The work of liver stem cell biologists, largely carried out in rodent models, has now started to manifest in human investigations and applications. We can now recognize complex regenerative processes in tissue specimens that had only been suspected for decades, but we also struggle to describe what we see in human tissues in a way that takes into account the findings from the animal investigations, using a language derived from species not, in fact, so much like our own. This international group of liver pathologists and hepatologists, most of whom are actively engaged in both clinical work and scientific research, seeks to arrive at a consensus on nomenclature for normal human livers and human reactive lesions that can facilitate more rapid advancement of our field.

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The stem cell factor (SCF)/c-kit ligand/receptor system has been implicated in stem (oval) cell activation following liver injury in the rat. The aim of this study was to determine the role of the SCF/c-kit system in pediatric human liver during acute and chronic liver injury. Tissue was obtained from hepatectomy specimens of patients undergoing liver transplantation for extrahepatic biliary atresia (EHBA) and fulminant hepatic failure (FHF). Specific expression of mRNA for c-kit and ␤-actin was measured by ribonuclease protection and by immunohistochemistry to localize c-kit in tissue sections. Expression of c-kit was detected at relatively consistent levels in normal and cirrhotic (

Fas-mediated mechanisms of apoptosis are thought to be involved in the bile duct loss that characterizes diseases such as primary biliary cirrhosis (PBC). We have previously shown that activation of CD40 on hepatocytes can amplify Fas-mediated apoptosis; in the present study, we investigated interactions between CD40 and Fas in biliary epithelial cells (BEC). We report that the bile ducts in PBC liver tissue frequently express increased levels of Fas, Fas ligand (FasL), and CD40 associated with apoptotic BEC. The portal mononuclear infiltrate contains CD40L+ve T cells and macrophages, thereby demonstrating a potential mechanism for CD40 engagement in vivo. Primary cultures of human BEC also expressed Fas, FasL, and CD40 but not CD40L protein or mRNA. Activation of CD40 on BEC using recombinant CD40L increased transcriptional expression of FasL and induced apoptosis, which was inhibited by neutralizing antibodies to either Fas or FasL. Thus, CD40-induced apoptosis of BEC is mediated through Fas/FasL. We then investigated the intracellular signals and transcription factors activated in BEC and found that NF-kappaB and AP-1 were both activated after CD40 ligation. Increased functional NF-kappaB was seen early after CD40 ligation, but returned to baseline levels after 4 h. In contrast, the rapid up-regulation of AP-1 was sustained over 24 h. This study provides further functional evidence of the ability of CD40 to induce Fas/FasL-dependent apoptosis of liver epithelial cells supporting the importance of cross-talk between tumor necrosis factor (TNF) receptor family members as an amplification step in apoptosis induction. Sustained activation of AP-1 in the absence of NF-kappaB signaling may be a critical factor in determining the outcome of CD40 engagement.

It has recently been shown that reactive bile ductules display neuroendocrine features, including immunoreactivity for the neural cell adhesion molecule (NCAM). In this study we have compared the immunohistochemical expression of NCAM with that of HEA-125 (biliary specific) and LKM-1 (hepatocyte specific) and other markers relevant to morphogenesis (Bcl-2, EMA) and cell proliferation (Ki-67) in cryostat sections from different chronic liver diseases and from fetal livers at different gestational ages. In parallel, viable NCAM-positive ductular cells were purified from collagenase digests of cirrhotic livers by immunomagnetic separation and characterized by immunocytochemistry and transmission electron microscopy. We demonstrated that reactive ductules with atypical morphology coexpressed NCAM and Bcl-2 and were found mainly in congenital diseases associated with ductal plate malformation and in primary cholangiopathies. On the contrary, reactive ductules with typical morphology were negative for NCAM/Bcl-2 and positive for EMA. Reactive ductules coexpressing NCAM/Bcl-2 were negative for the proliferation marker Ki-67 and appeared to be directly connected with periportal hepatocytes. In fetal livers NCAM/Bcl-2 was transiently expressed during the early developmental stages of ductal plate (10-16 weeks) and started to disappear as the ductal plate began duplicating. NCAM-positive ductal plate cells were Ki-67 negative, becoming positive in duplicated segments. Thus the histogenesis of ductular reactive cells seems to recapitulate the early stages of biliary ontogenesis. In primary cholangiopathies and ductal plate malformations, these cells do not appear to maturate further, and thus abundant ductular structures coexist with vanishing mature ducts. These NCAM-positive ductular cells were immunopurified from patients with chronic cholestatic liver diseases and showed ultrastructural features consistent with a less differentiated phenotype than mature cholangiocytes. These isolated cells represent a useful model for in vitro studies.

The existence of progenitor (stem) cells in the human liver remains a matter of debate. In rodent models of hepatocarcinogenesis and injury, oval cells proliferate in the periportal regions of the portal tracts and are suggested to derive from a stem cell compartment, because they are capable of differentiating into hepatocytes or biliary epithelial cells. In this study, the rat oval cell marker, OV-6 has been used to investigate the hypothesis that there are stem cells present in fetal and pediatric human liver. The pattern of OV-6 expression was compared with the established adult biliary cell markers human epithelial antigen-125 (HEA-125) and cytokeratin-19 (CK-19). In normal pediatric liver (n ‫؍‬ 7), bile ducts and ductules were immunostained with CK-19 and HEA-125, whereas OV-6 staining was consistently negative. In fetal tissue (n ‫؍‬ 10), ductal plate cells, primitive bile ducts, and hepatoblasts were stained with CK-19 and HEA-125 although only some of the ductal plate cells and hepatoblasts were OV-6 positive. In biliary atresia (n ‫؍‬ 6) and ␣1, anti-trypsin deficiency (␣1,AT) (n ‫؍‬ 4), CK-19 and HEA-125 immunostained ductular proliferative cells that tended to form finely anastomosing ductules, whereas OV-6 staining was found more on discrete cells confined to portal tract margins. Additionally, in diseased liver, OV-6 was strongly positive in hepatocyte lobules with greatest intensity in the periseptal regions. This widespread hepatocyte OV-6 positivity suggests that the antibody may identify cells of a less differentiated phenotype (transitional hepatocytes) that have replaced the mature cells. Therefore, it is proposed that in human liver, OV-6 is recognizing cells with a progenitor stem cell-like phenotype with the capacity to differentiate into OV-6 positive ductular cells or lobular hepatocytes. (HEPATOLOGY 1998;28:980-985.)Human liver organogenesis begins during the third or fourth week of gestation from endodermal cells lining the foregut. These endodermal cells give rise to hepatic cords representing liver progenitor cells (hepatoblasts), which are able to differentiate along biliary or hepatocyte lineages. 1

Notch signaling is an evolutionarily conserved mechanism, used to regulate cell fate decisions. Four Notch receptors have been identified in man (Notch‐1 to ‐4). In this study, semiquantitative reverse transcription polymerase chain reaction (RT‐PCR) and immunohistochemistry were used to examine the expression pattern of Notch receptor genes in whole adult human liver and isolated liver cell preparations. All 4 receptors were expressed in the adult liver, with no significant differences in the levels of Notch‐1, ‐2, and ‐4 messenger RNA (mRNA) between normal and diseased liver. However, Notch‐3 expression appeared to be increased in diseased tissue. The distribution of Notch‐1 and ‐4 in normal tissue was similar, with Notch‐1 also detectable at low levels in the sinusoidal endothelium. Notch‐2 expression was more widely distributed, and detectable in hepatocytes, medium‐sized bile ducts, and the sinusoidal endothelium. Notch‐3 expression was seen on hepatocytes, with weaker expression detectable in portal veins, hepatic arteries, and the sinusoids. In normal liver tissue Notch‐1, ‐2, and ‐3 were found to be coexpressed on bile duct epithelium; however, with the exception of Notch‐3 in primary sclerosing cholangitis (PSC) livers, expression was absent on proliferating ductules in all disease states examined. Interestingly, the expression of Notch‐2 and ‐3 was associated with numerous small vessels within the portal tract septa of diseased tissue. The absence of Notch receptor expression on proliferating bile ductules and its presence on neovessels suggests that Notch signaling may be important for normal bile duct formation and the aberrant neovascularization seen in diseased liver tissue.

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