Abstract Number: PB1599
Meeting: ISTH 2020 Congress
Background: Platelets are critical to hemostasis and platelet transfusion prevents and treats bleeding in thrombocytopenic patients. Limited availability and functional variability inherent to donor platelets may be overcome by transfusion of human induced pluripotent stem cell (hiPSC)‐derived platelets. To enable cost-effective megakaryocyte (MK) expansion and differentiation, conditionally immortalized megakaryocytic progenitor cell lines (imMKCLs) were developed (Cell stem cell. 2014; 14: 535) that can produce functional platelets. However, like many other in vitro platelet production systems, the imMKCL system suffers from low yields (~10 platelets/MK or about 1×109 platelets per 10-liter shaker flask culture volume).
Aims: To identify novel compounds and pathways to improve efficiency of megakaryocytic in vitro maturation and platelet production.
Methods: We developed a confocal live-cell imaging platform that allowed us to assess the rate and extent of megakaryocte-maturation and pro-platelet production in high-throughput manner and used it to screen chemical libraries.
Results: We identified two structurally related compounds (targeting the same pathway) that robustly induce in vitro (pro)-platelet production. We also identified additional compounds (acting through a distinct pathway) that promote imMKCL-maturation and/or endomitosis. Sequential exposure of imMKCLs to the maturation- and (pro)-platelet formation promoting compounds in a PBS-Mag type bioreactor system enabled generation of ≥1×1011 platelets per 10 liters, satisfying clinical production requirements. These imMKCL-derived platelets are structurally and functionally similar to normal human platelets as judged by 1) size, morphology and ultrastructure (electron microscopy and immunofluorescence), 2) resting and agonist-stimulated surface expression of activation biomarkers (flow cytometry), and 3) platelet aggregation. In vivo studies in non-thrombocytopenic mouse indicate that exogenous bioreactor-produced platelets are incorporated into the developing thrombus at a rate similar to that of normal human platelets during laser-induced thrombus formation.
Conclusions: Using a novel image based chemical-genetics screen, inducers of imMKCL-maturation as well as platelet release were identified. This method enables cost-efficient clinical scale production of functional platelets.
To cite this abstract in AMA style:Bhan A, Kareddy N, Forde E, Merrill-Skoloff G, Furie B, Frelinger A, Daley G, Schlaeger T. A Novel Image-Based Chemical-Genetics Screen Identifies Inducers of Platelet Release from Immortalized Megakaryocytic Cell Lines, Enabling Clinical-Scale Production of Functional Platelets [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/a-novel-image-based-chemical-genetics-screen-identifies-inducers-of-platelet-release-from-immortalized-megakaryocytic-cell-lines-enabling-clinical-scale-production-of-functional-platelets/. Accessed August 15, 2022.
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