Thesis Giulia Iacono

Although red blood cell (RBC) transfusion is one of the most common clinical practice, its dependency on donor blood presents challenges related to supply, storage, and infection risk. Alternatives to blood transfusions are being explored, among which, in-vitro RBC production is one of the most promising. Nevertheless, scaling manufacturing to transfusion-relevant quantities requires dynamic bioreactors that expose cells to mechanical forces like shear-stress. This thesis investigates how shear-stress influences erythroid differentiation to advance both the biological understanding of erythropoiesis and the optimization of large-scale RBC production. Erythroblasts cultured under moderate levels of shear-stress showed accelerated maturation, achieving enucleated CD49d⁻/CD235a⁺ reticulocytes four days earlier than under static conditions. Transcriptomic analyses revealed the downregulation of DNA replication genes and overexpression of cholesterol biosynthesis and uptake pathways, leading to transient membrane lipid remodeling that possibly enhances mechanical resilience. Functional studies identified mechanosensitive channel PIEZO1 and its downstream effector, the Gárdos channel, as active during differentiation, suggesting a feedback mechanism linking membrane lipid composition, ion flux, and cell volume regulation. Moreover, in-vitro RBC differentiation can provide models to study pathological erythropoiesis, as it has been shown in this thesis by the characterization of a novel β-spectrin mutation (SPTBc.6219G>A) associated with hereditary elliptocytosis. In summary, this work elucidates the mechanotransductive responses of erythroid precursors to shear-stress, demonstrating their impact on lipid metabolism, ion channel activity, and differentiation kinetics. These findings provide foundations for optimizing bioprocess parameters in RBC biomanufacturing and for implementing dynamic culture systems as models to investigate both normal and diseased erythropoiesis.