Expansion Of Liver Progenitor Cells Research Articles

Yes-associated protein promotes the proliferation and differentiation of liver progenitor cells during liver fibrosis.

Liver fibrosis is closely related to the proliferation and differentiation of liver progenitor cells (LPCs). Yes-associated protein (YAP) is a key effector molecule of the Hippo signaling pathway and plays an important role in regulating cell proliferation and liver homeostasis. However, its role in LPCs proliferation and differentiation during liver fibrosis are not well understood. Using immunohistochemistry, immunofluorescence staining, quantitative PCR and Western blotting, we discovered that LPCs expansion and enhanced YAP expression in LPCs in either choline-deficient, ethionine-supplemented (CDE) diet or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced fibrotic mice, as well as in patients with liver fibrosis. By injecting adeno-associated virus vectors under the transcriptional control of Lgr5 promoter, we found that targeted knockdown of YAP in LPCs attenuated the CDE/DDC diet-induced ductular reaction and liver fibrosis. Using EdU incorporation and Cell Counting Kit-8 assays, we demonstrated that YAP can modulate LPCs proliferation. Importantly, spleen transplantation of YAP-overexpressing LPCs improved their ability to differentiate into hepatocytes and alleviated carbon tetrachloride-induced liver fibrosis. Collectively, our findings indicate that LPCs expansion and differentiation during liver fibrosis could be modulated by YAP, further suggesting the possibility of manipulating YAP expression in LPCs as a potential treatment for chronic liver diseases.

Vertebrate lonesome kinase modulates the hepatocyte secretome to prevent perivascular liver fibrosis and inflammation.

ABSTRACTVertebrate lonesome kinase (VLK) is the only known extracellular tyrosine kinase, but its physiological functions are largely unknown. We show that VLK is highly expressed in hepatocytes of neonatal mice, but downregulated during adulthood. To determine the role of VLK in liver homeostasis and regeneration, we generated mice with a hepatocyte-specific knockout of the VLK gene (Pkdcc). Cultured progenitor cells established from primary hepatocytes of Pkdcc knockout mice produced a secretome, which promoted their own proliferation in 3D spheroids and proliferation of cultured fibroblasts. In vivo, Pkdcc knockout mice developed liver steatosis with signs of inflammation and perivascular fibrosis upon aging, combined with expansion of liver progenitor cells. In response to chronic CCl4-induced liver injury, the pattern of deposited collagen was significantly altered in these mice. The liver injury marker alpha-fetoprotein (AFP) was increased in the secretome of VLK-deficient cultured progenitor cells and in liver tissues of aged or CCl4-treated knockout mice. These results support a key role for VLK and extracellular protein phosphorylation in liver homeostasis and repair through paracrine control of liver cell function and regulation of appropriate collagen deposition. This article has an associated First Person interview with the first author of the paper.

Addressing the liver progenitor cell response and hepatic oxidative stress in experimental non-alcoholic fatty liver disease/non-alcoholic steatohepatitis using amniotic epithelial cells

BackgroundNon-alcoholic fatty liver disease is the most common liver disease globally and in its inflammatory form, non-alcoholic steatohepatitis (NASH), can progress to cirrhosis and hepatocellular carcinoma (HCC). Currently, patient education and lifestyle changes are the major tools to prevent the continued progression of NASH. Emerging therapies in NASH target known pathological processes involved in the progression of the disease including inflammation, fibrosis, oxidative stress and hepatocyte apoptosis. Human amniotic epithelial cells (hAECs) were previously shown to be beneficial in experimental models of chronic liver injury, reducing hepatic inflammation and fibrosis. Previous studies have shown that liver progenitor cells (LPCs) response plays a significant role in the development of fibrosis and HCC in mouse models of fatty liver disease. In this study, we examined the effect hAECs have on the LPC response and hepatic oxidative stress in an experimental model of NASH.MethodsExperimental NASH was induced in C57BL/6 J male mice using a high-fat, high fructose diet for 42 weeks. Mice received either a single intraperitoneal injection of 2 × 106 hAECs at week 34 or an additional hAEC dose at week 38. Changes to the LPC response and oxidative stress regulators were measured.ResultshAEC administration significantly reduced the expansion of LPCs and their mitogens, IL-6, IFNγ and TWEAK. hAEC administration also reduced neutrophil infiltration and myeloperoxidase production with a concurrent increase in heme oxygenase-1 production. These observations were accompanied by a significant increase in total levels of anti-fibrotic IFNβ in mice treated with a single dose of hAECs, which appeared to be independent of c-GAS-STING activation.ConclusionsExpansion of liver progenitor cells, hepatic inflammation and oxidative stress associated with experimental NASH were attenuated by hAEC administration. Given that repeated doses did not significantly increase efficacy, future studies assessing the impact of dose escalation and/or timing of dose may provide insights into clinical translation.

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.

Growth differentiation factor 11 attenuates liver fibrosis via expansion of liver progenitor cells

Growth differentiation factor 11 attenuates liver fibrosis via expansion of liver progenitor cells

Growth differentiation factor 11 attenuates liver fibrosis via expansion of liver progenitor cells

ObjectiveLiver fibrosis and cirrhosis resulting from chronic liver injury represent a major healthcare burden worldwide. Growth differentiation factor (GDF) 11 has been recently investigated for its role in rejuvenation of...

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)

DJ-1 deficiency attenuates expansion of liver progenitor cells through modulating the inflammatory and fibrogenic niches.

Our previous study suggested that DJ-1 has a critical role in initiating an inflammatory response, but its role in the liver progenitor cell (LPC) expansion, a process highly dependent on the inflammatory niche, remains elusive. The objective of this study is to determine the role of DJ-1 in LPC expansion. The correlation of DJ-1 expression with LPC markers was examined in the liver of patients with hepatitis B or hepatitis C virus (HBV and HCV, respectively) infection, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), nonalcoholic fatty liver disease (NAFLD), cirrhosis or hepatocellular carcinoma (HCC), respectively. The role of DJ-1 in LPC expansion and the formation of LPC-associated fibrosis and inflammation was examined in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced liver injury murine model. We also determined the ability of hepatic stellate cells (HSCs) in recruiting macrophages in DJ-1 knockout (KO) mice. The expression levels of DJ-1 were upregulated in the liver of HBV, HCV, PBC and PSC patients and DDC-fed mice. Additionally, DJ-1 expression was positively correlated with LPC proliferation in patients with liver injury and mice with DDC exposure. DJ-1 has no direct effect on LPC proliferation. Reduced activation of HSCs and collagen deposition were observed in DJ-1 KO mice. Furthermore, infiltrated CD11b+Gr-1low macrophages and pro-inflammatory factors (IL-6, TNF-α) were attenuated in DJ-1 KO mice. Mechanistically, we found that HSCs isolated from DJ-1 KO mice had decreased secretion of macrophage-mobilizing chemokines, such as CCL2 and CX3CL1, resulting in impaired macrophage infiltration. DJ-1 positively correlates with LPC expansion during liver injury. DJ-1 deficiency negatively regulates LPC proliferation by impairing the formation of LPC-associated fibrosis and inflammatory niches.

Adaptive remodeling of the biliary architecture underlies liver homeostasis.

Serving as the center for metabolism and detoxification, the liver is inherently susceptible to a wide variety of damage imposed by toxins or chemicals. Induction of cell populations with biliary epithelial phenotypes, which include progenitor-like cells and are referred to as liver progenitor cells, is often observed in histopathological examination of various liver diseases in both human patients and animal models and has been implicated in regeneration. However, the tissue dynamics underlying this phenomenon remains largely unclear. We have developed a simple imaging technique to reveal the global and fine-scale architecture of the biliary tract spreading in the mouse liver. Using this novel method, we show that the emergence and expansion of liver progenitor cells actually reflect structural transformation of the intrahepatic biliary tree in mouse liver injury models. The biliary branches expanded their area gradually and contiguously along with the course of chronic injury. Relevant regulatory signals known to be involved in liver progenitor cell regulation, including fibroblast growth factor 7 and tumor necrosis factor-like weak inducer of apoptosis, can modulate the dynamics of the biliary epithelium in different ways. Importantly, the structural transformations of the biliary tree were diverse and corresponded well with the parenchymal injury patterns. That is, when chronic hepatocyte damage was induced in the pericentral area, the biliary branches exhibited an extended structure from the periportal area with apparent tropism toward the distant injured area. The hepatobiliary system possesses a unique and unprecedented structural flexibility and can remodel dynamically and adaptively in response to various injury conditions; this type of tissue plasticity should constitute an essential component to maintain liver homeostasis.

Adaptive remodeling of the biliary tree: the essence of liver progenitor cell expansion.

The liver progenitor cell population has long been thought to exist within the liver. However, there are no standardized criteria for defining the liver progenitor cells, and there has been intense debate about the origin of these cells in the adult liver. The characteristics of such cells vary depending on the disease model used and also on the method of analysis. Visualization of three-dimensional biliary structures has revealed that the emergence of liver progenitor cells essentially reflects the adaptive remodeling of the hepatic biliary network in response to liver injury. We propose that the progenitor cell exists as a subpopulation in the biliary tree and show that the appearance of liver progenitor cells in injured parenchyma is reflective of extensive remodeling of the biliary structure.

Hepatic cell proliferation plays a pivotal role in the prognosis of alcoholic hepatitis

The role of liver progenitor cell (LPC) expansion, known as a marker of disease severity, as well as the impact of macrophage activation on liver regeneration remains unclear in humans. We aimed to characterize the LPC and macrophage compartments in alcoholic hepatitis (AH), as well as gene expression patterns to identify predictors of a good prognosis in this setting. Immunohistochemical studies for macrophages, proliferative hepatocytes, total and proliferative LPC, as well as whole liver microarray gene expression were performed on baseline liver biopsies of 58 AH patients early after admission. Abstinent cirrhotic patients were used as controls. Patients were qualified as "improvers" or "non-improvers" based on the change in MELD score three months after baseline. Compared to controls, AH patients demonstrated a significant expansion of macrophages, invasion of LPC and a higher number of proliferating hepatocytes and LPC. In AH patients, total LPC expansion (total Keratin7(+) cells) was associated with liver disease severity. The group of improvers (n=34) was characterized at baseline by a higher number of proliferating hepatocytes, proliferative LPC (double Keratin7(+)Ki67(+) cells) and liver macrophages as compared to non-improvers (n=24), despite similar clinical and biological variables. Upregulated genes in improvers were associated with cell cycle mitosis together with a major expression of SPINK1. Higher liver macrophage expansion, increased proliferative hepatocyte but also LPC number, as well as an upregulation of cell proliferation-related genes are associated with a favourable outcome. These new findings open novel therapeutic targets in AH.

The biliary epithelium gives rise to liver progenitor cells

Severe liver diseases are characterized by expansion of liver progenitor cells (LPC), which correlates with disease severity. However, the origin and role of LPC in liver physiology and in hepatic injury remains a contentious topic. We found that ductular reaction cells in human cirrhotic livers express hepatocyte nuclear factor 1 homeobox B (HNF1β). However, HNF1β expression was not present in newly generated epithelial cell adhesion molecule (EpCAM)-positive hepatocytes. In order to investigate the role of HNF1β-expressing cells we used a tamoxifen-inducible Hnf1βCreER/R26R(Yfp/LacZ) mouse to lineage-trace Hnf1β(+) biliary duct cells and to assess their contribution to LPC expansion and hepatocyte generation. Lineage tracing demonstrated no contribution of HNF1β(+) cells to hepatocytes during liver homeostasis in healthy mice or after loss of liver mass. After acute acetaminophen or carbon tetrachloride injury no contribution of HNF1β(+) cells to hepatocyte was detected. We next assessed the contribution of Hnf1β(+) -derived cells following two liver injury models with LPC expansion, a diethoxycarbonyl-1,4-dihydro-collidin (DDC)-diet and a choline-deficient ethionine-supplemented (CDE)-diet. The contribution of Hnf1β(+) cells to liver regeneration was dependent on the liver injury model. While no contribution was observed after DDC-diet treatment, mice fed with a CDE-diet showed a small population of hepatocytes derived from Hnf1β(+) cells that were expanded to 1.86% of total hepatocytes after injury recovery. Genome-wide expression profile of Hnf1β(+) -derived cells from the DDC and CDE models indicated that no contribution of LPC to hepatocytes was associated with LPC expression of genes related to telomere maintenance, inflammation, and chemokine signaling pathways. HNF1β(+) biliary duct cells are the origin of LPC. HNF1β(+) cells do not contribute to hepatocyte turnover in the healthy liver, but after certain liver injury, they can differentiate to hepatocytes contributing to liver regeneration.

Targeted Deletion of ER Chaperone GRP94 in the Liver Results in Injury, Repopulation of GRP94-Positive Hepatocytes, and Spontaneous Hepatocellular Carcinoma Development in Aged Mice

Hepatocellular carcinoma (HCC) often results from chronic liver injury and severe fibrosis or cirrhosis, but the underlying molecular pathogenesis is unclear. We previously reported that deletion of glucose regulated protein 94 (GRP94), a major endoplasmic reticulum chaperone, in the bone marrow and liver leads to progenitor/stem cell expansion. Since liver progenitor cell (LPC) proliferation can contribute to liver tumor formation, here we examined the effect of GRP94 deficiency on spontaneous liver tumorigenesis. Utilizing liver-specific Grp94 knockout mice driven by Albumin-Cre (cGrp94f/f), we discovered that while wild-type livers are tumor free up to 24 months, cGrp94f/f livers showed abnormal small nodules at 15 months and developed HCC and ductular reactions (DRs) by 21 months of age, associating with increased liver injury, apoptosis and fibrosis. cGrp94f/f livers were progressively repopulated by GRP94-positive hepatocytes. At 15 months, we observed expansion of LPCs and mild DRs, as well as increase in cell proliferation. In examining the underlying mechanisms for HCC development in cGrp94f/f livers, we detected increase in TGF-β1, activation of SMAD2/3, ERK, and JNK, and cyclin D1 upregulation at the premalignant stage. While epithelial-mesenchymal transition (EMT) was not evident, E-cadherin expression was elevated. Correlating with the recurrence of GRP94 positive-hepatocytes, the HCC was found to be GRP94-positive, whereas the expanded LPCs and DRs remained GRP94-negative. Collectively, this study uncovers that GRP94 deficiency in the liver led to injury, LPC expansion, increased proliferation, activation of oncogenic signaling, progressive repopulation of GRP94-positive hepatocytes and HCC development in aged mice.

Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy.

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.

121 HIGH LIVER EXPRESSION OF SPINK-1 IS ASSOCIATED WITH PROGENITOR CELL AND HEPATOCYTE PROLIFERATION AND DETERMINES MELD SCORE IMPROVEMENT IN DECOMPENSATED ALCOHOLIC LIVER DISEASE

Background and aims: The prognostic significance of liver progenitor cell (LPC) and macrophage expansion in the regeneration of decompensated alcoholic liver disease (ALD) remain ill defined. In a well-characterized population of patients with acutely decompensated ALD (Hepatology 2011, A62), we analysed macrophage infiltration, proliferative LPC and differential expression of hepatic genes at baseline in relation to outcome at 3 months follow up. Methods: Fifty-eight patients (MELD 20) were included. Liver biopsy at inclusion was used for (1) immunohistological analysis of proliferative LPC (MIB1+/CK7+), proliferative hepatocytes (MIB1+/CK7- parenchymal cells), morphometric analysis of macrophage infiltration (CD68) and LPC expansion (CK7), and (2) transcriptome profiling using Affymetrix GeneChip Human arrays. Serum levels of HGF were determined by immunoassay. A ≥ 3 points decrease in MELD at 3 months as compared to baseline defined the improvers. Fifteen abstinent cirrhotics served as controls. CD68 and SPINK3 mRNA expression was determined in various mice models of liver injury. Results: At baseline, patients with decompensated ALD presented a significant expansion of CD68+ macrophages and CK7+ cells compared to abstinent cirrhotics. Patients who will improve (n=34) were characterized at baseline by a higher number of CK7+/MIB1+ cells (1.9 ± 1.5 versus 0.9 ± 0.9 cells/field, p<0.01), MIB1+ hepatocytes (4.1 ± 3.6 versus 1.8 ± 1.4 cells/field, p<0.01), an increased expansion of liver macrophages (4.4% versus 3.3% of surface area, p<0.05) and a higher level of serum HGF (p<0.05), compared to those who will not (n=24). Transcriptome analysis revealed that the first pathways upregulated in improvers were related to cell cycle and a 7-fold increase of liver serine peptidase inhibitor Kazal type I (SPINK1) compared with non-improvers (p=0.005). SPINK1 liver expression positively correlated with CD68 (r=0.46) and cyclinE1 (r=0.6). In mice, a 20-fold increase in liver SPINK3 expression, the homolog of human SPINK1, was evidenced following partial hepatectomy, concurrent with hepatocyte proliferation. Conclusions: Baseline markers of liver macrophages and liver cell proliferation predict the clinical outcome in decompensated ALD. This study reveals an unexpected implication of SPINK1, an acute phase reactant, in liver regeneration and human ALD.

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.

Correspondence

We agree with Dr. Coombes and Dr. Syn that Hedgehog (Hh) signaling may be an important factor influencing liver regeneration, and in particular the development of the ductular reaction and progenitor cell expansion. We have not particularly assessed the Hh signaling in patients with alcoholic hepatitis. Studies have shown that hedgehog-responsive progenitor cells proliferate in liver injury and accumulate in humans with alcoholic liver disease correlating with disease severity.1, 2 This observation is particularly interesting because Hh signaling has been shown to participate in progenitor cell activation and proliferation in adults and to induce hepatoblast proliferation during embryogenesis, but has to be shut off to allow hepatoblast commitment and differentiation.3 These findings suggest that Hh signaling may be implicated in the accumulation of progenitor cells in the liver, promoting proliferation and preventing differentiation. Although inhibition of Hh signaling seems to reduce progenitor cell response and liver regeneration in animal models of liver injury, the role of these cells in liver regeneration is not yet completely understood, and the contribution of Hh-responsive progenitor cells to newly generated hepatocytes has not been elucidated. Further studies are warranted to elucidate this question. The association of inflammation with progenitor cell proliferation has been described but has never been investigated in alcoholic hepatitis. We agree that inflammation and progenitor cell proliferation are key events in alcoholic hepatitis, thus its relationship should be specifically investigated. In our study it was not possible to investigate the inflammatory cell populations infiltrating the damaged liver, but the overall quantification of inflammatory cells by standard histological methods did not show a positive correlation with progenitor cell expansion and mortality. The results of our study raise the question whether liver progenitor cell expansion is a marker of liver injury in acute-on-chronic conditions or the result of an inefficient liver regeneration attempt. The assessment of liver progenitor cell expansion and differentiation in human samples together with mechanistic studies in relevant animal models of liver injury will help in understanding the role of progenitor cells in liver regeneration and disease outcome and its contribution to liver repair. Pau Sancho-Bru M.D.*, José Altamirano M.D.*, Ramon Bataller M.D.*, * Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Barcelona, Spain.

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.