Natural killer (NK) cells differentiated from hematopoietic stem cells (HSCs) may have significant clinical benefits over NK cells from adult donors, including the ability to choose alloreactive donors and potentially more robust in vivo expansion. Stromal-based methods have been used to study the differentiation of NK cells from HSCs. Stroma and cytokines support NK cell differentiation, but may face considerable regulatory hurdles. A recently reported clinical-grade, heparin-based method could serve as an alternative. How the stromal-based and heparin-based approaches compare in terms of NK cell generating efficiency or function is unknown. We show that compared with heparin-based cultures, stroma significantly increases the yield of HSC-derived NK cells by differentiating less-committed progenitors into the NK lineage. NK cells generated by both approaches were similar for most NK-activating and -inhibiting receptors. Although both approaches resulted in a phenotype consistent with CD56bright stage IV NK cells, heparin-based cultures favored the development of CD56+CD16+ cells, whereas stroma produced more NK cell immunoglobulin-like receptor-expressing NK cells, both of which are markers of terminal maturation. At day 21, stromal-based cultures demonstrated significantly more IL-22 production, and both methods yielded similar amounts of IFN-γ production and cytotoxicity by day 35. These findings suggest that heparin-based cultures are an effective replacement for stroma and may facilitate clinical trials testing HSC-derived NK cells.
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Financial disclosure: In this work, ex vivo generation of NK cells was done using GBGM Medium, a commercially available cell culture medium previously developed by Glycostem Therapeutics. The purchase and/or use of GBGM medium is not restricted by patent rights. Glycostem Therapeutics has acted as a sponsor of this study by providing media. All experiments were conducted and interpreted by the Verneris laboratory staff. Jan Spanholtz is an employee of Glycostem, which is scientifically directed by Harry Dolstra. Dolstra has been compensated by Glycostem previously. Neither Dolstra nor Radboud University Medical Center has a financial interest in Glycostem. The authors agree to make freely available any materials and information associated with their publication that are reasonably requested by others for the purpose of academic, noncommercial research. This work was supported by funding from the American Cancer Society (to M.R.V.) and the Children’s Cancer Research Fund (to M.R.V.) and by National Institutes of Health Grants PO1 CA65493 (to J.S.M.), PO1 111412 (to M.R.V. and J.S.M.), RO1 HL55417 (to J.S.M.), and PO1 067493 (to J.S.M.). The authors have no conflicts of interest to disclose.
- NK differentiation