In vitro expansion of cirrhosis derived liver epithelial cells with defined small molecules.

BACKGROUND & AIMS: Mature hepatocytes have limited expansion capability in culture and rapidly loose key functions. Recently however, tissue culture conditions have been developed that permit rodent hepatocytes to proliferate and transform into progenitor-like cells with ductal characteristics in vitro. Analogous cells expressing both hepatic and duct markers can be found in human cirrhotic liver in vivo and may represent an expandable population. METHODS: An in vitro culture system to expand epithelial cells from human end stage liver disease organs was developed by inhibiting the canonical TGF-ß, Hedgehog and BMP pathways. RESULTS: Human cirrhotic liver epithelial cells became highly proliferative in vitro. Both gene expression and DNA methylation site analyses revealed that cirrhosis derived epithelial liver cells were intermediate between normal hepatocytes and cholangiocytes. Mouse hepatocytes could be expanded under the same conditions and retained the ability to re-differentiate into hepatocytes upon transplantation. In contrast, human cirrhotic liver derived cells had only low re-differentiation capacity. CONCLUSIONS: Epithelial cells of intermediate ductal-hepatocytic phenotype can be isolated from human cirrhotic livers and expanded in vitro. Unlike their murine counterparts they have limited liver repopulation potential.

Identification and Isolation of Clonogenic Cholangiocyte in Mouse.

Cholangiocytes are proliferative and are one of the sources for liver progenitor cells. Clonogenic cholangiocytes are defined as cells capable of clonally proliferating and differentiating cholangiocytes both in vitro and in vivo. In this protocol, we describe the method for isolation of primary cholangiocytes from mouse. To study the heterogeneity of cholangiocytes, we used flow cytometry-based cell sorting to isolate different subsets of cholangiocytes. Organoid-forming efficiencies from sorted single cells are compared within different cholangiocyte populations to identify clonogenic cholangiocytes.