Abstract

Megakaryopoiesis is an elaborate biological process that primarily occurs in the bone marrow. To gain deeper insights into molecular mechanisms driving normal megakaryopoiesis, we utilized an in vitro human megakaryocytic culture system based on mobilized peripheral blood-derived CD34 + cells. Following fluorescence-activated cell sorting (FACS) isolation of CD41 high and CD41 dim megakaryocyte (MK) subsets, mature MKs were confirmed through characterization of MK-specific surface markers, ploidy analysis, Giemsa staining, and immunofluorescence. Subsequent bulk RNA sequencing of these distinct populations enabled the identification of differentially expressed genes (DEGs) and enriched pathways. Based on our CD34 + -derived MK differentiation model, the expression of CD41 was found robustly induced by day 4 and further elevated by day 10. The CD41 high population exhibited marked co-expression of CD42b and CD61, a significantly higher proportion of polyploid cells (≥16 N), along with characteristic morphological features of mature MKs, including proplatelet formation, cytoplasmic maturation, and cell size enlargement compared to the CD41 dim subset. Transcriptomic profiling of these two populations identified 1877 up-regulated and 1817 down-regulated DEGs in CD41 high MKs. Protein-protein interaction (PPI) network analysis of the key DEGs revealed hub genes including VWF, PF4V1, SELP, PF4, GP1BA, CD40LG, PPBP, CLEC1B, P2RY12, and THBS1. Functional enrichment underscored the acquisition of migratory, adhesive, and secretory capacities, marked by significant upregulation of platelet activation and wound healing signatures. Pathway analysis further indicated coordinated activation of focal adhesion, cytoskeletal reorganization, glycerolipid metabolism, and neuroactive ligand-receptor interaction during maturation. This study provides an integrative transcriptomic blueprint of human MK maturation and highlights the novel candidate targets for thrombopoiesis.