erythropoiesis.