1). These results demonstrate that the human iPSCs exhibit pluripotent properties before hepatogenic differentiation. It is imperative to ensure the differentiation abilities of the human iPSCs prior to therapeutic application. Here, we developed a three-step protocol by modifying the culture condition described by Hay et

al.,10 and Kuo et al.,26 in order to bring about the rapid generation of hepatocyte-like cells from human iPSCs. In this protocol, which is described in the Materials and Methods section and Table 1, the human iPSCs were allowed to reach approximately 70% confluence in feeder cell-free culture system over 4 days, and this was followed by treatment with endodermal induction medium on day 0 (Fig. 2A, panel i) in the presence of activin A, Wnt3a, and HGF. This produced a human Small molecule library iPSC morphology with a spiky shape due to the loss of ES cell structure that occurred after dissociation from cell–cell contact (Fig. 2A,

panel ii). Immunostaining revealed that most of the cells were positive for the definitive endoderm click here marker Sox17 (sex-determining region Y box 17; Fig. 2B), indicating that the human iPSCs efficiently differentiated into definitive endoderm during the endodermal induction step. Following the endodermal induction step, cells were treated with the hepatic commitment medium for 3 days; this changed the cell morphology from a spiky shape to a polygonal shape that had tight cell–cell contact (Fig. 2A, panel iii). Finally, the medium was changed to maturation medium, which resulted in the human iPSC morphology changing into a cuboidal shape (Fig. 2A, panel iv). Immunostaining of these cells confirmed that these hepatocyte-like cells were positive for alpha-fetoprotein (AFP) and albumin (ALB) (Fig. 2C). HGF has multiple effects on target cells in culture and has been demonstrated to be involved in liver development.19 In our endodermal induction step, we were interested in how HGF acted synergistically with activin A and Wnt3a to accelerate definitive endoderm formation. To confirm

this process, human iPSCs were induced mafosfamide in endodermal induction medium with or without HGF for 3 days. Consistent with definitive endoderm marker Sox17 expression, we observed that forkhead box a2 (Foxa2), which is another endodermal marker, could be detected after the endodermal induction step (Fig. 3A). Moreover, differentiation into Foxa2+ cells was detected in 39.35% ± 0.98% of iPSCs treated with HGF, compared to 14.18% ± 0.54% of iPSCs that did not have HGF treatment during the endodermal induction step (Fig. 3B). To further investigate whether HGF treatment results in increased formation of hepatic lineage cells, we examined the expression of Sox17 and Foxa2 expression at day 5. The results showed that Sox17 and Foxa2 coexisted during the hepatic commitment step (Fig. 3C).