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Hepatocyte‐like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells are expected to be applied for regenerative medicine. In this study, we attempted to generate safe and therapeutically effective human iPS‐HLCs for hepatocyte transplantation. First, human iPS‐HLCs were generated from a human leukocyte antigen‐homozygous donor on the assumption that the allogenic transplantation might be carried out. Highly efficient hepatocyte differentiation was performed under a feeder‐free condition using human recombinant laminin 111, laminin 511, and type IV collagen. The percentage of asialoglycoprotein receptor 1‐positive cells was greater than 80%, while the percentage of residual undifferentiated cells was approximately 0.003%. In addition, no teratoma formation was observed even at 16 weeks after human iPS‐HLC transplantation. Furthermore, harmful genetic somatic single‐nucleotide substitutions were not observed during the hepatocyte differentiation process. We also developed a cryopreservation protocol for hepatoblast‐like cells without negatively affecting their hepatocyte differentiation potential by programming the freezing temperature. To evaluate the therapeutic potential of human iPS‐HLCs, these cells (1 × 10 6  cells/mouse) were intrasplenically transplanted into acute liver injury mice treated with 3 mL/kg CCl 4  only once and chronic liver injury mice treated with 0.6 mL/kg CCl 4  twice weekly for 8 weeks. By human iPS‐HLC transplantation, the survival rate of the acute liver injury mice was significantly increased and the liver fibrosis level of chronic liver injury mice was significantly decreased.  Conclusion : We were able to generate safe and therapeutically effective human iPS‐HLCs for hepatocyte transplantation. ( Hepatology Communications  2017;1:1058–1069)

Generation of safe and therapeutically effective human induced pluripotent stem cell‐derived hepatocyte‐like cells for regenerative medicine