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Title: Development, characterization and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19

Author
item Talbot, Neil
item Caperna, Thomas
item Garrett, Wesley

Submitted to: Journal of Animal Science
Publication Type: Review Article
Publication Acceptance Date: 10/28/2012
Publication Date: 1/20/2013
Citation: Talbot, N.C., Caperna, T.J., Garrett, W.M. 2013. Development, characterization and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19. Journal of Animal Science. 91:66-77.

Interpretive Summary: Embryonic stem cell lines have not been established from farm animals, however, we have developed several somatic cell lines from the in vitro culture of early embryonic cells of the pig. One such cell line, PICM-19, was isolated and found to differentiate into two liver cell types, viz., hepatocytes and bile duct cells. The morphology, cell structure, biochemistry and gene expression patterns of the cells as grown in culture were similar to the cells as they exist in live piglets. Several sublines of the PICM-19 cell line have also been developed whereby the cells will differentiate into either hepatocytes or bile duct cells, but not both. In addition new culture techniques have been developed that simplify the culture of these unique cells which will allow better characterization of their growth and cellular differentiation. In addition, the PICM-19 cell line has been used for several biomedical/agricultural related purposes such as the in vitro replication of Hepatitis E virus, a virus of pigs that can spread to people, and a spaceflight experiment to evaluate cell differentiation and liver cell function in microgravity. The cell line was also evaluated for its use in pharmacology/toxicology testing and has been utilized in a commercial artificial liver device. The availability of these cells will provide a better model system to investigate pig liver metabolism and may provide a useful tool to for the biomedical community to study and treat human liver diseases.

Technical Abstract: Totipotent embryonic stem cell lines have not been established from ungulates, however, we have developed several somatic cell lines from the in vitro culture of pig epiblast cells. One such cell line, PICM-19, was isolated via colony-cloning and was found to spontaneously differentiate into hepatic parenchymal epithelial cell types, viz., hepatocytes and bile duct cells. Hepatocytes form as monolayers and bile duct cells as 3D bile ductules. Transmission electron microscopy revealed that the ductules were composed of radially arranged, monociliated cells with their cilia projecting into the ductule’s lumen, whereas hepatocytes were arranged in monolayers with lateral canalicular structures containing numerous microvilli and connected by tight junctions and desmosomes. Extensive Golgi and rough endoplasmic reticulum networks were also present, indicative of active protein synthesis. Analysis of conditioned media by 2D-electrophoresis and mass spectrometry indicated a spectrum of serum-protein secretion by the hepatocytes. The PICM-19 cell line maintains a range of inducible cytochrome P450 activities, and, most notably, is the only non-transformed cell line that synthesizes urea in response to ammonia challenge. The PICM-19 cell line has been used for several biomedical/agricultural related purposes such as the in vitro replication of Hepatitis E virus (HEV), a zoonotic virus of pigs, and a spaceflight experiment to evaluate somatic stem cell differentiation and liver cell function in microgravity. The cell line was also evaluated as a platform for toxicity testing and has been utilized in a commercial artificial liver rescue device bioreactor (HepaLife Technologies, Inc.). A PICM-19 subclone, PICM-19H, which only differentiates into hepatocytes was isolated and methods are currently under development to grow PICM-19 cells without feeder-cells. Feeder-cell independent growth will facilitate the study of mesenchymal-parenchymal interactions that influence the divergent differentiation of the PICM-19 cells, will enhance our ability to genetically modify the cells, and will provide a better model system to investigate porcine hepatic metabolism.