Three-dimensional reconstructions of intrahepatic bile duct tubulogenesis in human liver

Protocols were developed to digitally visualize portal vessel branching and protein expression of hepatic cell lineage and extracellular matrix deposition markers in three dimensions. Samples from human prenatal livers ranging from 7 weeks + 2 days to 15？ weeks post conception as well as adult normal and acetaminophen intoxicated liver were used. The markers included cytokeratins (CK) 7 and 19, the epithelial cell adhesion molecule (EpCAM), hepatocyte paraffin 1 (HepPar1), sex determining region Y (SRY)-box 9 (SOX9), laminin, nestin, and aquaporin 1 (AQP1).Digital three-dimensional reconstructions using CK19 as a single marker protein disclosed a fine network of CK19 positive cells in the biliary tree in normal liver and in the extensive ductular reactions originating from intrahepatic bile ducts and branching into the parenchyma of the acetaminophen intoxicated liver. In the developing human liver, three-dimensional reconstructions using multiple marker proteins confirmed that the human intrahepatic biliary tree forms through several developmental stages involving an initial transition of primitive hepatocytes into cholangiocytes shaping the ductal plate followed by a process of maturation and remodeling where the intrahepatic biliary tree develops through an asymmetrical form of cholangiocyte tubulogenesis.The developed protocols provide a novel and sophisticated three-dimensional visualization of vessels and protein expression in human liver during development and disease.In the human embryo, the earliest morphological manifestation of the developing liver is the hepatic diverticulum. This structure is discernible as a thickening of endodermal cells in the embryonic foregut at the 17 somite stage, corresponding to 3 weeks + 5 days post conception [1]. Under the influence of mesodermal signaling, cords of endodermal cells expand from the cephalic part of the hepatic diverticulum into the septum transversum [2,3]. The parenchymal cords anastomose around pre-existin