In healthy humans over 2 million RBCs are produced per second in the process of erythropoiesis. The ex vivo generation of cultured RBCs (cRBCs) from haematopoietic stem cells [1] is key to disease and development modelling. However, more significantly, cRBCs offer vast potential in improving clinical transfusion practice. Advantages of cRBCs include the ability to provide matched blood components to patients with rare blood groups who can be challenging or impossible to provide for. Additionally, cRBCs are all at the beginning of their 120-day lifespan and survival comparisons in mice revealed cRBCs survive longer than donor red cells [2] suggesting that cRBCs may offer advantages for transfusion-dependent patients by reducing the volume and number of transfusions required.

Erythropoiesis, the terminal differentiation of hematopoietic stem cells into enucleated erythrocytes, is tightly regulated by a complex system of signalling molecules, both in vivo and in vitro. One of these mediators is the glycoprotein stem cell factor (SCF), a cytokine which activates the receptor tyrosine kinase glycoprotein, c-KIT (CD117). SCF glycosylation has been shown to modulate SCF/c-KIT activity in leukemic models, however its role in erythropoiesis remains unknown. SCF is a vital component of media formulations to grow cRBCs in the laboratory. In fact, most (if not all) in vitro studies of human erythropoiesis were modelled using nonglycosylated SCF, a form not found endogenously in humans.

Here, we investigate the impact of glycosylated and nonglycosylated SCF on erythroid development, at two defined concentrations, using our ex vivo model for human erythropoiesis. We compared erythroid expansion and viability across 18 days in culture and evaluated the extent of differentiation and enucleation using erythroid cell morphology and flow cytometry. We utilised a liquid chromatography (LC)-tandem-mass spectrometry (MS/MS) based, label-free quantitative workflow using an Orbitrap FusionTM TribridTM mass spectrometer (MS) to compare erythroid proteomes at defined timepoints [3]. In addition, we performed relative quantitation of the erythroid N- and O-glycome using porous graphitised carbon-LC technologies with an amaZon Iontrap MS.

Our system-wide analyses reveal, for the first time, that glycosylation is key regulator of SCF activity in human erythropoiesis. We show that glycosylated SCF promotes significant erythroid expansion (up to 3 x 104-fold) compared to their nonglycosylated counterpart at minimal concentrations. We tracked the expression of > 1,200 protein groups throughout RBC development and identified SCF glycosylation dependent changes in the erythroid proteome at key stages of development. Notably, this study also provides the first characterisation of the erythroid glycome across stages of maturation, highlighting dynamic remodelling of > 60 N- and O-glycan structures towards terminal erythroid differentiation.

Our findings recapitulate the importance of glycosylation in protein function and highlight the pivotal role of SCF glycosylation in erythropoiesis. These understandings are key to increasing yield and efficiency in the creation of the optimal product in the quest to deliver transfusable ex vivo generated RBCs.

  1. Griffiths, R.E., et al., Maturing reticulocytes internalize plasma membrane in glycophorin A-containing vesicles that fuse with autophagosomes before exocytosis. Blood, 2012. 119(26): p. 6296-306.

  2. Kupzig, S., et al., Superior survival of ex vivo cultured human reticulocytes following transfusion into mice. Haematologica, 2017. 102(3): p. 476-483.

  3. Oliveira, T., et al., Glycoproteome remodeling in MLL-rearranged B-cell precursor acute lymphoblastic leukemia. Theranostics, 2021. 11(19): p. 9519-9537.

Disclosures

No relevant conflicts of interest to declare.

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