A scalable, spin‐free approach to generate enhanced induced pluripotent stem cell–derived natural killer cells for cancer immunotherapy

Abstract Natural killer (NK) cells play a vital role in innate immunity and show great promise in cancer immunotherapy. Traditional sources of NK cells, such as the peripheral blood, are limited by availability and donor variability. In addition, in vitro expansion can lead to functional exhaustion and gene editing challenges. This study aimed to harness induced pluripotent stem cell (iPSC) technology to provide a consistent and scalable source of NK cells, overcoming the limitations of traditional sources and enhancing the potential for cancer immunotherapy applications. We developed human placental–derived iPSC lines using reprogramming techniques. Subsequently, an optimized two‐step differentiation protocol was introduced to generate high‐purity NK cells. Initially, iPSCs were differentiated into hematopoietic‐like stem cells using spin‐free embryoid bodies (EBs). Subsequently, the EBs were transferred to ultra‐low attachment plates to induce NK cell differentiation. iPSC‐derived NK (iNK) cells expressed common NK cell markers (NKp46, NKp30, NKp44, CD16 and eomesodermin) at both RNA and protein levels. iNK cells demonstrated significant resilience to cryopreservation and exhibited enhanced cytotoxicity. The incorporation of a chimeric antigen receptor (CAR) construct further augmented their cytotoxic potential. This study exemplifies the feasibility of generating iNK cells with high purity and enhanced functional capabilities, their improved resilience to cryopreservation and the potential to have augmented cytotoxicity through CAR expression. Our findings offer a promising pathway for the development of potential cellular immunotherapies, highlighting the critical role of iPSC technology in overcoming challenges associated with traditional NK cell sources.