|CAPERNA, THOMAS - Retired ARS Employee|
|Blomberg, Le Ann|
Submitted to: International Journal of Biochemistry and Cell Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/24/2017
Publication Date: N/A
Interpretive Summary: Baby pig liver cells were grown in vitro, that is, outside of the body, in culture dishes for a period of 2-3 weeks. Two types of liquid media were used to maintain the cells in culture. One of the liquid media contained a steroid and the other did not. When the pig liver cells were cultured in the steroid containing medium the cell culture became a layer of “hepatocytes”, the major cell type making up the liver. When the pig liver cells were cultured in medium without steroid the pig liver cells grew a little and self-organized into bile ducts, the ducts that drain bile out of the liver and into the gallbladder. The two cell cultures were analyzed for what genes they each turned on or off in order to be hepatocytes or bile ducts. This “gene expression” analysis showed that the cultured hepatocytes and bile ducts in the dish were very similar to the same cells in the actual liver of a pig. The work further defined this liver cell in vitro model. This liver cell model is unique, for any species, in showing the creation of actual bile ducts in vitro. The work demonstrates that the in vitro model will be useful for studying the functions of the pig’s liver, diseases of the pig’s liver, and the effects of changing the gene expression of the pig’s liver. The model will be applicable to human biomedical studies too.
Technical Abstract: A serum-free, feeder-cell-dependent, inductive differentiation culture system of porcine hepatocytes and bile ductules was analyzed for differential gene expression on a porcine genome microarray. Primary cultures of baby pig hepatocytes (BPH) were matured in culture as a monolayer of hepatocytes when cultured in serum-free (SF) medium containing dexamethazone (DEX) and 1% dimethylsulfoxide (DMSO) over a 2-3 week period. Primary cultures of BPH were induced to differentiate into cholangiocytes and self-organize into 3D bile ductules by culture in SF medium containing 0.5% DMSO. The transcriptional profile of the hepatocyte cultures were compared to the cultures of bile ductules. Several thousand pig genes were assayed for their expression level, and a review of 2.5-fold or greater differences reflected the known biological functions and gene expression markers of the two cell types, hepatocyte vs. cholangiocyte. Semi-quantitative RT-PCR analysis confirmed the differential gene expression found in the BPH differential differentiation culture system. Hepatocytes (DEX/DMSO culture conditions) expressed serum protein mRNA levels at significantly elevated levels in comparison to the bile ductule-differentiated cultures (0.5% DMSO culture condition). Other markers of hepatocytes or hepatocyte precursors, such as cytochrome P450, CD14, growth hormone receptor (GRH), and hepatic nuclear factor-1 alpha (HNF1A) were more highly expressed in hepatocyte cultures. Conversely mRNAs specific to cholangiocyte differentiation and function were significantly elevated in cultures that differentiated into 3D bile ductules in comparison to hepatocyte monolayers. The results further demonstrate the validity of the BPH culture system for modeling hepatocyte and cholangiocyte biology in vitro. New genes of potential importance to in vivo parenchymal liver tissue growth and differentiation were identified.