Aglialoro, F. | Search bioRxiv

bioRxiv2023

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.

cell biology↗

bioRxiv2018

Circular RNA expression in human hematopoietic cells is widespread and cell-type specific

Hematopoietic stem cells differentiate into a broad range of specialized blood cells. This process is tightly regulated and depends on transcription factors, micro-RNAs, and long non-coding RNAs. Recently, also circular RNA (circRNA) were found to regulate cellular processes. Their expression pattern and their identity is however less well defined. Here, we provide the first comprehensive analysis of circRNA expression in human hematopoietic progenitors, and in differentiated lymphoid and myeloid cells. We here show that the expression of circRNA is cell-type specific, and increases upon maturation. circRNA splicing variants can also be cell-type specific. Furthermore, nucleated hematopoietic cells contain circRNA that have higher expression levels than the corresponding linear RNA. Enucleated blood cells, i.e. platelets and erythrocytes, were suggested to use RNA to maintain their function, respond to environmental factors or to transmit signals to other cells via microvesicles. Here we show that platelets and erythrocytes contain the highest number of circRNA of all hematopoietic cells, and that the type and numbers of circRNA changes during maturation. This cell-type specific expression pattern of circRNA in hematopoietic cells suggests a hithero unappreciated role in differentiation and cellular function.

immunology↗