Conditional CRISPR-Mediated Deletion of Lyn Kinase Promotes Megakaryocyte Differentiation, Platelet Production and Function

Thrombocytopenia leading to life-threatening excessive bleeding can be caused by defects in megakaryocyte/platelet production, platelet depletion by platelet-specific auto- or allo-antibodies, blood loss following trauma or surgery, or as a result of damage to bone marrow blood stem cells induced by chemo- or radiation therapy. Currently, platelets available for transfusion are entirely dependent on volunteer blood donation, which are often in short supply, carry a risk of infection, and often unable to be antigen-matched for alloimmunized patients. Thus, maintaining an adequate supply of platelets for transfusion is often a challenge. In response to this growing clinical need, a number of laboratories have started differentiating human induced pluripotent stem cells (iPSCs) into megakaryocytes, with the long-term goal of creating a readily available supply of antigen-matched, highly functional platelets. Current methods for generating platelets from iPS cell-derived megakaryocytes have been improved to produce enough platelets in vitro to begin phase I safety trials in humans; however, modifying the iPS cell genome has the potential to further improve both the yield and function of in vitro-generated platelets. The purpose of this investigation, therefore, was to combine recent advances in gene editing technology with iPS cell differentiation techniques to improve the yield and/or functionality of in vitro-derived platelet products. As a start, we sought to examine whether disruption of negative regulators of platelet production and/or activation might allow the generation of increased numbers of platelets with improved hemostatic effectiveness. One such negative regulator of platelet function is the Src family kinase, Lyn. Previous studies using pharmacologic inhibitors and global Lyn knockout mice have shown that that disrupting Lyn activity leads to mild thrombocytosis and megakaryocytosis. The effects of specifically targeting Lyn in human megakaryocytes and platelets, however, are not known. We hypothesized that conditionally disrupting Lyn during the process of iPSC to megakaryocyte differentiation might be an effective way to enhance megakaryocyte differentiation, platelet production, and hemostatic function. Using CRISPR/Cas9 gene editing technology, we generated a temporally controllable, tamoxifen-inducible Lyn-KO iPS cell line system that allowed us to conditionally delete Lyn at any desired stage of the iPSC→hematopoietic stem cell (HSC)→megakaryocyte→platelet differentiation process. We found tamoxifen efficiently disrupted Lyn expression at any of these stages within 24-48 hours after addition of the drug to the cell culture, and also improved the efficiency and yield of the iPSC→megakaryocyte→proplatelet differentiation process. Lyn-deficient human megakaryocytes prepared in this fashion exhibited enhanced reactivity, as reported by increased Erk phosphorylation in response to thrombopoietin stimulation, and by an improved ability to support hemostasis in human blood in vitro, as determined using thromboelastometry (ROTEM). Taken together, these data suggest that temporal disruption of Lyn kinase might represent an effective strategy for the engineering and manufacture of in vitro-derived platelets for both diagnostic and future therapeutic use.