Red blood cells (RBCs, also known as erythrocytes) are essential for almost all animal lifeforms on earth. Clinically, ex vivo generation of RBCs offers great potential, not only for transfusion purposes, but also for disease and development modelling. 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 glycosylated receptor tyrosine kinase, c-KIT (CD117). SCF glycosylation has been shown to modulate SCF/c-KIT activity in leukemic models, however its role in erythropoiesis remains unknown. 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 investigated 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 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. In addition, we performed relative quantitation of the erythroid N- and O-glycome using porous graphitised carbon-LC technology with an amaZon Iontrap MS.
Our system-wide analyses revealed, for the first time, that glycosylation is key regulator of SCF activity in human erythropoiesis. We showed 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 erythroid proteome at key stages of development. Notably, this study also provided 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. Together, our findings recapitulate the importance of glycosylation in protein function and highlights the pivotal role of SCF glycosylation in the quest to deliver transfusable ex vivo generated RBCs.