Rare antigen‐negative red blood cells from pluripotent stem cells for precision transfusion medicine

BACKGROUND: Blood bank identification of antibodies against high-prevalence antigens remains a challenge due to the scarcity of antigen-negative reagent red cells sourced from blood donors. The MAM antigen, encoded by EMP3, is one such antigen associated with red cell alloimmunization and hemolytic disease of the fetus and newborn. STUDY DESIGN AND METHODS: We used CRISPR-Cas9 gene editing to generate an EMP3 knockout (EMP3KO) induced pluripotent stem cell (iPSC) line from a type O, Rh null parent line, enabling production of rare MAM-negative red blood cells. Since a prior study suggested that loss of EMP3 may enhance erythroid proliferation, we hypothesized that EMP3KO could both yield a rare reagent cell and potentially improve erythroid expansion to support scalable production. Transcriptomic analysis allowed us to further investigate the effect of EMP3 loss in late erythroblasts. RESULTS: EMP3KO cells differentiated efficiently into erythroid cells, showing >95% CD235/CD71 co-expression and orthochromatic erythroblast morphology. Compared to unedited cells, no proliferative advantage was observed, contrasting with prior non-isogenic cell models. Agglutination assays confirmed complete loss of MAM antigen and demonstrated the diagnostic utility for identifying MAM antibodies. Transcriptomic profiling of EMP3KO erythroblasts revealed expression of key erythroid genes, as well as regulators of proliferation and heme metabolism, was comparable to the parent line. DISCUSSION: This study demonstrates that iPSC technology combined with gene editing can generate rare antigen-negative RBCs for immunohematology applications. Beyond MAM, this platform offers a strategy to create additional rare RBC phenotypes, advancing precision transfusion medicine and improving antibody identification against high-prevalence antigens.