Generation and characterization of HLA-Universal, iPSC-derived platelets

Abstract

Platelet demand has been increased around the world resulting from many factors including the rise of aging population, hematological malignancy incidence, and hematopoietic stem cell transplantation. In addition, inadequacy of donors or matched donors, time consuming, cost, the risk of transfusion-transmitted infections and transfusion-associated reactions, and platelet transfusion refractoriness are limits of this approach. To solve these problems, here we propose to generate platelets in vitro which do not induce alloimmunity to HLA class I, a major cause of immune factors in platelet transfusion refractoriness. In this study, we have knocked out β2-microglobulin gene (β2m) in iPSCs using paired CRISPR/Cas9 nickases then differentiated to hematopoietic stem cells, megakaryocytes (MKs) and platelets via ES-sac method. Silencing of HLA class I expression on cell surface of β2m-knocked out iPSCs, iPSC-derived HSCs, MKs and platelets were observed. Moreover, all lines of iPSC-derived MKs showed large size with multilobed nucleus, polyploidy with higher than 4n of DNA contents, proplatelet formation and they gave rise to functional platelets with lower activity when compared with peripheral blood-derived platelets using platelet activation and platelet aggregation assay based on flow cytometry. In summary, we generated in vitro functional iPSC-derived platelets with HLA class I deficiency by knocking out β2m gene using paired CRISPR/Cas9nickases. The genome editing system which is highly efficient on target and barely detected off-target, can be applied for regenerative medicine field. Further studies to produce large-scale iPSC-derived platelets and tests about safety profile are needed before the practical use of this approach to prevent or treat the patients of all HLA class I type from risk of life-threatening hemorrhage.