Abstract
Abstract
To generate a mature red blood cell, erythropoiesis requires the committed erythroid progenitor cell and later erythroblast stages to undergo a continuum of extreme morphological and functional remodeling.
In vitro
culture has been crucial to understand the extensive proteome dynamics that underlie this process. However, the millions of cells required for conventional proteomics technologies preclude the study of erythropoiesis within the steady-state human bone marrow (BM). Thus, culture-induced stress may shape our understanding of erythroid maturation. To address this knowledge gap, we developed an optimized methodology for low-cell proteomics (pauciproteomics) and pursued total proteomic analysis of paired steady-state BM and culture-derived erythroid precursor stages from 11 healthy donors. This approach defines four main protein expression patterns during BM erythroid maturation which are only partially recapitulated by cultured cells. Culture-induced stress rewires the erythroid proteome from early stages of differentiation, reshaping major transcription factor networks, kinase pathways, metabolic enzymes and erythrokine regulation. Notably, single-cell RNA sequencing analysis of steady-state healthy and myelodysplastic syndromes (MDS) BM demonstrates that the cultured cell proteome parallels gene expression dynamics within the MDS BM, modelling the regulation of
in vivo
stress erythropoiesis. Culture-specific alterations were mostly restricted to extensive carbonic anhydrase dysregulation and embryofetal globin switching, both potentially linked to altered gas exchange dynamics in culture. In conclusion, we reveal the steady-state erythroid proteome and its substantial remodeling by culture-induced stress, reframing existing knowledge on erythroid differentiation, defining new elements of stress erythropoiesis and guiding future studies in the field.