Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration

Recent evidence has contradicted the prevailing view that homeostasis and regeneration of the adult liver are mediated by self duplication of lineage-restricted hepatocytes and biliary epithelial cells. These new data suggest that liver progenitor cells do not function solely as a backup system in chronic liver injury; rather, they also produce hepatocytes after acute injury and are in fact the main source of new hepatocytes during normal hepatocyte turnover. In addition, other evidence suggests that hepatocytes are capable of lineage conversion, acting as precursors of biliary epithelial cells during biliary injury. To test these concepts, we generated a hepatocyte fate-tracing model based on timed and specific Cre recombinase expression and marker gene activation in all hepatocytes of adult Rosa26 reporter mice with an adenoassociated viral vector. We found that newly formed hepatocytes derived from preexisting hepatocytes in the normal liver and that liver progenitor cells contributed minimally to acute hepatocyte regeneration. Further, we found no evidence that biliary injury induced conversion of hepatocytes into biliary epithelial cells. These results therefore restore the previously prevailing paradigms of liver homeostasis and regeneration. In addition, our new vector system will be a valuable tool for timed, efficient, and specific loop out of floxed sequences in hepatocytes.     

On the origin of the liver

While it has been well established that the fetal liver originates from foregut endoderm, the identity of the mechanisms that maintain liver mass under both basal and injury conditions remains controversial. Dramatically different models have been proposed based on the experimental design employed. In this issue of the JCI, Malato and colleagues report their elegant new model for genetic lineage tracing of mature mouse hepatocytes using an adenoassociated virus-driven Cre recombinase. They show convincingly that maintenance of liver mass during normal turnover or in response to mild injury is achieved by mature hepatocytes, rather than cholangiocytes or specialized progenitor cells, as has been suggested by others.     

Hepatocellular carcinoma originates from hepatocytes and not from the progenitor/biliary compartment

In many organs, including the intestine and skin, cancers originate from cells of the stem or progenitor compartment. Despite its nomenclature, the cellular origin of hepatocellular carcinoma (HCC) remains elusive. In contrast to most organs, the liver lacks a defined stem cell population for organ maintenance. Previous studies suggest that both hepatocytes and facultative progenitor cells within the biliary compartment are capable of generating HCC. As HCCs with a progenitor signature carry a worse prognosis, understanding the origin of HCC is of clinical relevance. Here, we used complementary fate-tracing approaches to label the progenitor/biliary compartment and hepatocytes in murine hepatocarcinogenesis. In genotoxic and genetic models, HCCs arose exclusively from hepatocytes but never from the progenitor/biliary compartment. Cytokeratin 19–, A6- and α-fetoprotein–positive cells within tumors were hepatocyte derived. In summary, hepatocytes represent the cell of origin for HCC in mice, and a progenitor signature does not reflect progenitor origin, but dedifferentiation of hepatocyte-derived tumor cells.     

TWEAK induces liver progenitor cell proliferation

Progenitor ("oval") cell expansion accompanies many forms of liver injury, including alcohol toxicity and submassive parenchymal necrosis as well as experimental injury models featuring blocked hepatocyte replication. Oval cells can potentially become either hepatocytes or biliary epithelial cells and may be critical to liver regeneration, particularly when hepatocyte replication is impaired. The regulation of oval cell proliferation is incompletely understood. Herein we present evidence that a TNF family member called TWEAK (TNF-like weak inducer of apoptosis) stimulates oval cell proliferation in mouse liver through its receptor Fn14. TWEAK has no effect on mature hepatocytes and thus appears to be selective for oval cells. Transgenic mice overexpressing TWEAK in hepatocytes exhibit periportal oval cell hyperplasia. A similar phenotype was obtained in adult wild-type mice, but not Fn14-null mice, by administering TWEAK-expressing adenovirus. Oval cell expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was significantly reduced in Fn14-null mice as well as in adult wild-type mice with a blocking anti-TWEAK mAb. Importantly, TWEAK stimulated the proliferation of an oval cell culture model. Finally, we show increased Fn14 expression in chronic hepatitis C and other human liver diseases relative to its expression in normal liver, which suggests a role for the TWEAK/Fn14 pathway in human liver injury. We conclude that TWEAK has a selective mitogenic effect for liver oval cells that distinguishes it from other previously described growth factors.     

Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary malignancy in the liver. ICC has been classified as a malignant tumor arising from cholangiocytes; however, the co-occurrence of ICC and viral hepatitis suggests that ICC originates in hepatocytes. In order to determine the cellular origin of ICC, we used a mouse model of ICC in which hepatocytes and cholangiocytes were labeled with heritable, cell type–specific reporters. Our studies reveal that ICC is generated by biliary lineage cells derived from hepatocytes, rather than cholangiocytes. Additionally, we found that Notch activation is critical for hepatocyte conversion into biliary lineage cells during the onset of ICC and its subsequent malignancy and progression. These findings will help to elucidate the pathogenic mechanism of ICC and to develop therapeutic strategies for this refractory disease.     

Cholangiocarcinomas can originate from hepatocytes in mice

Intrahepatic cholangiocarcinomas (ICCs) are primary liver tumors with a poor prognosis. The development of effective therapies has been hampered by a limited understanding of the biology of ICCs. Although ICCs exhibit heterogeneity in location, histology, and marker expression, they are currently thought to derive invariably from the cells lining the bile ducts, biliary epithelial cells (BECs), or liver progenitor cells (LPCs). Despite lack of experimental evidence establishing BECs or LPCs as the origin of ICCs, other liver cell types have not been considered. Here we show that ICCs can originate from fully differentiated hepatocytes. Using a mouse model of hepatocyte fate tracing, we found that activated NOTCH and AKT signaling cooperate to convert normal hepatocytes into biliary cells that act as precursors of rapidly progressing, lethal ICCs. Our findings suggest a previously overlooked mechanism of human ICC formation that may be targetable for anti-ICC therapy.     

Gene therapy progress and prospects: stem cell plasticity

With the identification of stem cell plasticity several years ago, multiple reports raised hopes that tissue repair by stem cell transplantation could be within reach in the near future. Krause et al reported that a single purified hematopoietic stem cell not only repopulated the bone marrow of a host animal, but also integrated into unrelated tissues. Lagasse et al demonstrated that in a genetic model of liver disease, purified hematopoietic stem cells can give rise to hepatocytes and rescue fatal liver damage. More recent work by Jiang et al demonstrated that cultured cells can retain their stem cell potential. There are a number of possible mechanisms that could explain these phenomena, and recent experiments have raised controversy about which mechanism is prevalent. One possibility is transdifferentiation of a committed cell directly into another cell type as a response to environmental cues. Transdifferentiation has been shown mainly in vitro, but some in vivo data also support this mechanism. Direct transdifferentiation would clinically be limited by the number of cells that can be introduced into an organ without removal of resident cells. If bone marrow cells could on the other hand give rise to stem cells of another tissue, then they could in theory repopulate whole organs from a few starting cells. This model of dedifferentiation is consistent with recent data from animal models. Genetic analysis of cells of donor origin in vivo and in vitro has brought to light another possible mechanism. The fusion of host and donor cells can give rise to mature tissue cells without trans- or dedifferentiation. The resulting heterokaryons are able to cure a lethal genetic defect and do not seem to be prone to give rise to cancer. All these models will clinically face the problem of accessibility of healthy primary cells for transplantation. This underlines the importance of the recent identification of a population of mesenchymal stem cells (MSCs) with stem cell properties similar to embryonic stem (ES) cells. These cells can be cultured and expanded in vitro without losing their stem cell potential making them an attractive target for cell therapy. Finally, it is still not clear if stem cells for various tissues are present in peripheral blood, or bone marrow and thus can be directly purified from these sources. Identification of putative tissue stem cells would be necessary before purification strategies can be devised. In this review, we discuss the evidence for these models, and the conflicting results obtained to date.     

异种肝前体细胞移植治疗急性肝损伤大鼠

背景：成体肝前体细胞可在受体肝脏内定植并分化为肝细胞。不过,异种肝前体细胞移植能否促进急性肝损伤的恢复,脾脏微环境能否促进移植物的存活和向肝细胞分化,尚没有研究。目的：评价异种肝前体细胞移植治疗急性肝损伤的作用;监测移植肝前体细胞在大鼠脾脏实质内的定植及向肝细胞的分化。方法：体外培养雄性小鼠来源的肝前体细胞系肝上皮样前体细胞。通过CCl4腹腔注射联合2/3肝切除构建急性肝损伤大鼠模型,进行肝上皮样前体细胞脾脏移植。在肝切除后1,5,14和21 d,苏木精-伊红染色观察肝脏病理改变,全自动生化分析仪监测血清转氨酶变化,PCR反应检测脾脏组织Y染色体特异性序列Sry,脾脏CK-19和Alb免疫组织化学追踪移植肝上皮样前体细胞的植入和肝细胞分化。结果与结论：肝上皮样前体细胞可在体外长期培养,保持增殖能力和双向分化潜能。肝上皮样前体细胞脾脏移植后,肝损伤大鼠肝细胞肿胀明显减轻,丙氨酸转氨酶和天门冬氨酸转氨酶下降更明显。移植后1,5,14和21 d,脾脏DNA中均能检测到Sry序列。在整个实验期间CK-19阳性细胞在大鼠脾脏实质内始终存在。Alb阳性细胞在移植后5 d在脾脏实质中出现,随后阳性细胞数逐渐增多。实验表明,移植肝前体细胞能在大鼠脾脏实质中植入,并分化为肝细胞,能有效促进CCl4腹腔注射联合2/3肝切除诱导的大鼠急性肝损伤的修复过程。     

Hedgehog signaling regulates epithelial-mesenchymal transition during biliary fibrosis in rodents and humans

Epithelial-mesenchymal transitions (EMTs) play an important role in tissue construction during embryogenesis, and evidence suggests that this process may also help to remodel some adult tissues after injury. Activation of the hedgehog (Hh) signaling pathway regulates EMT during development. This pathway is also induced by chronic biliary injury, a condition in which EMT has been suggested to have a role. We evaluated the hypothesis that Hh signaling promotes EMT in adult bile ductular cells (cholangiocytes). In liver sections from patients with chronic biliary injury and in primary cholangiocytes isolated from rats that had undergone bile duct ligation (BDL), an experimental model of biliary fibrosis, EMT was localized to cholangiocytes with Hh pathway activity. Relief of ductal obstruction in BDL rats reduced Hh pathway activity, EMT, and biliary fibrosis. In mouse cholangiocytes, coculture with myofibroblastic hepatic stellate cells, a source of soluble Hh ligands, promoted EMT and cell migration. Addition of Hh-neutralizing antibodies to cocultures blocked these effects. Finally, we found that EMT responses to BDL were enhanced in patched-deficient mice, which display excessive activation of the Hh pathway. Together, these data suggest that activation of Hh signaling promotes EMT and contributes to the evolution of biliary fibrosis during chronic cholestasis.     

Combined hepatocellular-cholangiocarcinoma: what the radiologist needs to know about biphenotypic liver carcinoma

Combined hepatocellular-cholangiocarcinoma (CHC), also referred to as primary liver carcinoma (PLC) with biphenotypic differentiation, is an increasingly recognized subtype of malignant PLC encompassing varying morphologic forms thought to arise either from progenitor cell lineage or dedifferentiation of mature liver cells. Tumor cells express both biliary and hepatocellular markers by immunohistochemistry, and may also express progenitor cell and stem cell markers. Due to the relative rarity of this tumor type, little is known about the risk factors, imaging appearance, or prognosis. Few studies have demonstrated risk factors that overlap with hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), though not all appear to arise in the background of cirrhosis. The imaging appearances of these tumors may overlap with those of HCC and CC and discriminating features such as classic enhancement patterns and biliary ductal dilation are not universally present. Serum tumor markers, such as alpha-fetoprotein and carbohydrate antigen 19-9, may be helpful when they are discordant with imaging or if both are elevated to a significant degree. In regards to management and prognosis, most studies demonstrate worse outcomes compared with HCC or CC. In the United States, the diagnosis of HCC is frequently made with imaging alone, and subsequent management decisions, including organ allocation for transplantation, rely upon the radiological diagnosis. Given the importance of radiological diagnosis, awareness of this tumor type is essential for appropriate management.     

Hepatic lipid peroxidation in vivo in rats with chronic iron overload

Peroxidative decomposition of cellular membrane lipids is a postulated mechanism of hepatocellular injury in parenchymal iron overload. In the present study, we looked for direct evidence of lipid peroxidation in vivo (as measured by lipid-conjugated diene formation in hepatic organelle membranes) from rats with experimental chronic iron overload. Both parenteral ferric nitrilotriacetate (FeNTA) administration and dietary supplementation with carbonyl iron were used to produce chronic iron overload. Biochemical and histologic evaluation of liver tissue confirmed moderate increases in hepatic storage iron. FeNTA administration produced excessive iron deposition throughout the hepatic lobule in both hepatocytes and Kupffer cells, whereas dietary carbonyl iron supplementation produced greater hepatic iron overload in a periportal distribution with iron deposition predominantly in hepatocytes. Evidence for mitochondrial lipid peroxidation in vivo was demonstrated at all three mean hepatic iron concentrations studied (1,197, 3,231, and 4,216 micrograms Fe/g) in both models of experimental chronic iron overload. In contrast, increased conjugated diene formation was detected in microsomal lipids only at the higher liver iron concentration (4,161 micrograms Fe/g) achieved by dietary carbonyl iron supplementation. When iron as either FeNTA or ferritin was added in vitro to normal liver homogenates before lipid extraction, no conjugated diene formation was observed. We conclude that the presence of conjugated dienes in the subcellular fractions of rat liver provide direct evidence of iron-induced hepatic mitochondrial and microsomal lipid peroxidation in vivo in two models of experimental chronic iron overload.     

Molecular mimicry in primary biliary cirrhosis. Evidence for biliary epithelial expression of a molecule cross-reactive with pyruvate dehydrogenase complex-E2.

Sera from patients with primary biliary cirrhosis (PBC) react with enzymes of the 2-oxo dehydrogenase pathways, particularly PDC-E2. These enzymes are present in all nucleated cells, yet autoimmune damage is confined to biliary epithelial cells. Using a panel of eight mouse monoclonal antibodies and a human combinatorial antibody specific for PDC-E2, we examined by indirect immunofluorescence and confocal microscopy sections of liver from patients with PBC, progressive sclerosing cholangitis, and hepatocarcinoma. The monoclonal antibodies gave typical mitochondrial immunofluorescence on biliary epithelium and on hepatocytes from patients with either PBC, progressive sclerosing cholangitis, or hepatocarcinoma. However, one of eight mouse monoclonal antibodies (C355.1) and the human combinatorial antibody reacted with great intensity and specificity with the luminal region of biliary epithelial cells from patients with PBC. Simultaneous examination of these sections with an antiisotype reagent for human IgA revealed high IgA staining in the luminal region of biliary epithelial cells in patients with PBC. IgG and IgA antibodies to PDC-E2 were detected in the bile of patients with PBC but not normal controls. We believe that this data may be interpreted as indicating that a molecule cross-reactive with PDC-E2 is expressed at high levels in the luminal region of biliary epithelial cells in PBC.     

Localization of actin and cytokeratin in hepatocytes and biliary epithelial cells in normal human livers using a low temperature resin and post-embedding immunohistochemistry

Using a low temperature resin Lowicryl K4M and post-embedding immunogold labeling, localization of actin and cytokeratin was examined in hepatocytes and biliary epithelial cells in normal human livers. Preservation of the liver tissues was satisfactory at the electron microscopic level. In immunolabeling of actin, gold particles were mainly found on microvilli and around the junctional complexes and at cell borders in the hepatocytes as well as biliary epithelial cells. In immunolabeling of cytokeratin, the gold particles were rather preferentially found in the perinuclear cytoplasm and around the junctional complexes in the biliary epithelial cells and hepatocytes. These particles were mainly found to be located on the intermediate filaments at higher magnification, although a considerable number of intermediate filaments were not labeled in the hepatocytes and biliary epithelial cells. Double straining method using the gold particles of different sizes clearly confirmed the abovementioned distribution of actin and cytokeratin in the same cells. From these observations, it was suggested that post-embedding immunoelectron microscopy using Lowicryl K4M is a useful tool for analysis of cytoskeletal organization in the liver tissue.     

New horizons for studying human hepatotropic infections

The liver serves as a target organ for several important pathogens, including hepatitis B and C viruses (HBV and HCV, respectively) and the human malaria parasites, all of which represent serious global health problems. Because these pathogens are restricted to human hepatocytes, research in small animals has been compromised by the frailty of the current mouse xenotransplantation models. In this issue of the JCI, Bissig et al. demonstrate robust HBV and HCV infection in a novel xenotransplantation model in which large numbers of immunodeficient mice with liver injury were engrafted with significant quantities of human hepatocytes. This technical advance paves the way for more widespread use of human liver chimeric mice and forms the basis for creating increasingly complex humanized mouse models that could prove useful for studying immunopathogenesis and vaccine development against hepatotropic pathogens. More than 800 million people are chronically infected with hepatitis B and C viruses (HBV and HCV, respectively) and malaria, causing over 1.5 million deaths annually (1, 2). Persistent HBV and HCV infections can lead to cirrhosis and/or hepatocellular carcinoma (HCC). Basic research, as well as the development of drugs and vaccines targeting human hepatotropic pathogens, has been handicapped by the lack of robust in vitro and in vivo platforms that mimic human liver biology and disease susceptibility. The narrow species tropism of HBV and HCV restricts preclinical studies to chimpanzees or human liver–chimeric mice. Large primate studies are often limited by financial and ethical concerns, and humanized mice are currently cumbersome and low-throughput. In culture, HCC-derived cell lines and immortalized hepatocytes have been useful for studying various aspects of HBV and HCV life cycles, but these cells differ phenotypically and functionally from human hepatocytes in vivo. Primary hepatocytes, which can be coaxed to maintain liver-specific functions by culture with fibroblasts, hold promise as a more physiologically relevant in vitro substrate for HCV infection (3). Addressing fundamental questions about hepatotropic pathogen biology in vivo, however, requires a suitable small animal model to complement and guide more challenging and expensive studies, including clinical trials.     

Tumor suppressor gene PTEN and non-alcoholic steatohepatitis (NASH)

Although hepatic steatosis had been considered to be a benign condition that does not deteriorate to either liver cirrhosis or hepatocellular carcinoma (HCC). Non-alcoholic steatohepatitis (NASH) is notable disease that has similar pathological features to alcoholic liver injury and progresses to liver cirrhosis and HCC. But the molecular mechanism for the onset of NASH, and for the transformation from steatosis to carcinogenesis are still unclear. Hepatocyte-specific PTEN deficient mice, we generated, have similar histological features to the patients of human NASH. These hepatocytes showed enhanced lipid accumulation, inflammatory change, and hyperoxidation. Moreover, they developed into HCC. Thus, impairment of PI3K/PTEN signaling may possibly be involved in a part of NASH/HCC cases in human.     

Epidermal growth factor improves lentivirus vector gene transfer into primary mouse hepatocytes

Partial resistance of primary mouse hepatocytes to lentiviral (LV) vector transduction poses a challenge for ex vivo gene therapy protocols in models of monogenetic liver disease. We thus sought to optimize ex vivo LV gene transfer while preserving the hepatocyte integrity for subsequent transplantation into recipient animals. We found that culture media supplemented with epidermal growth factor (EGF) and, to a lesser extent, hepatocyte growth factor (HGF) markedly improved transduction efficacy at various multiplicities of infection. Up to 87% of primary hepatocytes were transduced in the presence of 10 ng EGF, compared with ~30% in standard culture medium (SCMs). The increased number of transgene-expressing cells correlated with increased nuclear import and more integrated pro-viral copies per cell. Higher LV transduction efficacy was not associated with proliferation, as transduction capacity of gammaretroviral vectors remained low (<1%). Finally, we developed an LV transduction protocol for short-term (maximum 24 h) adherent hepatocyte cultures. LV-transduced hepatocytes showed liver repopulation capacities similar to freshly isolated hepatocytes in alb-uPA mouse recipients. Our findings highlight the importance of EGF for efficient LV transduction of primary hepatocytes in culture and should facilitate studies of LV gene transfer in mouse models of monogenetic liver disease.     

射频热凝家兔肝组织的热效应观察

目的研究射频热凝家兔肝组织的热效应范围，并观察肝细胞、肝内动脉、静脉及胆管组织的损伤情况。方法将40只家兔随机分为2组（实验组与对照组，每组20只），均分别剖腹暴露肝脏，于实验组家兔肝膈面插入射频针并给予射频热凝处理，对照组家兔手术操作与实验组相同，但不给予射频热凝处理。2组家兔分别于术后1，5，10，15d各处死5只，取肝脏不同部位进行常规切片染色，观察肝细胞、动脉、静脉及胆管组织的损伤情况。采用TUNEL法检测肝细胞、胆管上皮细胞的凋亡率，并测定家免术前、术后血清GPT、T-BIL及D-BIL水平。结果通过组织学观察，发现该射频热凝治疗仪（单电极）对家兔肝组织的最大热效应损伤半径为2．5cm，在坏死区域内可见肝细胞、血管及胆管等均有程度不一的损伤。距凝固坏死区外边缘2、0cm范围内的肝细胞、胆管上皮细胞的凋亡指数均高于对照组相应部位细胞，差异有统计学意义（P〈0.01）；另外还发现实验组家兔肝细胞的凋亡指数也高于同区域胆管上皮细胞凋亡指数（P〈0．01）。结论对射频热效应耐受性由高至低排列依次为：胆管〉动脉〉静脉〉肝细胞，正常家兔肝细胞对射频热效应的敏感性大于胆管上皮细胞，细胞凋亡可能与射频热效应相关，即射频热凝能引发肝细胞、胆管上皮细胞凋亡。     

Transplantation of endothelial cells corrects the phenotype in hemophilia A mice

BACKGROUND: The deficiency of factor VIII, a co-factor in the intrinsic coagulation pathway results in hemophilia A. Although FVIII is synthesized largely in the liver, the specific liver cell type(s) responsible for FVIII production is controversial. OBJECTIVE: This study aimed to determine the cellular origin of FVIII synthesis and release in mouse models. METHODS: We transplanted cells into the peritoneal cavity of hemophilia A knockout mice. Plasma FVIII activity was measured using a Chromogenix assay 2-7 days after cell transplantation, and phenotypic correction was determined with tail-clip challenge 7 days following cell transplantation. Transplanted cells were identified by histologic and molecular assays. RESULTS: Untreated hemophilia A mice, as well as mice treated with the hepatocyte-enriched fraction, showed extensive mortality following tail-clip challenge. In contrast, recipients of unfractionated liver cells (mixture of hepatocytes, liver sinusoidal endothelial cells (LSEC), Kupffer cells, and hepatic stellate cells) or of the cell fraction enriched in LSECs survived tail-clip challenge (P < 0.001). FVIII was secreted in the blood stream in recipients of unfractionated liver cells, LSECs and pancreatic islet-derived MILE SVEN 1 (MS1) endothelial cells. Although transplanted hepatocytes maintained functional integrity in the peritoneal cavity, these cells did not produce detectable plasma FVIII activity. CONCLUSIONS: The assay of cell transplantation in the peritoneal cavity showed that endothelial cells but not hepatocytes produced phenotypic correction in hemophilia A mice. Therefore, endothelial cells should be suitable additional targets for cell and gene therapy in hemophilia A.