Generation of HLA-Universal IPSC-Derived Megakaryocytes and Platelets for Survival Under Refractoriness Conditions

Platelet (PLT) transfusion is indispensable to maintain homeostasis in thrombocytopenic patients. However, PLT transfusion refractoriness is a common life-threatening condition observed in multitransfused patients. The most frequent immune cause for PLT transfusion refractoriness is the presence of alloantibodies specific for human leukocyte antigen (HLA) class I epitopes. Here, we have silenced the expression of HLA class I to generate a stable HLA-universal induced pluripotent stem cell (iPSC) line that can be used as a renewable cell source for the generation of low immunogenic cell products. The expression of HLA class I was silenced by up to 82% and remained stable during iPSC cultivation. In this study, we have focused on the generation of megakaryocytes (MK) and PLTs from a HLA-universal iPSC source under feeder- and xeno-free conditions. On d 19, differentiation rates of MKs and PLTs with means of 58% and 76% were observed, respectively. HLA-universal iPSC-derived MKs showed polyploidy with DNA contents higher than 4n and formed proPLTs. Importantly, differentiated MKs remained silenced for HLA class I expression. HLA-universal MKs produced functional PLTs. Notably, iPSC-derived HLA-universal MKs were capable to escape antibody-mediated complement- and cellular-dependent cytotoxicity. Furthermore, HLA-universal MKs were able to produce PLTs after transfusion in a mouse model indicating that they might be used as an alternative to PLT transfusion. Thus, produced low immunogenic MKs and PLTs may become an alternative to PLT donation in PLT-based therapies and an important component in the management of severe alloimmunized patients.