Tracing the in vivo behavior of nucleated cultured red blood cells generated from hematopoietic stem and progenitor cells

Red blood cells generated from stem cells, called cultured RBCs (cRBCs), may alleviate the shortage of blood supplies worldwide. The intermediate product, nucleated cRBCs, may further bypass the inconvenience of RBC manufacturing. Elucidating the in vivo behavior of cRBCs, especially the interaction between cRBCs and macrophages, will aid in understanding the effects of their transfusion and facilitate the clinical translation of these cells. In this study, we generated large-scale nucleated cRBCs from cord blood mononuclear cells using G-Rex bioreactors and evaluated their in vivo distribution, maturation and function by infusing these cells into irradiated NOD/SCID mice and comparing them with the properties of infused mature human RBCs. Similar to the infusion of mature human RBCs, in the absence of macrophage depletion, the infusion of cRBCs improved the maintenance of the red blood cell count and hemoglobin concentration, but the infused human cells were barely detected in the peripheral blood of the mice. Immature cRBCs accumulated in various organs, including the spleen, bone marrow and liver, at 2 h postinfusion, independent of macrophage depletion. Macrophages phagocytose mature foreign RBCs while nursing immature cRBCs until maturation; the difference in the innate expression of cRBCs during maturation might account for this effect. In the short term after transfusion, the spleen and bone marrow were the major sites of cRBC maturation. In the liver, macrophages primarily functioned to clear mature foreign RBCs. cRBCs matured, enucleated, and were released into the circulation within one to three days after infusion. Pulmonary accumulation of cRBCs and embolism were not observed after the cRBC infusion. Nucleated cRBCs can mature in vivo, and their transfusion advances our understanding of their potential as future transfusion products.