STEMdiff NK Cell Kit

STEMCELL Technologies provides a culture system for generating natural killer (NK) cells from human pluripotent stem cells (hPSCs) under serum- and feeder-free conditions, without the use of stromal cells. The STEMdiff NK Cell Kit combines STEMdiff™ Hematopoietic - EB reagents with the StemSpan™ NK Cell Generation Kit and is used in a four-stage protocol that spans 40 days of culture.

In the first two stages, hPSCs are differentiated into CD34+ hematopoietic progenitor cells. Cells are seeded into AggreWell™ plates and cultured for 3 days in EB Formation Medium and EB Medium A to induce mesoderm specification and form uniform 600-cell embryoid body aggregates. A further 9 days of culture in EB Medium B promotes hematopoietic specification, after which the embryoid bodies are dissociated and CD34+ cells are enriched using EasySep™ immunomagnetic positive selection. In the third stage, CD34+ cells are cultured for 14 days in StemSpan™ Lymphoid Progenitor Expansion Medium on plates coated with StemSpan™ Lymphoid Differentiation Coating Material. In the fourth stage, lymphoid progenitor cells are reseeded onto non-coated plates in StemSpan™ NK Cell Differentiation Medium and cultured for 14 days to generate CD56+ NK cells.

The small molecule UM729 is required for optimal NK cell differentiation during the fourth stage and must be purchased separately. Across multiple ES and iPS cell lines, approximately 230 NK cells can be generated per input hPSC-derived CD34+ cell under this protocol.