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 . 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-existing endothelial-lined spaces, increase in mass and become more organized at the expense of the septum transversum that eventually forms the liver capsule .
Several theories exist regarding the development of intrahepatic bile ducts from this early stage liver. Current understanding of the process is that primitive hepatocytes in contact with the mesenchyme surrounding developing hepatic portal veins form a single-layered structure known as the ductal plate. The ductal plate becomes bi-layered with a parenchymal and a mesenchymal facing sheet, respectively. The ductal plate consists of cuboidal cells with increased immunoreactivity for epithelial intermediate filaments such as cytokeratins (CK) 8, CK18 and CK19, relative to the surrounding parenchymal cells [4, 5]. Slit-like lumina form between the two layers of cells that migrate into the portal mesenchyme to form mature intrahepatic bile ducts [6, 7]. Thus, development of intrahepatic bile ducts by cholangiocyte tubulogenesis can be suggested to occur through a series of remodeling stages, i.e. the "ductal plate" stage, the "remodeling bile duct" stage, and the "remodeled bile duct" stage [8–10].
In adult liver, the intrahepatic bile duct is connected with the hepatocytic canaliculi via the Canal of Hering. Current consensus is that the Canal of Hering being the most distal part of the biliary tree constitutes the hepatic progenitor cell niche, a protective microenvironment in which hepatic progenitors reside [11–14]. In prenatal liver, the ductal plates are suggested to not only constitute the hepatic progenitor niche, but also to be directly antecedent to the canal of Hering [11, 13]. The postnatal liver has considerable inherent regenerative capacity. Following acute injury, the tissue mass is restored by mitotic division of mature hepatocytes and cholangiocytes. However, when this capacity is compromised during massive or chronic injury hepatic progenitor cells are recruited to restore a functional liver [15–18].
When analyzing human liver sections histologically, a somewhat constant architecture of hepatocytes, vessels and bile ducts can be demonstrated. This monotonous histological appearance actually reflects a highly complex tissue architecture that is not well understood and frequently debated [19–21]. Therefore, gaining knowledge of the mechanisms involved in human cholangiocyte differentiation is expected to enhance fundamental understanding of cholangiocyte tubulogenesis. This is obtained by visualizing not only ductal plate formation and the remarkable remodeling of the biliary tree during liver development, but also the branching network in adult normal and diseased liver.
The aims of the present study were to develop protocols for digital reconstruction of the human intrahepatic biliary system and the portal vessels in three dimensions (3D) from 2 dimensional photographs of serially sectioned liver, and to describe human intrahepatic cholangiocyte tubulogenesis. These 3D-protocols were based on two approaches: volumetric rendering and surface rendering through image segmentation. Volumetric renderings are constructed by calculating the brightness and color from each volumetric pixel element (voxel) based on the voxel color value and the voxel transparency . Thereby, a three dimensional picture can be created displaying the actual sections in colors without the need for segmenting the objects of interest on each photograph. Surface rendering based on image segmentation involves the process of outlining the objects of interest, thereby assigning each pixel to a certain object or structure and creating vectors for visualizing the objects in three dimensions.
To prove the validity of this 3D-approach in studies of human liver biology, we stained serial sections from adult normal and acetaminophen intoxicated livers with the cholangiocyte marker CK19 recapitulating a previous non-digitized 3D-study by Theise et al. . Next, in order to describe the structural development of the intrahepatic biliary tree, a unique collection of liver from adults as well as human embryos and fetuses ranging from 5 weeks + 6 days to 15½ weeks post conception were stained with markers previously used to identify the cholangiocytic or hepatocytic lineages in the adult liver [4, 23–26]. These markers included CK7 and CK19, epithelial cell adhesion molecule (EpCAM), hepatocyte paraffin 1 (HepPar1), sex-determining region Y (SRY)-box 9 (SOX9) and aquaporin 1 (AQP1). In addition, laminin and nestin were used as markers of extracellular matrix deposition during tubulogenesis. By means of the developed 3D-protocols, digital reconstructions were created of the three developmental stages, i.e. the "ductal plate", the "remodeling bile duct" and the "remodeled bile duct" stages, with emphasis on the localization of CK7, CK19, EpCAM, SOX9 and AQP1.
The digitized 3D-reconstruction study successfully depicted the spatial expression of marker proteins and the distribution of vessels in portal areas of the developing and adult human liver. It also demonstrated the tubular formation of cholangiocytes into a branching network that will eventually form the human intrahepatic biliary tree. The findings support the theory that human intrahepatic biliary tree development proceeds by an asymmetrical tubulogenesis, which has recently been described in mice . Finally, it was shown that the ductular reaction characteristic of acetaminophen intoxication forms a tortuous network of interconnected CK19 positive biliary structures extending from the bile duct epithelium into the parenchyma, as originally described by Theise et al. .