Stem/progenitors have been identified intrahepatically in the canals of Hering and extrahepatically in glands of the biliary tree. Glands of the biliary tree (peribiliary glands) are tubulo‐alveolar glands with mucinous and serous acini, located deep within intrahepatic and extrahepatic bile ducts. We have shown that biliary tree stem/progenitors (BTSCs) are multipotent, giving rise in vitro and in vivo to hepatocytes, cholangiocytes or pancreatic islets. Cells with the phenotype of BTSCs are located at the bottom of the peribiliary glands near the fibromuscular layer. They are phenotypically heterogeneous, expressing transcription factors as well as surface and cytoplasmic markers for stem/progenitors of liver (e.g. SOX9/17), pancreas (e.g. PDX1) and endoderm (e.g. SOX17, EpCAM, NCAM, CXCR4, Lgr5, OCT4) but not for mature markers (e.g. albumin, secretin receptor or insulin). Subpopulations co‐expressing liver and pancreatic markers (e.g. PDX1⁺/SOX17⁺) are EpCAM^+/−, and are assumed to be the most primitive of the BTSC subpopulations. Their descendants undergo a maturational lineage process from the interior to the surface of ducts and vary in the mature cells generated: pancreatic cells in hepatopancreatic ducts, liver cells in large intrahepatic bile ducts, and bile duct cells along most of the biliary tree. We hypothesize that there is ongoing organogenesis throughout life, with BTSCs giving rise to hepatic stem cells in the canals of Hering and to committed progenitors within the pancreas. The BTSCs are likely to be central to normal tissue turnover and injury repair and to be key elements in the pathophysiology of liver, pancreas and biliary tree diseases, including oncogenesis.

Hepatic progenitor cells (HPCs) play a major role in liver repair and regeneration. We evaluated HPC involvement in pediatric nonalcoholic fatty liver disease (pNAFLD). Thirty biopsies of consecutive children and adolescents with untreated NAFLD (19 with nonalcoholic steatohepatitis [NASH] and 11 without NASH) were studied using immunohistochemistry and immunofluorescence. HPCs and HPC-expressing adipokines (e.g., adiponectin, resistin, and glucagon-like peptide 1 [GLP-1]) were counted and correlated with steatosis, inflammation, hepatocyte ballooning, fibrosis, and NAFLD activity score (NAS). The HPC compartment was expanded in pNAFLD, especially in children with NASH, and was independently associated with degree of fibrosis (r = 0.303; P = 0.033). NASH livers were also characterized by increased hepatocyte apoptosis, cell-cycle arrest, and an expanded pool of intermediate hepatocytes. Adiponectin expression in HPCs of pNAFLD patients was down-regulated with respect to the healthy liver, and this expression was inversely correlated with NAS score (r = −0.792; P < 0.001) and steatosis (r = −0.769; P < 0.001). Resistin expression in HPCs increased in pNAFLD and was related to degree of fibrosis (r = 0.432; P < 0.05). GLP-1 was overexpressed in HPCs of pNAFLD patients, and GLP-1 expression was related to degree of steatosis (r = 0.577; P < 0.05) and NAS (r = 0.594; P < 0.01). Conclusions: HPC activation is involved in the response of the liver to oxidative stress in pNAFLD and is correlated with fibrosis and the progression toward NASH. HPCs express adiponectin, resistin, and GLP-1, which become available to resident liver cells and are strongly associated with the severity of NAFLD. These results may have important pathophysiological implications in the modulation of hepatic insulin resistance and the progression of liver injury. (HEPATOLOGY 2012;56:2142–2153)

Rat and human biliary epithelium is morphologically and functionally heterogeneous. As no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory, and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models. For morphometry, normal and bile duct ligation (BDL) mouse livers (C57/BL6) were dissected into blocks of 2–4 μm², embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte-specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl⁻/HCO₃⁻ AE2) by immunofluorescence and western blot; and intracellular cyclic adenosine 3′,5′-monophosphate (cAMP) levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after BDL, small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by fluorescence-activated cell sorting, and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections. In situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, with smaller cholangiocytes lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl⁻/HCO₃⁻ exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate. We conclude that similar to rats, mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous. The mouse is therefore a suitable model for defining the heterogeneity of the biliary tree.