| 摘要: | Cholangiocytes are epithelial cells that line the biliary system and the diseases that target cholangiocytes are known as cholangiopathies, which most still have unknown etiologies. Liver transplantation is the only treatment available if the patients fall into end-stage liver diseases. However, the limited accessibility of the bile duct precludes the modeling of several cholangiocyte diseases. In this study, by mimicking the embryonic development of cholangiocytes, we present a robust, feeder-free, serum-free protocol with 2D and 3D culture conditions to generate the cholangiocytes from the human PSCs. The expressions of mature cholangiocyte markers (CK7, CK19, CFTR) and their functional assays, including secretory potential, alkaline phosphatase, and CFTR activity, were used to evaluate the human PSC-derived cholangiocytes on the final day of differentiation (day-28). Further analysis showed that generated cholangiocyte organoids (COs) under 3D culture show regional-specific markers of intrahepatic cholangiocytes markers (YAP1, JAG1) and extrahepatic cholangiocytes (AQP1, MUC1), indicating the successful protocols in generating both the intra- and extrahepatic cholangiocytes, and that might be regulated by Notch and Wnt signaling with the extracellular matrix supports. In addition, the COs can resemble the tube-like structure after day 28 of differentiation. This study provides the step-wise cholangiocyte differentiation protocol from human PSCs and the advantages of the 3D culture model compared to the 2D system in functional cholangiocyte maturation. The extracellular matrix supports and epidermal growth factor receptor (EGFR)-mediated signaling regulation may contribute to cholangiocyte maturation and differentiation. Our results indicate the successful differentiation of intrahepatic and extrahepatic cholangiocytes using 3D systems, showcasing the benefits of our 3D culture system over the 2D culture system in promoting cholangiocyte functional differentiation and maturation. In conclusion, our study advances previous research by providing a concept of transdifferentiation of small-large cholangiocytes derived from human PSCs under cost-effective 3D culture conditions, with potential implications for developing effective cell-based therapies for patients with cholangiopathies
Cholangiocytes are epithelial cells that line the biliary system and the diseases that target cholangiocytes are known as cholangiopathies, which most still have unknown etiologies. Liver transplantation is the only treatment available if the patients fall into end-stage liver diseases. However, the limited accessibility of the bile duct precludes the modeling of several cholangiocyte diseases. In this study, by mimicking the embryonic development of cholangiocytes, we present a robust, feeder-free, serum-free protocol with 2D and 3D culture conditions to generate the cholangiocytes from the human PSCs. The expressions of mature cholangiocyte markers (CK7, CK19, CFTR) and their functional assays, including secretory potential, alkaline phosphatase, and CFTR activity, were used to evaluate the human PSC-derived cholangiocytes on the final day of differentiation (day-28). Further analysis showed that generated cholangiocyte organoids (COs) under 3D culture show regional-specific markers of intrahepatic cholangiocytes markers (YAP1, JAG1) and extrahepatic cholangiocytes (AQP1, MUC1), indicating the successful protocols in generating both the intra- and extrahepatic cholangiocytes, and that might be regulated by Notch and Wnt signaling with the extracellular matrix supports. In addition, the COs can resemble the tube-like structure after day 28 of differentiation. This study provides the step-wise cholangiocyte differentiation protocol from human PSCs and the advantages of the 3D culture model compared to the 2D system in functional cholangiocyte maturation. The extracellular matrix supports and epidermal growth factor receptor (EGFR)-mediated signaling regulation may contribute to cholangiocyte maturation and differentiation. Our results indicate the successful differentiation of intrahepatic and extrahepatic cholangiocytes using 3D systems, showcasing the benefits of our 3D culture system over the 2D culture system in promoting cholangiocyte functional differentiation and maturation. In conclusion, our study advances previous research by providing a concept of transdifferentiation of small-large cholangiocytes derived from human PSCs under cost-effective 3D culture conditions, with potential implications for developing effective cell-based therapies for patients with cholangiopathies |
