Natural Killer (NK) cells play an important role in innate immunity by secreting proinflammatory cytokines and killing tumor cells and virus-infected cells. Human NK cells can be generated by culturing CD34+ hematopoietic stem and progenitor cells (HSPCs) with stromal cells and cytokines. The use of stroma (and serum) is, however, not desirable in clinical applications. We developed a culture method for generating large numbers of NK cells from purified CD34+ cord blood (CB) HSPCs in the absence of serum and stromal cells. CD34+ CB cells were isolated by EasySep magnetic separation, seeded into culture plates coated with a Notch ligand and cultured for two weeks in StemSpan Serum-Free Expansion Medium (SFEM II) supplemented with SCF, TPO, Flt3L and IL-7. These conditions promote expansion of HSPCs and their differentiation into CD7+CD5+ lymphoid progenitors. Cells were then transferred to non-coated plates and cultured for two more weeks in StemSpan SFEM II supplemented with SCF, Flt3L, IL-7, IL-15 and a small molecule, UM729, to promote differentiation into NK cells. After culture, on average 76% (range: 54–90%, n=12) of cells expressed the NK cell marker CD56 and 58% (range: 25–80%) coexpressed CD56 and NKp46 (a NK cell activating receptor). A more mature CD56+CD16+ NK cell subset also emerged in these cultures (5%, range: 1–11%). The yield of CD56+ NK cells was ~13,000 (600–41,000) per initial CD34+ cell. The NK cells were functional and able to kill K562 target cells. These results show that HSPCs can expand and differentiate into NK cells under stroma- and serum-free culture conditions. This novel culture system will be useful for studies directed toward the development of cancer immunotherapies where large numbers of NK cells are needed.
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