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

In vitro culture of erythroblasts and production of mature erythrocytes for transfusion purposes requires upscaling in bioreactors with turbulent fluidic environments, resulting in membrane shear stress. For the implementation of erythroid cultures in bioreactors, further understanding of the effects of mechanical stress on terminal erythroblast differentiation are required. To this end, we investigated the effect of orbital shaking-induced shear stress on differentiation of CD49d+CD235low primary human erythroblast (EBL) towards enucleated reticulocytes at the molecular, cellular, and functional level. Orbital shaking at the onset of erythroblast differentiation enhanced cell maturation and increased enucleation percentage compared to static cultures, without loss of cell viability. Transcriptome analysis uncovered 505 genes to be differentially expressed between static and non-static cultures, where genes involved in lipids and cholesterol biosynthesis pathways where upregulated in non-static conditions. In line with this finding, cells differentiated in orbital shakers showed an increased cholesterol concentration and higher osmotic resistance compared to cells differentiated in static condition. HMGCR (3-Hydroxy-3-Methylglutaryl-CoA-Reductase), a rate limiting enzyme of the cholesterol biosynthesis pathway, is significantly increased and showed earlier induction during differentiation in shear stress environments. Inhibition of HMGCR using lovastatin led to severe loss of EBL differentiation in shear environments but not in static conditions. In conclusion, differentiating EBL display a dynamic capability to adapt to shear stress environments, by modulating their transcriptional program and consequently upregulating cholesterol biosynthesis, which is critical for their survival. This work sheds light into specific mechanisms which will assist on the successful upscaling of erythroid differentiation in turbulent bioreactors. Overall design: To understand the effect of mechanical forces on erythroblasts differentiation we performed RNAseq analysis of erythroblast differentiated in static and dynamic conditions. We then compared the RNA-seq data obtained from 4 different donors during 3 culture time points: day of differentiation 1, 2 and 4, determining 3 different expression profiles.