Optimizing the Method for Differentiation of Functional Platelets from Human Induced Pluripotent Stem Cells

Objective The global shortage of platelets presents a significant challenge in healthcare. Although human induced pluripotent stem cells (hiPSCs) offer a renewable source for ex vivo platelet production, the current approach remains constrained by heterogeneity, low yield, and high costs. This study introduces an optimized differentiation scheme (ODS) to improve ex vivo platelet differentiation from hiPSCs. Methods A systematically optimized culture protocol was developed, incorporating: (1) a higher initial dose of embryoid body (EB) cells, (2) refining culture medium, (3) substitution of cytokines with small molecules, and (4) enhancement of megakaryocyte (MK) polyploidization via small-molecule supplementation. Feasibility and effectiveness were evaluated using microscopy, cell counting, flow cytometry, Wright-Giemsa staining, immunofluorescence (IF), and transmission electron microscopy (TEM). Results Increasing the initial EB cell count significantly promoted megakaryocyte production and accelerated the process. A serum-free medium supplemented with human platelet lysate (HPL) was favorable for megakaryocyte generation. Small molecules 740Y-P and butyzamide effectively substituted SCF and TPO for differentiation, while the combination of blebbistatin and 616452 enhanced megakaryocyte maturation. Mature megakaryocytes continuously generated functional platelets that, upon thrombin activation, facilitated fibrin clot formation and contraction in vitro. This method shortened differentiation to 19 days, enhanced output to 1.42 CD41⁺ megakaryocytes and 14.9 platelets per iPSC, and reduced costs by 58.3%. Conclusion We have established a cost-effective strategy for platelet production via hiPSC differentiation, with potential applications in cell therapy and gene editing. Graphical