Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells

Abstract

Blood transfusion is a critical, lifesaving medical procedure for dogs. However, the limited availability of blood donors and ethical concerns highlight the need for alternative solutions, such as in vitro-produced red blood cells (RBCs), which remain unexplored in canines. This study aimed to produce canine erythrocytes in vitro from peripheral blood (PB) mononuclear cells (MNCs), optimize culture conditions using either human or canine reagents, and identify relevant cell markers. Results indicated that canine erythropoiesis can be induced by human or canine cytokines, producing RBCs within approximately 20 days. Although cell numbers decreased during the first seven days, immature erythroid cells proliferated, reaching peak expansion and RBC production by day 17. Despite the smaller cell size of the cultured RBCs than that of humans, the morphology at each stage of erythroid maturation was analogous to that of human erythropoiesis. Furthermore, the expression patterns of canine alpha hemoglobin stabilizing protein and erythropoietin receptor mirrored those observed in human erythropoiesis. Oxygen-hemoglobin (oxygen-Hb) association and dissociation curves of cultured RBCs closely resembled those of native canine RBCs, indicating an appropriate oxygen-carrying capacity. This study presents the first evidence of successful in vitro production of canine RBCs, offering a promising tool for research and potential therapeutic applications.