Chemically Defined, Efficient Megakaryocyte Production from Human Pluripotent Stem Cells

Abstract

Highlights What are the main findings? Butyzamide (MPL agonist) plus M-CSF efficiently generates hPSC-derived MKs, replacing TPO. What is the implication of the main finding? PSC-MKs can be used as a source for disease modeling, mechanistic studies, and in vitro platelet production.Highlights What are the main findings? Butyzamide (MPL agonist) plus M-CSF efficiently generates hPSC-derived MKs, replacing TPO. What is the implication of the main finding? PSC-MKs can be used as a source for disease modeling, mechanistic studies, and in vitro platelet production.Abstract Platelet shortage poses a significant barrier to research and transfusion therapies because native megakaryocytes (MKs) are scarce in blood. To overcome this limitation, pluripotent stem cell-derived MKs (PSC-MKs) offer a standardized, donor-independent platform for research and therapeutic development, including disease modeling and ex vivo platelet production. Here, we report a chemically defined, feeder-free protocol to generate MKs from human pluripotent stem cells (hPSCs). The protocol combines the small molecule MPL agonist Butyzamide, macrophage colony-stimulating factor (M-CSF), and three-dimensional (3D) suspension culture, achieving high efficiency and reproducibility. Butyzamide replaced recombinant thrombopoietin (TPO), yielding comparable CD41+/CD42b+ populations and enhanced polyploidization. M-CSF accelerated nuclear lobulation and induced 4N MKs, while 3D culture increased yield, cell size, and substrate detachment. Multiple independent assays confirmed mature MK hallmarks, multi-nuclei, demarcation membranes, granules, and elevated mitochondrial respiration. Single-cell RNA sequencing outlined a continuous trajectory from early progenitors to functionally specialized MK subsets. This platform enables reliable MK supply for mechanistic studies and in vitro platelet production, advancing both basic research and therapeutic development.