METTL3 positively regulates the development and cytotoxicity of human embryonic stem cells-derived NK cells

Human embryonic stem cell-derived NK (hESC-NK) cells or induced pluripotent stem cell derived NK cells have demonstrated efficacy and safety in clinical trials for cancer therapy and serve as a valuable tool for studying the mechanisms of human NK cell development and effector functions. We previously demonstrated that the methylase METTL3 was essential for the development and effector functions of murine NK cells, but its role in human NK cells remained unknown. Herein, we constructed an H1 ESC strain with reduced METTL3 expression using lentivirus-delivered short hairpin (sh) RNA and generated hESC-NK cells via a two-stage differentiation system. Our findings demonstrated that METTL3 knockdown in hESCs reduced the proportion of hematopoietic stem and progenitor cells (HSPCs, CD34⁺ cells) during embryoid bodies (EBs) formation, and impaired subsequent differentiation into mature NK cells. Moreover, ESC-NK cells derived from shMETTL3-ESC (called shMETTL3-ESC-NK) showed impaired anti-tumor activity, evidenced by downregulation of mRNA and protein levels of critical effectors (perforin, granzyme B and IFN-γ) and reduced cytotoxicity against target cells. Furthermore, both mRNA and protein levels of T-BET and EOMES were significantly down-regulated in shMETTL3-ESC-NK cells. These transcription factors are critical for NK cell development and cytotoxicity, and their downregulation may underlie the maturation defects of shMETTL3-ESC-NK cells. Collectively, our study elucidates that METTL3 promotes the development, maturation and cytotoxicity of hESC-NK cells, recapitulating previous reports in murine NK cells.