Differentiating erythroblasts adapt to turbulent flow by accelerating maturation and activating cholesterol biosynthesis.

In vitro culture of erythroblasts (EBL) and production of mature erythrocytes for transfusions requires upscaling in fluidic-turbulent bioreactors, resulting in membrane shear stress. For the implementation of erythroid cultures in bioreactors, understanding the effects of mechanical stress on terminal EBL differentiation is required. To this end, we investigated the effect of orbital shaking-induced shear stress on differentiating CD49d+CD235low primary human EBL towards enucleated reticulocytes at the molecular, cellular, and functional level. Orbital shaking at the onset of EBL differentiation enhanced cell maturation increasing enucleation percentage compared to static cultures, without cell viability loss. Transcriptome analysis uncovered 505 genes differentially expressed between static and dynamic cultures, with genes involved in lipid and cholesterol biosynthesis upregulated in dynamic conditions. In line with this, cells differentiated in orbital-shakers showed increased cholesterol concentration and osmotic resistance compared to static cultures. HMGCR (3-Hydroxy-3-Methylglutaryl-CoA-Reductase), rate-limiting enzyme of the cholesterol biosynthesis pathway, showed earlier and significantly higher induction during differentiation in dynamic. The severe loss of EBL in dynamic, but not in static conditions, due to HMGCR inhibition confirmed the ability of EBL to adapt to shear stress through modulating of their transcriptional program and upregulation of cholesterol biosynthesis. This work sheds light into specific mechanisms that will assist the successful upscaling of erythroid differentiation in turbulent bioreactors. In addition, as shear-stress on hematopoietic cells is also occurring within the bone marrow niche, these results introduces a potential novel signalling axis that need to be integrated into the known transduction pathways that control erythropoiesis.