Erythrocyte-mimetic oxygen carriers based on enucleated mesenchymal stem cells: A novel strategy for emergency blood substitution

Acute major hemorrhage triggered by a sudden reduction in blood volume, accompanied by coagulopathy, systemic hypoperfusion, and tissue hypoxia, is likely the underlying cause of death. Shortages in donor blood supplies limit the efficacy of conventional transfusion therapies, leading to heightened interest in blood substitutes as potential alternatives or supplements. To address his limitation, we developed a novel oxygen carrier based on enucleated bone marrow mesenchymal stem cells (BMSCs) in this study. A complete sequence encoding hemoglobin α/β subunits linked by a P2A sequence for tandem expression was integrated into the BMSCs genome via lentiviral transduction. Western blot and Native-PAGE analyses confirmed that these subunits were released as monomers and assembled into hemoglobin tetramers. Furthermore, ultraviolet-visible (UV-Vis) spectroscopy exhibited the characteristic dual peaks in the oxygenated state and a single peak in the deoxygenated state, indicating an oxygen-carrying capacity comparable to that of natural hemoglobin. Importantly, the resulting enucleated vesicles were comparable in size to erythrocytes, remained highly viable for up to 24 hours, and exhibited low cytotoxicity. In conclusion, this recombinant enucleated BMSC-derived hemoglobin (BMSC-Hb) shows promising oxygen transport capacity and low immunogenicity, positioning it as a potential blood substitute for emergency transfusion.