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

Abstract

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 in vivo transfusion in a mouse model indicating that they might be used as an alternative to PLT transfusion. Thus, in vitro 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.

Towards the Manufacture of Megakaryocytes and Platelets for Clinical Application

“…The application of donor-independent, infinite progenitor sources for optimized PLT production such as HLA-deficient iPSCs [61,63] or immortalized MKs [49] in close biomimetic reactors [69][70][71] or systems allowing a high production scalability, would enable one step further to clinical application [74,75]. Despite the recent advances in optimizing the features and methods for in vitro PLT production, so far the manufacture of PLTs remains ineffective, which may support the evaluation of alternative strategies.…”

“…Furthermore, Liu et al [62] achieved the differentiation of MKs from human iPSCs using FDA-approved pharmacological reagents. To reduce the risk of PLT transfusion refractoriness, iPSCs have been genetically engineered for the downregulation of HLA class I or PLT antigens using gene regulatory (RNA interference) [63] or editing (TALEN) strategies [61]. iPSC-derived HLA class I-silenced MKs and PLTs have shown to escape antibody-mediated cytotoxicity in vitro and in vivo [63].…”

“…To reduce the risk of PLT transfusion refractoriness, iPSCs have been genetically engineered for the downregulation of HLA class I or PLT antigens using gene regulatory (RNA interference) [63] or editing (TALEN) strategies [61]. iPSC-derived HLA class I-silenced MKs and PLTs have shown to escape antibody-mediated cytotoxicity in vitro and in vivo [63]. Also, CRISPR/Cas9 gene editing of PLT antigens in iPSCs was performed [64].…”

Large-Scale Cultures and Bioreactors for the Production of Megakaryocytes and Platelets

“…To circumvent an immunological reaction against in vitro generated MK or PLTs, alternatively a genetic knock out [14,15] or knockdown [16] of HLA class I in the source cells is possible, targeting the conserved domain beta2-microglobulin by TALEN, CRISPR/Cas9 or RNA interference, respectively. As the complete absence of HLA class I is activating the recognition by natural killer (NK) cells, an HLA knock out is useful for the production of PLTs only, since PLTs are not recognized by NK cells [17], in contrast to MKs.…”

“…However, a certain risk of MKs homing to secondary loci of thrombopoiesis such as the lung has likewise to be addressed. Available studies from mouse models reveal divergent results [16,32] which might be explained by a potentially abnormal behavior of human MKs within the murine circulation system.…”