The generation of stem cell-derived hepatocytes holds promise as a cell-based treatment for liver disease. However, the immune response to stem cell-derived grafts is poorly understood. The purpose of this study was to investigate the role of natural killer (NK) cells in recognition and killing of stem cells and their differentiated cellular products. Murine embryonic stem cells (ESC) were derived from FVB-Tg(CAG-luc,-GFP) L2G85Chco/J mice and were utilized to derive hepatoblasts using a two step, 13 day differentiation process. Differentiation was monitored by gene expression, flow cytometry and microscopy. At day 13 of differentiation, >90% of cells were positive for the hepatoblast marker CD326+cKIT, displayed morphologic features of the hepatocyte lineage including albumin and glycogen production, and expressed hepatoblast-associated genes. ESC-derived hepatoblasts were analyzed by qPCR for expression of NK cell ligand genes. ESC-derived hepatoblasts express higher levels of transcripts for the NK cell ligands Rae-1, H60, and MULT1, and lower levels of the MHC class 1 subunit, beta 2 microglobulin, than adult hepatocytes. In comparison to ESC, the expression of H60 was lower on hepatoblasts, while Rae-1 was similar, and MULT-1 was increased. The altered expression of NK cell activating and inhibitory ligands suggests that ESC and ESC-derived hepatoblasts may be vulnerable to NK cell mediated killing. To test this possibility NK cells were isolated from FVB or C57Bl/6 mice and used in in vitro cytotoxicity assays against the NK cell target YAC, or FVB ESC, at E:T of 1:1 to 16:1. Maximal killing of syngeneic FVB ESC targets by NK cells at an E:T of 16:1 reached 8.9%±0.3 whereas killing by allogeneic NK cells targets at E:T of 16:1 was 44.6%±0.9. At E:T of 16:1 killing of YAC targets was 82.1%±3.9. Together, these data indicate that ESC and ESC-derived hepatoblasts displayed increased expression of NK cell ligands and decreased expression of the NK cell inhibitory ligands compared to adult hepatoctes. Moreover, ESC are vulnerable to NK cell-mediated killing. Thus, the success of ESC-derived cellular grafts may require modulation or inhibition of NK cell mediated cytotoxicity.

CITATION INFORMATION: Cisneros T, Dillard D, Castro M, Arredondo-Guerrero J, Krams S, Esquivel C, Martinez O. The Role of Natural Killer Cells in Recognition and Killing of Stem Cells and Stem Cell-Derived Hepatoblasts. Am J Transplant. 2017;17 (suppl 3).

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