Abstract
Human induced pluripotent stem cell-derived hepatocyte-like cells are expected to be utilized in pharmaceutical research and regenerative medicine. In general, human induced pluripotent stem (iPS) cells are differentiated into hepatocyte-like cells through definitive endoderm cells and hepatoblast-like cells using various growth factors that are essential for liver development. Although recombinant bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) are widely used in the hepatoblast differentiation, hepatoblast differentiation process has not been fully modified. In this study, we examined the roles of BMPs and FGFs in the hepatoblast differentiation from human iPS cells. Surprisingly, the gene expression levels of hepatoblast markers were upregulated by the removal of FGFs. In addition, the percentages of hepatoblast markers-positive cells were increased by the removal of FGFs. Furthermore, the hepatocyte differentiation potency was also significantly increased by the removal of FGFs. To examine whether FGF signals are completely unnecessary for the hepatoblast differentiation, the expression levels of endogenous FGF ligands and receptors were examined. The definitive endoderm cells highly expressed the FGF ligand, FGF2, and the FGF receptor, FGFR1. To examine the role of endogenous FGF signals, an FGFR inhibitor was treated during the hepatoblast differentiation. The hepatoblast differentiation was promoted by using FGFR inhibitor, suggesting that endogenous FGF signals are also unnecessary for the hepatoblast differentiation. In conclusion, we found that FGF signals are not essential for hepatoblast differentiation. We believe that our finding will be useful for generating functional hepatocyte-like cells for medical applications.
Highlights
Human induced pluripotent stem cell-derived hepatocyte-like cells (HLCs) are expected to be utilized in pharmaceutical research and regenerative medicine
In the presence of BMP4, the gene expression levels of hepatoblast markers in the hepatoblast-like cells differentiated without fibroblast growth factors (FGFs) were higher than those in the hepatoblast-like cells differentiated with FGFs (Fig. 1b)
In the presence of BMP4, the gene expression levels of the pluripotent marker (octamer-binding transcription factor (OCT) 3/4) and the definitive endoderm marker (Sry-related HMG-box gene (SOX) 17) in hepatoblast-like cells differentiated without FGFs were decreased as compared with those of hepatoblast-like cells differentiated with FGF1/2/4 (Fig. S2)
Summary
Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells (HLCs) are expected to be utilized in pharmaceutical research and regenerative medicine. We expect that optimizing the hepatoblast differentiation protocol will be essential for the generation of functional and homogenous HLCs. Previous embryological studies of mouse, Xenopus, and zebrafish liver development have shown that bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signals are necessary for liver bud formation[7,8]. The expression levels of hematopoietically expressed homeobox and prospero-related homeobox 1 in hepatoblasts were significantly reduced when Bmp receptors or Fgf receptors were blocked by using dominant-negative forms of Bmp or Fgf receptors[9] From these studies, Bmp and Fgf signals have been considered to be essential for the hepatoblast differentiation from embryonic stem (ES) cells and iPS cells. The expression profiles of FGF and BMP receptors and their functions have not been fully examined in human iPS cell-derived definitive endoderm cells. We examined the expression profile and functions of FGF ligands and receptors in the hepatoblast differentiation
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