Producing red blood cells in the laboratory addresses a persistent difficulty: transfusion needs exceed the supply of donors, and the shortage weighs particularly on patients with rare blood groups. The team had previously established a three-step protocol yielding mature red blood cells from CD34+ cells obtained from bone marrow, peripheral blood, leukapheresis or cord blood, in a serum-free medium supplemented with cytokines. What remained was to establish that these cells are metabolically comparable to their native counterparts. That is the purpose of this work, which uses metabolomics to compare culture-derived reticulocytes and red blood cells with those obtained from cord blood.
The culture began with CD34+ cells of cord blood origin, steered towards the erythroid lineage and transferred onto murine MS-5 stroma, whose microenvironment carries differentiation through to enucleation. By day 15 the population was predominantly reticulocytic — 89% ± 10% on thiazole orange staining, the same figure for the transferrin receptor CD71. The comparator came from cord blood too: native reticulocytes isolated by immunomagnetic anti-CD71 selection, at better than 96% ± 2% purity. Both populations then went through two liquid chromatography systems coupled to high-resolution mass spectrometry, in positive and negative ionisation, hydrophilic interaction chromatography covering the highly polar compounds and reverse phase the fatty acids and acylcarnitines. Eighty-six compounds of biological interest were kept for analysis.
The central pathways of red cell function — glycolysis, glutathione metabolism — proved superimposable between the two types of reticulocyte. Twenty-one metabolites varied by at least twofold. Among those raised in culture were six amino acids (glutamine, alanine, serine, ornithine, threonine, asparagine), which their concentration in the medium and their surface transporters are enough to explain. The decrease in carnitine and its derivatives suggests lower membrane turnover than in circulation, where the stress on the membrane is greater. The decrease in malate, 2-oxoglutarate and succinate points to ATP production favouring glycolysis, encouraged by the glucose-rich medium. The two most depleted metabolites, ergothioneine and stachydrine, share the same transporter and are absent or barely present in the medium: ergothioneine, which human cells cannot synthesise, comes from maternal intake and fuels an efficient antioxidant pathway.
A further eight days of culture brought both populations to maturity. Most metabolites elevated in culture-derived reticulocytes had by then returned to native levels. Of the eighty-six metabolites followed, only seven remained attributable to culture conditions and four to sample origin, a covariance analysis having ruled out inter-individual variability for seven of the eleven metabolites concerned. Bio-engineered red blood cells therefore carry a metabolic signature almost identical to that of native red blood cells, and the residual differences do not affect essential functions — an argument for the robustness of a protocol intended for patients with rare blood groups.
This work is cited in our article Metabolomics.