Generation of iPSC-Derived Human Megakaryocytes for Flow Cytometric Detection of Platelet-Specific Alloantibodies

Antibodies that form against human platelet alloantigens (HPAs) are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia, posttransfusion purpura, and multitransfusion platelet refractoriness. Some of HPAs are relatively rare in the population, and difficult to obtain for purposes of transfusion therapy and diagnostic testing. In addition, HPA alloantisera often contain antibodies against human leucocyte antigen (HLA) class I, thereby limiting antibody detection to glycoprotein (GP)-specific assays such as the modified antigen capture enzyme-linked immunosorbent assay (MACE) and the monoclonal immobilization of platelet antigen (MAIPA), which are tedious and require solubilization of platelet GPs that may cause the loss of epitopes. In this study we aimed to generate gene-edited, HPA-specific, megakaryocytes (MKs) derived from human induced pluripotent stem cells (iPSCs) that could be used for simple flow cytometric detection of specific HPA alloantibodies present in patient sera. The HPA-3a/HPA-3b alloantigen system, also known as Bak^a/Bak^b, is caused by a single T13809G nucleotide substitution in the ITGA2B gene, resulting in an Ile874Ser amino acid polymorphism near the C terminus of the integrin αIIb subunit (GPIIb). Here we targeted HPA-3 system because alloantibodies targeting HPA-3 are often hard to detect with current detection methods, in part due to the requirement for cell type-specific glycosylation. To prevent interference of anti-A or anti-B antibodies in patient sera, a blood type O iPSC line (OT1-1) was generated from human peripheral blood mononuclear cells derived from a healthy donor using integration-free episomal vectors. The gene for β2 microglobulin (B2M) was first ablated from the OT1-1 iPS cell line using CRISPR/Cas9 to prevent binding of HLA class I alloantibodies. The resulting B2M knockout (B2MKO) cells were then additionally gene edited to convert the endogenous HPA-3a alloantigenic epitope present on B2MKO OT1-1 cells to HPA-3b. Two different guide RNAs targeting sequences that flank exon 26 of the ITGA2B gene were designed such that the entire exon harboring the HPA-3 polymorphic site was removed. A plasmid harboring a template replacing exon 26 with the G13809 mutation, flanked by 600 bp homology arms, was cotransfected into the B2MKO OT1-1 iPSCs together with the two CRISPR/Cas9 guide RNA constructs. iPSC clones containing the desired targeted T13809G mutation were identified by a diagnostic MfeI digestion specific for the G13089-bearing HPA-3b allele. Sequence analysis confirmed conversion of T13089 to G in these HPA-3b clones. Flow cytometric analysis showed the HPA-3a iPSCs, when differentiated into CD41⁺/CD42b⁺ MKs, specifically reacted with HPA-3a, but not HPA-3b, patient sera, while the HPA-3b iPSC-derived MKs lost reactivity with HPA-3a patient serum, and gained the reactivity with HPA-3b patient sera. Taken together, we have established genetically modified iPSC-derived MKs expressing specific HPAs that are suitable for simple flow cytometry-based detection of HPA alloantibodies in patient sera, with low non-specific background binding. This system provides intact antigens on the cell surface with carbohydrate moieties that likely mimic those found on human platelets, thus facilitating the detection of HPA alloantibodies that are normally hard to detect with current methods. Application of this strategy to genetically edit this and other clinically-important HPAs holds great potential for producing Designer Platelets for diagnostic, investigative and ultimately therapeutic use.