A Robust Protocol for Differentiation and Expansion of hPSC-derived NK Cells

Natural killer (NK) cells are critical innate lymphocytes that serve as the first line of defense against tumor and virus-infected cells by exerting major histocompatibility complex (MHC)-unrestricted cytotoxicity and regulating adaptive immunity through cytokine secretion. However, the clinical application of primary NK (pNK) cells is limited by low yield, inefficient gene transduction, and donor-to-donor variability. Human pluripotent stem cell (hPSC)-induced NK (iNK) cells represent a promising "off-the-shelf" cell source for cancer immunotherapy, enabling the scalable production of homogeneous iNK cells. Here, we present a standardized and reproducible protocol for the directed differentiation of hPSCs into functional iNK cells, along with their efficient ex vivo expansion. We define critical procedural steps, optimized culture conditions, key parameters, and quality control criteria to ensure consistency and functionality. This protocol offers a valuable resource for preclinical research and facilitates the development of standardized, off-the-shelf NK cell therapies for oncology applications.