No abstract

The intrahepatic bile duct (IHBD) is a highly organized tubular structure consisting of cholangiocytes, biliary epithelial cells, which drains bile produced by hepatocytes into the duodenum. Although several models have been proposed, it remains unclear how the three-dimensional (3D) IHBD network develops during liver organogenesis. Using 3D imaging techniques, we demonstrate that the continuous luminal network of IHBDs is established by 1 week after birth. Beyond this stage, the IHBD network consists of large ducts running along portal veins (PVs) and small ductules forming a mesh-like network around PVs. By analyzing embryonic and neonatal livers, we found that newly differentiated cholangiocytes progressively form a continuous and homogeneous luminal network. Elongation of this continuous network toward the liver periphery was attenuated by a potent Notch-signaling inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. Subsequent to this first step, the fine homogenous network is reorganized into the mature hierarchical network consisting of large ducts and small ductules. Between E17 and E18, when the homogenous network is radically reorganized into the mature hierarchical network, bile canaliculi rapidly extend and bile flow into IHBDs may increase. When formation of bile canaliculi was blocked between E16 and E18 by a multidrug resistance protein 2 inhibitor (benzbromarone), the structural rearrangement of IHBDs was significantly suppressed. Conclusion: Establishment of the mature IHBD network consists of two sequential events: (1) formation of the continuous luminal network regulated by the Notch-signaling pathway and (2) dynamic rearrangement of the homogeneous network into the hierarchical network induced by increased bile flow resulting from the establishment of hepatobiliary connections. (HEPATOLOGY 2016;64:175-188)

Late fetal and adult livers are reported to contain bipotential liver stem/ progenitor cells (LPCs), which share surface markers, including epithelial cell adhesion molecule (EpCAM), with cholangiocytes and differentiate into both hepatocytes and cholangiocytes. However, recent results do not necessarily support the idea that LPCs contribute significantly to cellular turnover and regeneration by supplying new hepatocytes. Here, we examined the colony-forming capability of EpCAM + cells isolated from mouse livers between E17 and 11 weeks of age. We found that the number of bipotential colonies was greatly reduced between 1 and 6 weeks, indicating that the number of LPCs decreases during postnatal development. Moreover, bipotential colonies derived from adult LPCs contained substantially fewer albumin + cells than those from neonatal LPCs. We further examined the differentiation potential of neonatal and adult LPCs by transplantation and found that neonatal cells differentiated into mature hepatocytes in recipient livers more frequently than adult LPCs. Since we previously reported that the transcription factor grainyhead-like 2 (GRHL2) expressed in EpCAM + cells inhibits hepatocytic differentiation, we examined whether targets of GRHL2 might block hepatocytic differentiation. DNA and microRNA microarrays revealed that miR122, the expression of which correlates with hepatocytic differentiation, was greatly reduced in adult as compared with neonatal EpCAM + cells. Indeed, GRHL2 negatively regulates the promoter/enhancer activity of the Mir122 gene. Our results indicate that neonatal but not adult EpCAM + LPCs have great potential to produce albumin + hepatocytes. GRHL2 suppresses transcription of miR122 and thereby restricts the differentiation potential of adult LPCs.

In galactosamine (GalN)-induced rat liver injury, hepatic stem/progenitor cells, small hepatocytes (SHs) and oval cells, transiently appear in the initial period of liver regeneration.

Oval cells and small hepatocytes (SHs) are known to be hepatic stem and progenitor cells. Although oval cells are believed to differentiate into mature hepatocytes (MHs) through SHs, the details of their differentiation process are not well understood. Furthermore, it is not certain whether the induced cells possess fully mature functions as MHs. In the present experiment, we used Thy1 and CD44 to isolate oval and progenitor cells, respectively, from D-galactosamine-treated rat livers. Epidermal growth factor, basic fibroblast growth factor, or hepatocyte growth factor could trigger the hepatocytic differentiation of sorted Thy1 1 cells to form epithelial cell colonies, and the combination of the factors stimulated the emergence and expansion of the colonies. Cells in the Thy1 1 -derived colonies grew more slowly than those in the CD44 1 -derived ones in vitro and in vivo and the degree of their hepatocytic differentiation increased with CD44 expression. Although the induced hepatocytes derived from Thy1 1 and CD44 1 cells showed similar morphology to MHs and formed organoids from the colonies similar to those from SHs, many hepatic differentiated functions of the induced hepatocytes were less well performed than those of mature SHs derived from the healthy liver. The gene expression of cytochrome P450 1A2, tryptophan 2,3-dioxygenase, and carbamoylphosphate synthetase I was lower in the induced hepatocytes than in mature SHs. In addition, the protein expression of CCAAT/enhancerbinding protein alpha and bile canalicular formation could not reach the levels of production of mature SHs. Conclusion: The results suggest that, although Thy1 1 and CD44 1 cells are able to differentiate into hepatocytes, the degree of maturation of the induced hepatocytes may not be equal to that of healthy resident hepatocytes. (HEPATOLOGY 2012; 57:1192-1202