Since the immunosuppressive regimen currently used after organ transplantation reduces the adaptive immune components but effectively maintains the innate components of cellular immunity, the augmentation of the natural killer (NK) cell response, which is thought to play a pivotal role in innate immunity, may be a promising immunotherapeutic approach to prevent infectious complications. Therapies involving NK cells under immunosuppression may be accomplished by transferring exogenous cells by adoptive cell therapy. We attempted to develop a technique to generate NK cells from human hematopoietic stem cells (HSCs) and induced pluripotent stem cells (iPSCs)

To differentiate NK cells, we incubated human CD34+ HSCs derived from either bone marrow or peripheral blood in X-VIVO-15 medium containing 5% serum, SCF, IL-7, IL-15, and Flt-3L for 14-25 days. After further incubation with IL-12/IL-18 for 3 days, the propagated NK cells expressed NKp46 and were phenotypically similar to the maturational stage IV NK cells, i.e. they expressed TRAIL, NKG2D, and CD226, and produced IFN-γ. In an experiment using HCV replicon cells, the propagated NK cells significantly inhibited HCV replication. In an experiment using human hepatocyte chimeric mice, which develop persistent HCV infection, administration of the propagated NK cells reduced the incidence of HCV infection after 1 week. The propagated NK cells exhibited vigorous cytotoxicity against hepatoma cells even in the presence of immunosuppressive drugs. This cytotoxicity was canceled by anti-TRAIL mAb and concanamycin A.

Additionally, undifferentiated human iPSCs were induced to hematopoietic differentiation by coculture with the mouse bone marrow-derived stromal cells. After 28 days, the propagated CD34+ hematopoietic cells were isolated and transferred to the mouse liver-derived stromal cells. Further 21-28 days passed, proliferated cells displayed the typical NK cell phenotype with cytotoxicity and anti HCV activities, i.e. they expressed NKp46, CD16, CD94, and CD226 in addition to IFN-γ.

In conclusion, HSCs effectively differentiated into IFN-γ-producing NK cells that potentially inhibited HCV RNA replication and exhibited anti-HCC activities. We also demonstrated the derivation of NK cells from iPSCs that had the potential of anti-HCV and anti-HCC activities. The adaptive immunotherapy with these cells may be a promising modality to promote biophylaxis activities after organ transplantation (e.g. to prevent recurrence of HCV/HCC after liver transplantation).