Introduction
Liver transplantation is currently the only curative therapy for end-stage liver failure; however, establishment of alternative treatments is required owing to the serious donor organ shortage. Here, we propose a novel model of hybrid three-dimensional artificial livers using both human induced pluripotent stem cells (hiPSCs) and a rat decellularized liver serving as a scaffold.
Methods
Rat liver harvesting and decellularization were performed as reported in our previous studies. The decellularized liver scaffold was recellularized with hiPSC-derived hepatocyte-like cells (hiPSC-HLCs) through the biliary duct. The recellularized liver graft was continuously perfused with the culture medium using a pump at a flow rate of 0.5 mL/min in a standard CO
2
(5%) cell incubator at 37 °C.
Results
After 48 h of continuous perfusion culture, the hiPSC-HLCs of the recellularized liver distributed into the parenchymal space. Furthermore, the recellularized liver expressed the albumin (
ALB
) and
CYP3A4
genes, and secreted human ALB into the culture medium.
Conclusion
Novel hybrid artificial livers using hiPSCs and rat decellularized liver scaffolds were successfully generated, which possessed human hepatic functions.
Liver fibrosis is characterized by the progressive accumulation of extracellular matrix (ECM) and is a strong predictor of hepatocellular carcinoma (HCC) development and progression. However, the effect of ECM in fibrotic livers on HCC cells is poorly understood. The aims of this study were to create a new culture system that retained the natural ECM of fibrotic model livers and to establish whether natural ECM regulated the characteristics of HCC cells. Using an organ decellularization technique, we created a new culture system that preserved the tissue-specific ECM of fibrotic model livers from CCl4-treated rats. The content of ECM in fibrotic model liver scaffolds was increased and the ECM microstructure was distorted. Quantitative polymerase chain reaction and immunofluorescence assays of HCC cells cultured in fibrotic model liver scaffolds for 7 days showed an epithelial-mesenchymal transition phenotype. Moreover, the ECM of fibrotic model livers promoted proliferation and chemoresistance of HCC cells. These results showed a novel effect of natural ECM in fibrotic model livers on the malignant behaviour of HCC cells. This new culture system will be useful for both understanding the cell biology of fibrotic livers and developing novel anti-cancer drugs.
The current lack of an easily measurable surrogate marker of cancer stem cells (CSCs) prevents the clinical application of CSCs for hepatocellular carcinoma (HCC). We previously reported that keratin 19 (K19) is a novel HCC‐CSC marker associated with transforming growth factor beta (TGFβ)/Smad signaling, and that K19+ HCC‐CSCs could be a new therapeutic target of TGFβ receptor 1 inhibitor LY2157299. In this study, we examined whether K19+ HCC‐CSCs can be tracked using cytokeratin 19 fragment CYFRA 21‐1. In 147 HCC patients who underwent curative resection and evaluated K19 expression by immunohistochemistry, preoperative serum CYFRA 21‐1 levels were significantly higher in K19+ patients than in K19− patients (P < 0.01). Receiver operating characteristic analyses revealed that serum CYFRA 21‐1 was the statistically significant and the most sensitive predictor of tumor K19 expression among preoperative laboratory test values (P < 0.001). In HCC cells encoding with a K19 promoter‐driven enhanced green fluorescent protein, fluorescence‐activated cell sorting (FACS)‐isolated K19+ cells displayed significantly higher levels of supernatant CYFRA 21‐1 than K19− cells (P < 0.01). Gain/loss of K19 function experiments confirmed that CYFRA 21‐1 levels were regulated by K19 function in HCC cells. Furthermore, CYFRA 21‐1 levels reflected the treatment efficacy of LY2157299 in K19+ cells. In conclusion, CYFRA 21‐1 can be used to predict K19 expression in HCC, and should thereby aid in the development of novel therapeutic strategies targeting K19+ HCC‐CSCs.
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