Natural Killer Cell Differentiation from Hematopoietic Stem Cells: A Comparative Analysis of Heparin- and Stromal Cell–Supported Methods

Abstract

Natural killer (NK) cells differentiated from hematopietic stem cells (HSCs)may have significant clinical benefits over those from adult donors, including the ability to choose allo-reactive donors and potentially more robust in vivo expansion. Stromal-based methods have been used to study NK differentiation from HSCs. Stroma and cytokines support NK differentiation, but may have considerable regulatory hurdles. Recently, a clinical grade heparin-based method has been reported and could serve as an alternative. How these two compare in NK generating efficiency or functionis unknown. We show that compared to heparin-based cultures, stromasignificantly 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 or inhibitor receptors. While both approaches resulted in a phenotype consistent with CD56brightstage IV NK cells, heparin-based cultures favored the development of CD56+CD16+ cells, while stromaproducedmore KIR-expressing NK cells, both markers of terminal maturation. At day 21, stromal-based cultures showed significantly more IL-22 production and both methods yielded similar amounts of IFN-γ production and cytotoxicity (day 35). Thus, heparin-based cultures are an effective replacement for stroma and may facilitate clinical trials testing HSC-derived NK cells.

Frozen Cord Blood Hematopoietic Stem Cells Differentiate into Higher Numbers of Functional Natural Killer Cells In Vitro than Mobilized Hematopoietic Stem Cells or Freshly Isolated Cord Blood Hematopoietic Stem Cells

“…It could be that additional signals are required in our system for KIR expression. In the same study the authors found higher CD16 expression on NK cells generated using a heparin-based system compared to those generated using the EL08.1D2 feeder layer [14]. The generation of CD56 + CD16 + cells from either PBSC (100% CD16 + ) [43] or CBSC (24% CD16 + ) has been reported [44].…”

Combined IL-15 and IL-12 drives the generation of CD34+-derived natural killer cells with superior maturation and alloreactivity potential following adoptive transfer

“…In particular, expansion and differentiation of HPCs allows the generation of clinically applicable NK cell products with high purity and functionality. 25 Nevertheless, we and others have observed that NK cells generated under stroma-free culture conditions using IL-15 and IL-2 have low surface expression of CD16 and only a subset (5-10%) express KIRs, 23,37 raising questions about NK cell potency and alloreactivity following adoptive transfer. Moreover, clinical studies reported so far show that adoptively transferred allogeneic NK cells have a relatively short lifespan in patients.…”

Bringing natural killer cells to the clinic: ex vivo manipulation

“…Dezell et al have shown that NK cell expansion from hematopoietic progenitors using stromal cells appears to increase the yield of NK cells compared with stromal-free conditions, perhaps by differentiating less committed progenitors into the NK cell lineage. 62 Despite this potential advantage, stromal-based expansions are logistically more challenging to use under GMP conditions, potentially limiting their application in the clinical setting.…”