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Starting with human PBMCs, the purities of the start and final isolated fractions in the above example are 6.7% and 91.0%, respectively, using a FITC-conjugated anti-human CD19 antibody and EasySep™ Human FITC Positive Selection Kit II.
Conditional CRISPR-mediated deletion of Lyn kinase enhances differentiation and function of iPSC-derived megakaryocytes. A. J. Moroi and P. J. Newman Journal of thrombosis and haemostasis : JTH 2022 jan
Abstract
BACKGROUND Thrombocytopenia leading to life-threatening excessive bleeding can be treated by platelet transfusion. Currently, such treatments are totally dependent on donor-derived platelets. To support future applications in the use of in vitro-derived platelets, we sought to identify genes whose manipulation might improve the efficiency of megakaryocyte production and resulting hemostatic effectiveness. Disruption of Lyn kinase has previously been shown to improve cell survival, megakaryocyte ploidy and TPO-mediated activation in mice, but its role in human megakaryocytes and platelets has not been examined. METHODS To analyze the role of Lyn at defined differentiation stages during human megakaryocyte differentiation, conditional Lyn-deficient cells were generated using CRISPR/Cas9 technology in iPS cells. The efficiency of Lyn-deficient megakaryocytes to differentiate and become activated in response to a range of platelet agonists was analyzed in iPSC-derived megakaryocytes. RESULTS Temporally controlled deletion of Lyn improved the in vitro differentiation of hematopoietic progenitor cells into mature megakaryocytes, as measured by the rate and extent of appearance of CD41+ CD42+ cells. Lyn-deficient megakaryocytes also demonstrated improved hemostatic effectiveness, as reported by their ability to mediate clot formation in rotational thromboelastometry. Finally, Lyn-deficient megakaryocytes produced increased numbers of platelet-like particles (PLP) in vitro. CONCLUSIONS Conditional deletion of Lyn kinase increases the hemostatic effectiveness of megakaryocytes and their progeny as well as improving their yield. Adoption of this system during generation of in vitro-derived platelets may contribute to both their efficiency of production and their ability to support hemostasis.
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