Activation Of Liver Progenitor Cells Research Articles

The protease-inhibitor SerpinB3 as a critical modulator of the stem-like subset in human cholangiocarcinoma.

Background and aimsCholangiocarcinoma (CCA) is a form of primary liver cancer with limited therapeutic options. Recently, cancer stem cells (CSCs) have been proposed as a driving force of tumour initiation and dissemination, thus representing a crucial therapeutic target. The protease inhibitor SerpinB3 (SB3) has been identified in several malignancies including hepatocellular carcinoma. SB3 has been involved in the early events of hepatocarcinogenesis and is highly expressed in hepatic progenitor cells and in a mouse model of liver progenitor cell activation. However, only limited information on the possible role of SB3 in CCA stem‐like compartment is available.MethodsEnrichment of CCA stem‐like subset was performed by sphere culture (SPH) in CCA cell lines (CCLP1, HUCCT1, MTCHC01 and SG231). Quantitative RT‐PCR and Western blotting were used to detect SB3 in both SPH and parental monolayer (MON) cells. Acquired CSC‐like features were analysed using an endogenous and a paracrine in vitro model, with transfection of SB3 gene or addition of recombinant SB3 to cell medium respectively. SB3 tumorigenic role was explored in an in vivo mouse model of CCA by subcutaneous injection of SB3‐transfected MON (MONSB3+) cells in immune‐deficient NOD‐SCID/IL2Rg null (NSG) mice. SB3 expression in human CCA sections was investigated by immunohistochemistry. Overall survival (OS) and time to recurrence (TTR) analyses were carried out from a transcriptome database of 104 CCA patients.ResultsSB3, barely detected in parental MON cells, was overexpressed in the same CCA cells grown as 3D SPH. Notably, MONSB3+ showed significant overexpression of genes associated with stemness (CD24, CD44, CD133), pluripotency (c‐MYC, NOTCH1, STAT3, YAP, NANOG, BMI1, KLF4, OCT4, SOX2), epithelial mesenchymal transition (β‐catenin, SLUG) and extracellular matrix remodelling (MMP1, MMP7, MMP9, ADAM9, ADAM10, ADAM17, ITGB3). SB3‐overexpressing cells showed superior spherogenic capacity and invasion ability compared to control. Importantly, MONSB3+ exhibited activation of MAP kinases (ERK1/2, p38, JNK) as well as phosphorylation of NFκB (p65) in addition to up‐regulation of the proto‐oncogene β‐catenin. All these effects were reversed after transient silencing of SB3. According to the in vitro finding, MONSB3+ cells retained high tumorigenic potential in NSG mice. SB3 overexpression was observed in human CCA tissues and analysis of OS as well as TTR indicated a worse prognosis in SB3+ CCA patients.ConclusionThese findings indicate a SB3 role in mediating malignant phenotype of CCA and identify a new therapeutic target.

Abstract 5728: Liver progenitor cells induces fibrosis via RAGE signaling upon liver injury

Abstract Liver fibrosis is characterized by hepatic stellate cell activation and extracellular matrix deposition upon persistent injury and inflammation, which can impair hepatic function and its ability to regenerate. The origin and physiological role of facultative liver progenitor cells (LPCs) have been a controversial issue as it was found to be a major player to regenerate the damaged liver, but it is also associated with fibrosis, disease progression or tumor initiation. The receptor for advanced glycation end products (RAGE) signaling axis is often associated with chronic inflammation-associated tissue damage and plays an essential role in modulating the tumor microenvironment. Our previous data suggested that RAGE mediates LPC expansion, onset of liver fibrosis and HCC formation (Pusterla et al. Hepatology. 2013.) Hence, in this study, we seek to delineate the functional role and underlying mechanism of RAGE activity in LPC activation in response to inflammation-associated liver injury. R26TomHnf1β-CreER transgenic mice were crossed with Rage flox/flox (Ragefl/fl) mice to generate tamoxifen-inducible LPC-specific RAGE knockout mice (RAGEΔLPC). They were exposed to a choline-deficient ethionine-supplemented (CDE) diet for three weeks to induce liver damage. Ablation of RAGE in LPCs strongly impairs LPC expanding capacities in CDE diet-treated mice. Strikingly, this is accompanied by reduction of activated hepatic stellate cells and bridging fibrosis. This demonstrated that RAGE signaling in LPCs is a mediator of liver fibrosis in vivo. Necroinflammation is known to be associated with liver fibrosis. To investigate the role of RAGE in LPCs in the context of necroinflammation in vitro, primary LPCs were isolated from CDE-treated Ragefl/fl C57BL/6 mouse. Wildtype and knockout Rage cell lines were established. LPCs were stimulated with supernatants from necrotic hepatocytes followed by whole transcriptome sequencing to identify downstream targets of RAGE-dependent pathways. Stress response, inflammatory and pro-fibrotic pathways were enriched in LPCs upon treatment with necrotic medium. Most interestingly, signaling pathways that regulate organ size, tissue homeostasis and cell survival were found to be RAGE-dependent. Moreover, clusters of stem cell renewal-related genes were deregulated upon ablation of RAGE. In line with the whole transcriptome profile, we demonstrated that ablation of RAGE attenuates LPCs organoid-forming ability, implying that RAGE regulates stemness properties of LPCs. Our recent results demonstrated that RAGE is required for LPCs activation and proliferation, as well as the crosstalk with stellate cells in supporting fibrogenesis. Taken together, our data uncovers a potential mechanistic insight on the role of RAGE in LPCs in association with fibrosis upon chronic liver injury. Citation Format: Wai Ling Macrina Lam, Gisela Gabernet, Tanja Poth, Aurora De Ponti, Anna Saborowski, Sven Nahnsen, Doris Schneller, Peter Angel. Liver progenitor cells induces fibrosis via RAGE signaling upon liver injury [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5728.

A pivotal role of BEX1 in liver progenitor cell expansion in mice

BackgroundThe activation and expansion of bipotent liver progenitor cells (LPCs) are indispensable for liver regeneration after severe or chronic liver injury. However, the underlying molecular mechanisms regulating LPCs and LPC-mediated liver regeneration remain elusive.MethodsHepatic brain-expressed X-linked 1 (BEX1) expression was evaluated using microarray screening, real-time polymerase chain reaction, immunoblotting and immunofluorescence. LPC activation and liver injury were studied following a choline-deficient, ethionine-supplemented (CDE) diet in wild-type (WT) and Bex1−/− mice. Proliferation, apoptosis, colony formation and hepatic differentiation were examined in LPCs from WT and Bex1−/− mice. Peroxisome proliferator-activated receptor gamma was detected in Bex1-deficient LPCs and mouse livers, and was silenced to analyse the expansion of LPCs from WT and Bex1−/− mice.ResultsHepatic BEX1 expression was increased during CDE diet-induced liver injury and was highly elevated primarily in LPCs. Bex1−/− mice fed a CDE diet displayed impaired LPC expansion and liver regeneration. Bex1 deficiency inhibited LPC proliferation and enhanced LPC apoptosis in vitro. Additionally, Bex1 deficiency inhibited the colony formation of LPCs but had no effect on their hepatic differentiation. Mechanistically, BEX1 inhibited peroxisome proliferator-activated receptor gamma to promote LPC expansion.ConclusionOur findings indicate that BEX1 plays a pivotal role in LPC activation and expansion during liver regeneration, potentially providing novel targets for liver regeneration and chronic liver disease therapies.

Interleukins-17 and 27 promote liver regeneration by sequentially inducing progenitor cell expansion and differentiation.

Liver progenitor cells (LPCs)/ductular reactions (DRs) are associated with inflammation and implicated in the pathogenesis of chronic liver diseases. However, how inflammation regulates LPCs/DRs remains largely unknown. Identification of inflammatory processes that involve LPC activation and expansion represent a key step in understanding the pathogenesis of liver diseases. In the current study, we found that diverse types of chronic liver diseases are associated with elevation of infiltrated interleukin (IL)‐17‐positive (+) cells and cytokeratin 19 (CK19)+ LPCs, and both cell types colocalized and their numbers positively correlated with each other. The role of IL‐17 in the induction of LPCs was examined in a mouse model fed a choline‐deficient and ethionine‐supplemented (CDE) diet. Feeding of wild‐type mice with the CDE diet markedly elevated CK19+Ki67+ proliferating LPCs and hepatic inflammation. Disruption of the IL‐17 gene or IL‐27 receptor, alpha subunit (WSX‐1) gene abolished CDE diet‐induced LPC expansion and inflammation. In vitro treatment with IL‐17 promoted proliferation of bipotential murine oval liver cells (a liver progenitor cell line) and markedly up‐regulated IL‐27 expression in macrophages. Treatment with IL‐27 favored the differentiation of bipotential murine oval liver cells and freshly isolated LPCs into hepatocytes. Conclusion: The current data provide evidence for a collaborative role between IL‐17 and IL‐27 in promoting LPC expansion and differentiation, respectively, thereby contributing to liver regeneration. (Hepatology Communications 2018;2:329‐343)

Aging-associated oxidative stress inhibits liver progenitor cell activation in mice.

Recent studies have discovered aging-associated changes of adult stem cells in various tissues and organs, which potentially contribute to the organismal aging. However, aging-associated changes of liver progenitor cells (LPCs) remain elusive. Employing young (2-month-old) and old (24-month-old) mice, we found diverse novel alterations in LPC activation during aging. LPCs in young mice could be activated and proliferate upon liver injury, whereas the counterparts in old mice failed to respond and proliferate, leading to the impaired liver regeneration. Surprisingly, isolated LPCs from young and old mice did not exhibit significant difference in their clonogenic and proliferative capacity. Later, we uncovered that the decreased activation and proliferation of LPCs were due to excessive reactive oxygen species produced by neutrophils infiltrated into niche, which was resulted from chemokine production from activated hepatic stellate cells during aging. This study demonstrates aging-associated changes in LPC activation and reveals critical roles for the stem cell niche, including neutrophils and hepatic stellate cells, in the negative regulation of LPCs during aging.

A disintegrin and metalloprotease 10 (ADAM10) is a central regulator of murine liver tissue homeostasis.

A Disintegrin And Metalloprotease (ADAM) 10 exerts essential roles during organ development and tissue integrity in different organs, mainly through activation of the Notch pathway. However, only little is known about its implication in liver tissue physiology.Here we show that in contrast to its role in other tissues, ADAM10 is dispensable for the Notch2-dependent biliary tree formation. However, we demonstrate that expression of bile acid transporters is dependent on ADAM10. Consequently, mice deficient for Adam10 in hepatocytes, cholangiocytes and liver progenitor cells develop spontaneous hepatocyte necrosis and concomitant liver fibrosis.We furthermore observed a strongly augmented ductular reaction in 15-week old ADAM10Δhep/Δch mice and demonstrate that c-Met dependent liver progenitor cell activation is enhanced. Additionally, liver progenitor cells are primed to hepatocyte differentiation in the absence of ADAM10.These findings show that ADAM10 is a novel central node controlling liver tissue homeostasis.Highlights:Loss of ADAM10 in murine liver results in hepatocyte necrosis and concomitant liver fibrosis.ADAM10 directly regulates expression of bile acid transporters but is dispensable for Notch2-dependent formation of the biliary system.Activation of liver progenitor cells is enhanced through increased c-Met signalling, in the absence of ADAM10.Differentiation of liver progenitor cells to hepatocytes is augmented in the absence of ADAM10.

Liver progenitor cells-mediated liver regeneration in liver cirrhosis.

Cirrhosis is defined as the histological development of regenerative nodules surrounded by fibrous bands in response to chronic liver injury. In cirrhotic liver where hepatocytes proliferation is compromised, liver progenitor cells (LPCs) are activated and then differentiated into hepatocytes and cholangiocytes, leading to the generation of regenerative nodules and functional restoration. Here, we summarize and discuss recent findings on the mechanisms underlying LPCs-mediated regeneration in liver cirrhosis. Firstly, we provide recent research on the mechanism underlying LPCs activation in severe or chronic liver injury. Secondly, we present new and exciting data on exploring the origin of LPCs, which reveal that the hepatocytes give rise to duct-like progenitors that then differentiate back into hepatocytes in chronic liver injury or liver cirrhosis. Finally, we highlight recent findings from the literature exploring the role of LPCs niche in directing the behavior and fate of LPCs. This remarkable insight into the cellular and molecular mechanisms of LPCs-mediated regeneration in liver cirrhosis will provide a basis for translating this knowledge into clinical application.

Environmental Ligands of the Aryl Hydrocarbon Receptor and Their Effects in Models of Adult Liver Progenitor Cells.

The toxicity of environmental and dietary ligands of the aryl hydrocarbon receptor (AhR) in mature liver parenchymal cells is well appreciated, while considerably less attention has been paid to their impact on cell populations exhibiting phenotypic features of liver progenitor cells. Here, we discuss the results suggesting that the consequences of the AhR activation in the cellular models derived from bipotent liver progenitors could markedly differ from those in hepatocytes. In contact-inhibited liver progenitor cells, the AhR agonists induce a range of effects potentially linked with tumor promotion. They can stimulate cell cycle progression/proliferation and deregulate cell-to-cell communication, which is associated with downregulation of proteins forming gap junctions, adherens junctions, and desmosomes (such as connexin 43, E-cadherin, β-catenin, and plakoglobin), as well as with reduced cell adhesion and inhibition of intercellular communication. At the same time, toxic AhR ligands may affect the activity of the signaling pathways contributing to regulation of liver progenitor cell activation and/or differentiation, such as downregulation of Wnt/β-catenin and TGF-β signaling, or upregulation of transcriptional targets of YAP/TAZ, the effectors of Hippo signaling pathway. These data illustrate the need to better understand the potential role of liver progenitors in the AhR-mediated liver carcinogenesis and tumor promotion.

Two sides of one coin: massive hepatic necrosis and progenitor cell-mediated regeneration in acute liver failure.

Massive hepatic necrosis is a key event underlying acute liver failure, a serious clinical syndrome with high mortality. Massive hepatic necrosis in acute liver failure has unique pathophysiological characteristics including extremely rapid parenchymal cell death and removal. On the other hand, massive necrosis rapidly induces the activation of liver progenitor cells, the so-called “second pathway of liver regeneration.” The final clinical outcome of acute liver failure depends on whether liver progenitor cell-mediated regeneration can efficiently restore parenchymal mass and function within a short time. This review summarizes the current knowledge regarding massive hepatic necrosis and liver progenitor cell-mediated regeneration in patients with acute liver failure, the two sides of one coin.

Time-dependent effect of hypoxia on tumor progression and liver progenitor cell markers in primary liver tumors.

Background & AimsExpression of liver progenitor cell (LPC) characteristics has been proposed as a negative prognostic marker in primary liver tumors. Hypoxia has been linked to activation of the Notch pathway which is responsible for activation and proliferation of LPCs and hypoxia-induced LPC activation has been shown in hepatocellular carcinoma. Our aim was to elucidate the time-dependent effects of hypoxia on the LPC niche in hepatocellular carcinoma which could aid in determining a safe time frame for use of hypoxia inducing therapies.MethodsWe used dimethyloxaloylglycine to mimic a hypoxic reaction in mice by stabilizing hypoxia-inducible factor 1 alpha at three distinct time points in diethylnitrosamine induced hepatocarcinogenesis. LPC, metastasis and Notch pathway markers were determined by quantitative PCR and (immune)histochemistry (heamatoxillin-eosin, reticulin, Sirius red and cytokeratin 19 staining).ResultsActivating the hypoxia inducible pathway early in hepatocarcinogenesis resulted in an increased incidence of both cholangioma and hepatocellular lesions, associated with high expression of LPC, metastatic and Notch pathway markers. Adversely, activating the hypoxic response during tumor development resulted in decreased incidence of hepatocellular lesions and increased cholangioma incidence, with an unaltered gene expression profile of LPC-, Notch pathway- and metastatic markers. A hypoxic insult at advanced stages of hepatocarcinogenesis severely increased the expression of LPC characteristics, however without increased expression of actors of the Notch pathway and metastatic markers and minor changes in incidence of hepatocellular and cholangioma lesions.ConclusionOur results indicate that increased hypoxia at the onset of tumor development has detrimental effects on tumor progression; patients with HCC developed in a background of fibrosis/cirrhosis might therefore represent a more difficult treatment group. In contrast, hypoxia during tumor development appears to favor tumor outcome, highlighting the importance of early detection. Finally, hypoxia in advanced stages resulted in increased expression of LPC characteristics indicating poor outcome.

Fatty acid synthase-positive hepatocytes and subsequent steatosis in rat livers by irinotecan.

Using a rat model, we investigated factors contributing to the pathogenesis of irinotecan-associated fatty liver disease. Male Sprague-Dawley rats were administered 200 mg/kg irinotecan by intraperitoneal injection on days 1–4, but not on days 5–7. This schedule was repeated 3 times. Rats were sacrificed 4, 18 and 25 days after the last injection, and liver steatosis was evaluated by hematoxylin and eosin (H&E) staining, microarray analysis and immunohistochemistry. Panacinar intrahepatocyte vacuoles were absent on days 4 and 25, but present on day 18, and this alteration was more prominent around the bile ducts than the central veins. Microarray analysis showed that the expression of genes involved in the synthesis of cholesterol and fatty acids was upregulated on day 4. Immunohistochemistry detected fatty acid synthase (Fasn)-strongly positive hepatocytes as well as the activation of liver progenitor cells on day 4, whereas intracellular vacuoles were evident in carbonic anhydrase 3 (CA3)-positive hepatocytes on day 18. Thus, irinotecan-induced liver steatosis was preceded by Fasn-strongly-positive hepatocytes and liver progenitor cell activation. The magnitude of the decrease in the number of Fasn-strongly positive hepatocytes between days 4 and 18 was similar to that of the increase in the number of CA3-positive hepatocytes accompanying vacuoles.

The roles of transforming growth factor-β, Wnt, Notch and hypoxia on liver progenitor cells in primary liver tumours

Primary liver tumours have a high incidence and mortality. The most important forms are hepatocellular carcinoma and intrahepatic cholangiocarcinoma, both can occur together in the mixed phenotype hepatocellular-cholangiocarcinoma. Liver progenitor cells (LPCs) are bipotential stem cells activated in case of severe liver damage and are capable of forming both cholangiocytes and hepatocytes. Possibly, alterations in Wnt, transforming growth factor-β, Notch and hypoxia pathways in these LPCs can cause them to give rise to cancer stem cells, capable of driving tumourigenesis. In this review, we summarize and discuss current knowledge on the role of these pathways in LPC activation and differentiation during hepatocarcinogenesis.

IFN-γ inhibits liver progenitor cell proliferation in HBV-infected patients and in 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet-fed mice

Proliferation of liver progenitor cells (LPCs) is associated with inflammation and fibrosis in chronic liver diseases. However, how inflammation and fibrosis affect LPCs remains obscure. We examined the role of interferon (IFN)-γ, an important pro-inflammatory and anti-fibrotic cytokine, in LPC expansion in HBV-infected patients and in mice challenged with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)- or choline-deficient, ethionine-supplemented (CDE) diet as well as in primary LPCs and LPC cell line. The CK19 staining scores correlated with inflammation and fibrosis grades in the livers from 110 HBV-infected patients. Nine-month IFN-γ treatment decreased LPC numbers, inflammation, and fibrosis in these HBV-infected patients. Similarly, a two-week IFN-γ treatment also decreased LPC activation in DDC-treated mice. Disruption of IFN-γ or its signaling components (e.g., IFNGR, STAT1, and IRF-1) increased LPC proliferation and liver fibrosis in DDC-fed mice. In contrast, deletion of IFN-γ did not increase, but rather slightly reduced LPC proliferation in CDE-fed mice. In vitro, IFN-γ attenuated proliferation of the LPC cell line BMOL and of primary LPCs from wild type mice, but not STAT1(-/-) or IRF-1(-/-) mice. Furthermore, co-culture assays suggest that IFN-γ can indirectly promote LPC proliferation via the activation of macrophages but attenuate it via the inhibition of hepatic stellate cells. IFN-γ inhibits LPC expansion via the direct inhibition of LPC proliferation and indirect attenuation of liver fibrosis in the DDC model, but it may also enhance LPC expansion via the promotion of inflammation in the CDE model; thereby playing dual roles in regulating LPC proliferation in vivo.

FGF7 is a functional niche signal required for stimulation of adult liver progenitor cells that support liver regeneration

The liver is a unique organ with a remarkably high potential to regenerate upon injuries. In severely damaged livers where hepatocyte proliferation is impaired, facultative liver progenitor cells (LPCs) proliferate and are assumed to contribute to regeneration. An expansion of LPCs is often observed in patients with various types of liver diseases. However, the underlying mechanism of LPC activation still remains largely unknown. Here we show that a member of the fibroblast growth factor (FGF) family, FGF7, is a critical regulator of LPCs. Its expression was induced concomitantly with LPC response in the liver of mouse models as well as in the serum of patients with acute liver failure. Fgf7-deficient mice exhibited markedly depressed LPC expansion and higher mortality upon toxin-induced hepatic injury. Transgenic expression of FGF7 in vivo led to the induction of cells with characteristics of LPCs and ameliorated hepatic dysfunction. We revealed that Thy1(+) mesenchymal cells produced FGF7 and appeared in close proximity to LPCs, implicating a role for those cells as the functional LPC niche in the regenerating liver. These findings provide new insights into the cellular and molecular basis for LPC regulation and identify FGF7 as a potential therapeutic target for liver diseases.

Dissecting the role of the immune system in liver injury and hepatocarcinogenesis”

Background: Activation of the immune system by tumor cells might result in eradication of malignant cells. In this regard, modulation of the immune system has been shown to increase carcinogenesis in several knock-out models. However, there is increasing evidence that chronic activation of the immune system in pre-malignant tissues might also promote tumor development. In order to characterize the role of the immune system in chronic liver injury and hepatocarcinogenesis mouse model of hereditary tyrosinemia type 1 (HT1) was used. HT1 is a genetic defect of tyrosine catabolism and is characterized by an extremely high susceptibility for liver cancer. The only effective therapy is 2-(2-nitro-4-trifluor-methylbenzyl)-1,3 cyklohexanedion-(NTBC)-treatment that blocks an upstream enzyme in the tyrosine catabolism. Methods: To induce flares of liver injury NTBC was repeatedly withdrawn for 21 days followed by 5-day-NTBC-treamtent to allow partial liver regeneration. Livers were harvested and liver injury was analyzed by immunohistochemistry and biochemistry. Bi-potent progenitor cells and inflammatory cells in the liver were analyzed and quantified by FACS analysis and immunohistochemistry. Gene expression arrays and RT-PCR arrays has been performed to reveal changes in gene expression profile. Results: Flares of liver injury induced a severe hepatitis and significantly accelerated liver tumor development. Liver regeneration occurred by several mechanism including proliferation of differentiated hepatocytes, increase in size of hepatocytes and activation of liver progenitor cells. Liver injury was accompanied by a massive infiltration of T-cells, Monocytes and Macrophages in the liver. To determine the role of the immune system, Fah-/- Rag-2-/- IL-2-/- mice (FCR) were treated in the same way. Interestingly, survival of alymphoid Fah-/- mice was dramatically shorter than that of Fah-/- mice. Almost 70% of the FCR mice died within the first 2 months whereas over 90% of Fah-/- mice survived the FAA-induced hepatitis suggesting that the immune system plays an important pro-survival role during chronic liver injury. Interestingly however, liver tumor development was completely suppressed in surviving FCR mice. To better understand the role of the immune system in the Fah-/- model, extensive molecular (Microarrays, WB) and immunological (RT-PCR arrays/FACS) profiling, hepatocyte and hematopoietic stem cell transplantations, adoptive transfers of specific immune cell populations were performed and additional immune-suppressed mice were generated. Conclusion: Our analysis identifies distinct functions of the immune system, which are required for liver regeneration and survival and for hepatocarcinogenesis on the other hand. Targeting specifically the tumor-promoting pathways may be an attractive chemopreventive strategy for patients at risk.

P21 regulates liver progenitor cell activation and hepatocarcinogenesis in inflammation-associated liver disease

Introduction: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Given the poor prognosis in metastatic stage of the disease, new therapies are urgently needed. Previously it has been shown that loss of the cell-cycle regulator p21 dramatically accelerates hepatocarcinogenesis suggesting that p21 acts as tumor suppressor. In order to determine the impact of p21 in inflammation-induced carcinogenesis the Mdr2 mouse model was used. In Mdr2-/- mice, loss of the Abc4 protein leads to regurgitation of bile acids into the liver causing inflammation, fibrosis and tumor development.

Hepatic transforming growth factor beta gives rise to tumor-initiating cells and promotes liver cancer development

Liver cirrhosis is a predominant risk factor for hepatocellular carcinoma (HCC). However, the mechanism underlying the progression from cirrhosis to HCC remains unclear. Herein we report the concurrent increase of liver progenitor cells (LPCs) and transforming growth factor-β (TGF-β) in diethylnitrosamine (DEN)-induced rat hepatocarcinogenesis and cirrhotic livers of HCC patients. Using several experimental approaches, including 2-acetylaminofluorene/partial hepatectomy (2-AAF/PHx) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-elicited murine liver regeneration, we found that activation of LPCs in the absence of TGF-β induction was insufficient to trigger hepatocarcinogenesis. Moreover, a small fraction of LPCs was detected to coexpress tumor initiating cell (T-IC) markers during rat hepatocarcinogenesis and in human HCCs, and TGF-β levels were positively correlated with T-IC marker expression, which indicates a role of TGF-β in T-IC generation. Rat pluripotent LPC-like WB-F344 cells were exposed to low doses of TGF-β for 18 weeks imitating the enhanced TGF-β expression in cirrhotic liver. Interestingly, long-term treatment of TGF-β on WB-F344 cells impaired their LPC potential but granted them T-IC properties including expression of T-IC markers, increased self-renewal capacity, stronger chemoresistance, and tumorigenicity in NOD-SCID mice. Hyperactivation of Akt but not Notch, signal transducer and activator of transcription 3 (STAT3), or mammalian target of rapamycin (mTOR) was detected in TGF-β-treated WB-F344 cells. Introduction of the dominant-negative mutant of Akt significantly attenuated T-IC properties of those transformed WB-F344 cells, indicating Akt was required in TGF-β-mediated-generation of hepatic T-ICs. We further demonstrate that TGF-β-induced Akt activation and LPC transformation was mediated by microRNA-216a-modulated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) suppression. Hepatoma-initiating cells may derive from hepatic progenitor cells exposed to chronic and constant TGF-β stimulation in cirrhotic liver, and pharmaceutical inhibition of microRNA-216a/PTEN/Akt signaling could be a novel strategy for HCC prevention and therapy targeting hepatic T-ICs.

Liver progenitor cell interactions with the extracellular matrix

Liver progenitor cells (LPCs) are a promising source of cells to treat liver disease by cell therapy, due to their capability for self-replication and bipotentiality. In order to establish useful culture systems of LPCs and apply them to future clinical therapies, it is necessary to understand their interactions with their microenvironment and especially with the extracellular matrix (ECM). There is considerable evidence from in vivo studies that matrix proteins affect the activation, expansion, migration and differentiation of LPCs, but the information on the role that specific ECMs play in regulating LPCs in vitro is more limited. Nevertheless, current studies suggest that laminin, collagen type III, collagen type IV and hyaluronic acid help to maintain the undifferentiated phenotype of LPCs and promote their proliferation when cultured in media supplemented with growth factors chosen for LPC expansion, whereas collagen type I and fibronectin are generally associated with a differentiated phenotype under the same conditions. Experimental evidence suggests that α6β1 and α5β1 integrins as well as CD44 on the surface of LPCs, and their related downstream signals, are important mediators of interactions between LPCs and the ECM. The interactions of LPCs with the ECM form the focus of this review and the contribution of ECM molecules to strategies for optimizing in vitro LPC cultures for therapeutic applications is discussed.

Tumor necrosis factor-like weak inducer of apoptosis is a mitogen for liver progenitor cells

Liver progenitor cells (LPCs) represent the cell compartment facilitating hepatic regeneration during chronic injury while hepatocyte-mediated repair mechanisms are compromised. LPC proliferation is frequently observed in human chronic liver diseases such as hereditary hemochromatosis, fatty liver disease, and chronic hepatitis. In vivo studies have suggested that a tumor necrosis factor family member, tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is promitotic for LPCs; whether it acts directly is not known. In our murine choline-deficient, ethionine-supplemented (CDE) model of chronic liver injury, TWEAK receptor [fibroblast growth factor-inducible 14 (Fn14)] expression in the whole liver is massively upregulated. We therefore set out to investigate whether TWEAK/Fn14 signaling promotes the regenerative response in CDE-induced chronic liver injury by mitotic stimulation of LPCs. Fn14 knockout (KO) mice showed significantly reduced LPC numbers and attenuated inflammation and cytokine production after 2 weeks of CDE feeding. The close association between LPC proliferation and activation of hepatic stellate cells in chronic liver injury prompted us to investigate whether fibrogenesis was also modulated in Fn14 KO animals. Collagen deposition and expression of key fibrogenesis mediators were reduced after 2 weeks of injury, and this correlated with LPC numbers. Furthermore, the injection of 2-week-CDE-treated wildtype animals with TWEAK led to increased proliferation of nonparenchymal pan cytokeratin-positive cells. Stimulation of an Fn14-positive LPC line with TWEAK led to nuclear factor kappa light chain enhancer of activated B cells (NFkappaB) activation and dose-dependent proliferation, which was diminished after targeting of the p50 NFkappaB subunit by RNA interference. TWEAK acts directly and stimulates LPC mitosis in an Fn14-dependent and NFkappaB-dependent fashion, and signaling via this pathway mediates the LPC response to CDE-induced injury and regeneration.

In Vitro Differentiation of Liver Progenitor Cells Derived from Healthy Dog Livers

Naturally occurring liver disease in dogs resemble human liver disease in great detail; including the activation of liver progenitor cells (LPC) in acute and chronic liver disease. The aim of the present study was to isolate, culture, and characterize progenitor cells derived from healthy mature dog livers. A nonparenchymal cell fraction enriched with small hepatocytes was isolated and cultured in Hepatozyme-serum-free media (SFM) to stimulate the growth of colony-forming small epithelial cells. After 2 weeks of culturing, clonal expansion of keratin 7 (K7) immunopositive small cells with a large nucleus/cytoplasm ratio emerged in the hepatocyte monolayer. These colonies expressed genes of several hepatocyte (CYP1A1, ALB, and KRT18), cholangiocyte/LPC (KRT7 and KRT19), and progenitor cell markers (alpha-fetoprotein, CD44, prominin1, KIT, THY1, and neural cell adhesion molecule 1), indicating their immature and bipotential nature. Gene-expression profiles indicated a more pronounced hepatic differentiation in Hepatozyme-SFM compared to William's Medium E (WME). Furthermore, colony-forming cells differentiated toward intermediate hepatocyte-like cells with a more pronounced membranous K7 immunostaining. In conclusion, colony-forming small epithelial cells in long-term canine liver cell cultures express LPC markers and have differentiating capacities. These cells may therefore be considered as progenitor cells of the liver.