An Optimized Human Erythroblast Differentiation System Reveals Cholesterol‐Dependency of Robust Production of Cultured Red Blood Cells Ex Vivo

Abstract

The generation of cultured red blood cells (cRBCs) ex vivo represents a potentially unlimited source for RBC transfusion and other cell therapies. Human cRBCs can be generated from the terminal differentiation of proliferating erythroblasts derived from hematopoietic stem/progenitor cells or erythroid precursors in peripheral blood mononuclear cells. Efficient differentiation and maturation into cRBCs highly depend on replenishing human plasma, which exhibits variable potency across donors or batches and complicates the consistent cRBC production required for clinical translation. Hence, the role of human plasma in erythroblast terminal maturation is investigated and uncovered that 1) a newly developed cell culture basal medium mimicking the metabolic profile of human plasma enhances cell growth and increases cRBC yield upon erythroblast terminal differentiation and 2) LDL‐carried cholesterol, as a substitute for human plasma, is sufficient to support erythroid survival and terminal differentiation ex vivo. Consequently, a chemically‐defined optimized medium (COM) is developed, enabling robust generation of cRBCs from erythroblasts of multiple origins, with improved enucleation efficiency and higher reticulocyte yield, without the need for supplementing human plasma or serum. In addition, the results reveal the crucial role of lipid metabolism during human terminal erythropoiesis.

Production and stability of cultured red blood cells depends on the concentration of cholesterol in culture medium

“…The addition of LDL, which is taken up by the LDL-R, abrogated the requirement of plasma or serum at early stages of terminal differentiation 50 . All our differentiation experiments are performed in the presence of 5% plasma that is critically required for enucleation in our cultures.…”

“…Wang et al suggest that supplementation of cholesterol is not required during late terminal differentiation. We specifically increased the cholesterol concentration in our media at late stages for more efficient recovery of enucleated cells after filtration, which is not tested in their report 50 .…”

“…Whereas early addition inhibited enucleation, the addition of up to 5 mg/dl cholesterol to culture medium at day 5 of differentiation, at the point of enucleation, enhanced osmotic stability and filtration efficiency of enucleated cRBC. We used Bodipy-labelled-Cholesterol to track the uptake and exchange of Cholesterol from the cRBC membrane by flow cytometry 50 .…”