Stem Cell Biology
Article first published online: 15 OCT 2013
Copyright © 2013 American Association for the Study of Liver Diseases
Special Issue: The 64th Annual Meeting of the American Association for the Study of Liver Diseases: The Liver Meeting 2013
Volume 58, Issue S1, pages 1001A–1010A, October 2013
How to Cite
(2013), Stem Cell Biology. Hepatology, 58: 1001A–1010A. doi: 10.1002/hep.26875
- Issue published online: 1 OCT 2013
- Article first published online: 15 OCT 2013
Decellularized extracellular matrix promotes proliferation and differentiation of bone marrow mesenchymal stem cells into hepatocyte-like cells
Hongliang He, Liang Peng, Yujie Su, Qiyi Zhao, Ke Wang, Zhiliong Goo; Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
Background and Aim: Interactions between stem cells and extracellular matrix (ECM) are reguisite for inducing lineagespecific differentiation and maintaining biological functions of mesenchymal stem cells by providing a composite set of chemical and structural signals; thus our study aims to characterize the microstructure and biological nature of decellularized ECM deposited by bone marrow mesenchymal stem cells (BMSCs) and to investigate the effect on the BMSCs proliferation and differentiation into hepatocyfe-like cells. Methods: The morphology and matrix composition of decellularized ECM deposited by BMSCs were revealed by scanning electron microscopy and immunofluorescence staining. BMSCs were seeded in two different conditions: conventional tissue culture polystyrene (TCPS) and decellularized ECM under the same differentiation medium. Proliferative ability of BMSCs was determined by DNA assay. Production of reactive oxygen species (ROS) was measured by flow cytometry. BMSCs were induced to differentiation into hepatogenic lineage and glycogen storage was detected by Periodic acid-Schiff staining. Hepatocyte-specific gene expression was guantified by real-time PCR. Results: Decellularized ECM was detectable for multiple matrix proteins, including type I collagen, type Ill collagen, fibronectin and laminin that are identical to the matrix compositions of native liver. Compared with the cells on TCPS, BMSCs cultured on decellularized ECm showed a spindle-like shape, a robust proliferative capacity and suppressed level of intracellular ROS, accompanied with up-regulation of superoxide dismutase genes. In contrast to hepatocyte-like cells differentiated from BMSCs on TCPS, those on decellularized ECM were determined with a more intensive staining of glycogen storage, the urea concentration of differentiated BMSCs of ECM group was 8.7% higher than that of TCPS group at 21(10.5 ± 0.2 μg/mL/24h vs.9.7 ± 0.1 μg/mL/24h, p < 0.05) and 7.3% higher on Day 28 (10.9 ± 0.2 μg/mL/24h vs.10.2 ± 0.2μg/mL/24h, p< 0.05) and higher expressions of hepatocytespecific genes including albumin, tryptophan 2, 3-dioxygenase, cytochrome P450 7A1, cytochrome P45。3A4, cytokeratin 18 and hepatocyte nuclear factor 4 alpha on day 7,14,21 and 28 (p < 0.05). Conclusions: Decellularized ECM deposited by BMSCs can be an effective method to facilitate in vitro expansion and hepatic maturation of BMSCs and promote developments in stem cell-based liver regenerative medicine.
The following people have nothing to disclose: Hongliang He, Liang Peng, Yujie Su, Qiyi Zhao, Ke Wang, Zhiliang Gao
Epiregulin Promotes the Emergence and Proliferation of Adult Liver Progenitor Cells
Kyoko Tomita1, Hiroaki Haga1,2, Kei Mizuno1, Tomohiro Katsumi1, Chikako Sato1, Kazuo Okumoto1, Yuko Nishise1, Hisayoshi Watanabe1,Takafumi Saito1,Yoshiyuki Ueno1
1Yamagata Univer sity, Yamagata, Japan; 2Mayo Clinic, Jacksonville, L
[Aim] The appearance and proliferation of liver progenitor cells (LPCs) are considered to be important steps necessary for liver regeneration. Previously, we have demonstrated the differentiation of LPCs into hepatocytes during co-culture with bone marrow cells. Moreover, FGF2 was found to be a critical factor for migration of LPCs. Using DNA microarray analysis, we identified epiregulin, a growth factor belonging to the EGF family, as a candidate growth factor acting on LPCs. However, the relationship between epiregulin and LPCs is still unclear. The aim of the present study was to clarify the role of epiregulin during liver regeneration. [Methods] A liver injury model was developed using C57BL/6 mice fed a diet containing 0.1% 3.5diethoxycarbonyI-1.4-dihydrocoIIidine (DDC) to induce LPCs. We evaluated the expression of epiregulin in this DDC mouse model using immunohistochemistry (IHC) and RT-gPCR. Serum epiregulin levels were also examined in both DDC mice and patients with acute liver failure. The proliferation of an EpCAMpositive LPC cell line cultured with recombinant epiregulin was examined in vitro. Finally, overexpression of epiregulin was induced in mice by the hydrodynamic tail vein injection (HTVi) method to evaluate its effects in vivo. The expression of epiregulin, PCNA and CK19 in the mouse liver was investigated by IHC. [Results] In patients with acute liver failure, serum epiregulin levels were significantly increased in comparison with the healthy controls. Expression of the epiregulin gene in DDC mouse liver was significantly increased in comparison with control mice at 1 to 4 weeks after introduction of the DDC diet. CK19-positive LPCs were detected around the portal area as early as 1 week. Along with the emergence of LPCs, epiregulin was also detected around the portal area. Serum epiregulin levels in DDC mice were significantly increased relative to the controls at 4 weeks. Recombinant epiregulin dose-dependently augmented the proliferative capacity of the LPC cell line. Epiregulin expression in the liver after HTVi gene delivery was confirmed by IHC from 3 to 21 days after gene injection, reaching a peak at 3 days. Expression of PCNA on hepatocytes was increased significantly at 3,7, and 14 days. Finally, CK19-positive LPCs emerged around the portal area from 3 to 21 days. [Conclusions] Epiregulin expression promotes the proliferation of LPCs and DNA synthesis in hepatocytes, and is upregulated in sera from both patients and mice with liver injury. Furthermore, epiregulin induction leads to the appearance of LPCs. Taken together, the data indicate that epiregulin would be a useful biomarker of liver regeneration.
Yoshiyuki Ueno - Advisory Committees or Review Panels: Jansen
The following people have nothing to disclose: Kyoko Tomita, Hiroaki Haga, Kei Mizuno, Tomohiro Katsumi, Chikako Sato, Kazuo Okumoto, Yuko Nishise, Hisayoshi Watanabe, Takafumi Saito
Activation of the nucleosome remodelling and histone deacetylase complex in EpCAM+ hepatic cancer stem cells
Kouki Nio, Taro Yamashita, Mitsumasa Kondo, Mariko Yoshida, Hikari Okada, Tomoyuki Hayashi, Takehiro Hayashi, Yoshimoto Nomura, Yasumasa Hara, Naoki Oishi, Hajime Sunagozaka, Hajime Takatori, Masao Honda, Shuichi Kaneko
Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, Kanazawa, Japan
Background: Recent evidence suggests that the malignant nature of hepatocellular carcinoma (HCC) is closely related to the existence of cancer cells with stem/progenitor cell features (CSCs). The nucleosome remodeling and histone deacetylase (NuRD) complex is known to regulate oncogenesis and cancer progression, but its role in CSCs is obscure. Here we explore the expression of genes encoding the NuRD complex in epithelial cell adhesion molecule (EpCAM)+ HCC cells and investigate the potential mechanisms of CSC maintenance by the NuRD complex. Methods: Gene expression profiling analysis and immunohistochemistry (IHC) were used to characterize 383 surgically resected HCC tissue samples. Fluorescence-activated cell sorting was used to isolate EpCAM+ cells from aHuH7HCC cell line. The effects of RNA interference, plasmid transfection, histone deacetylase (HDAC) inhibitors suberohydroxamic acid (SBHA) and suberoylanilidehydroxamic acid (SAHA), and a poiy(ADP-ribose) polymerase (PARP) inhibitorAG-014699 on EpCAM+ CSCs were evaluated. Results: HCC microarray data indicated that genes encoding the NuRD complex (MTA1, MTA2, HDAC1, HDAC2, GATAD2A, and CHD4) were coordinately activated in EpCAM+ HCCs. Among them, CHD4 was confirmed to be strongly expressed in EpCAM+ HCC cells sorted from the HuH7 cell line and in surgically resected specimens. IHC analysis indicated poor prognosis forCHD4high HCCs with advanced pathological stage, large tumor size, and EpCAM expression. CHD4 overexpression in HuH1 cells resulted in chemoresistance to epirubicin, whereas knockdown of CHD4 decreased the population of EpCAM+ CSCs accompanied by increased sensitivity to epirubicin, suggesting the role of CHD4 in regulating the maintenance of EpCAM+ CSCs via the NuRD complex. To block NuRD complex activity, we treated HuH7 cells with HDAC inhibitors (SBHA and SAHA) or a PARP inhibitor (AG-014699) to identify the reduction of the EpCAM+ CSCs population, suggesting that NuRD participates in the maintenance of stemness and chemoresistance of EpCAM+ CSCs. Combination therapy of SBHA and AG-014699 successfully inhibited HCC growth in vivo in a mouse xenograft model. Conclusion: The NuRD complex mediates the maintenance of stemness and chemoresistance of EpCAM+ CSCs through interaction with HDAC and PARP. Combination therapy of HDAC and PARP inhibitors may offer novel therapeutic possibilities to patients with advanced HCC.
Mariko Yoshida - Grant/Research Support: Bayer
Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, Bayer Japan
The following people have nothing to disclose: Kouki Nio, Taro Yamashita, Mitsumasa Kondo, Hikari Okada, Tomoyuki Hayashi, Takehiro Hayashi, Yoshimoto Nomura, Yasumasa Hara, Naoki Oishi, Hajime Sunagozaka, Hajime Takatori, Masao Honda
Fas/Fas ligand apoptosis pathway underlies immunomodulatory properties of Human Biliary Tree Stem/Progenitor Cells
Massimo Riccio1, Vincenzo Cardinala2, Gianluca Carnevale1, Lara Gibellini1, Sara De Biasi1, Alessandra Pisciotta1, Guido Corpino3,4, Raffaele Gentile1,Lola M. Reid5, Andreo Cossarizza1, Eugenio Gaudio3, Domenico Alvaro2, Anto De Pol1
1Department of Surgery, Medicine, Dentistry and Morphological Sciencesy, University of Modena and Reggio Emilio, Modena, Italy; 2Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, Italy; 3Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza University of Rome, Rome, Italy; 4Foro Italico University of Rome' Rome, Italy; 5UNC School of Medicine' Chapel Hill' NC
Human biliary tree stem/progenitor cells (hBTSCs), obtained from human adult biliary tree tissue are multipotent endodermalderived epithelial stem cells, able to differentiate in vitro and in vivo towards hepatocytes, cholangiocytes or pancreatic islets. They reside in peribiliary glands (PBGs) of large intrahepatic and extrahepatic bile ducts and furnish maturational lineages within the bile duct walls sustaining tissue renewal. The ability of stem cells to control T-cells immune-response was recently described for mesenchymal stem cells and dental pulp stem cells. So far this property has never been investigated for adult endodermal-derived cell populations. AIM. To evaluate whether hBTSCs are able to modulate immune response via activating the Fas/Fas ligand (FasL) apoptosis signaling pathway in human lymphocytes. The in vitro interaction between hBTSC primary cultures and human lymphocytes isolated from different donors has been evaluated. Fluorescence-activated cell sorting analysis of T-cells co-cultured with hBTSCs through different time points indicated that hBTSCs induce apoptosis in activated CD4+ and CD8+ T-cell populations. Moreover, Fas receptor appeared to be more expressed in T-cells co-cultured with hBTSCs with respect to resting T-cells. Western blot data demonstrated that hBTSCs constitutively express high levels of FasL that markedly increased after co-culture with T-cells. Confocal microscopy carried out on the first cell seeding (after isolation) demonstrates that FasL was expressed only by epithelial cells and not by fibroblast-like cell population. Furthermore, triple immunofluorescence labeling with anti-Leucine-rich repeat-containing G protein-coupled receptor 5/anti-Epithelial cell adhesion molecule/anti-FasL demonstrated that the epithelial cell population expressing FasL corresponds to endodermal stem cell population identifiable with hBTSCs. In situ study by immunohistochemistry and immunofluorescence on the biliary tree of normal liver donors confirmed that the hBTSCs residing in the PBG niche constitutively express FasL. Our data suggest that hBTSCs may modulate the T-cells response through the production of FasL that in turn activate the lymphocyte Fas/FasL pathway which induces “premature” apoptosis of CD4+ and CD8+ T-cells. In conclusion, these results disclose an immunomodulatory property of hBTSCs which could have important implications in the regenerative medicine of liver and pancreas and in the pathogenesis of immune-mediated bile duct diseases, such as primary sclerosing cholangitis.
Lola M. Reid - Consulting: PhoenixSongs Biologicals; Grant/Research Support: Vesta Therapeutics, NIH, The Hamner Institute
The following people have nothing to disclose: Massimo Riccio, Vincenzo Cardinale, Gianluca Carnevale, Lara Gibellini, Sara De Biasi, Alessandra Pisciotta, Guido Carpino, Raffaele Gentile, Andrea Cossarizza, Eugenio Gaudio, Domenico Alvaro, Anto De Pol
Engraftment and in vivo HCV infection of human hepatocytes differentiated from embryonic and induced pluripotent stem cells
Arnaud Carpentier1, Abeba Tesfaye2, Virginia Chu1, Marian E. Major2, Stephen Feinsfone2, T Joke Liang1
1NIDDK, Liver Diseases Branch, NIH, Bethesda, MD; 2Division of Viral Products, Center for Biologics Evaluation and Research, FDA, Bethesda, MD
Using an established protocol with modifications, we were able to differentiate both human embryonic and patient-derived induced pluripotent stem cells (hESCs and hiPSCs) into hepatocyte-like cells, which functionally resembled primary human hepatocytes. We also showed that these differentiated human hepatocytes (DHHs) could be infected in vitro with JFH1-HCVcc and HCV(+) sera of different genotypes. The guestion remains whether it is possible to successfully engraft these cells and establish functional human hepatocytes in vivo. It is known that in vitro hepatic differentiation leads to monolayer of DHHs, which poorly reproduce the 3D architecture of native liver, and may be a reason for the incomplete differentiation of DHHs to mature hepatocytes. In this context, we engrafted, via intrasplenic injection, 2-4 millions DHHs into the liver parenchyma of immune-deficient transgenic mice carrying the urokinase-type plasminogen activator gene driven by the major urinary protein promoter (MUP-uPA/SCID/Bg). Human albumin (hALB) could be detected in the serum of the engrafted mice by ELISA as early as day 10 post-engraftment, with concentrations ranging from 0.4 to 2.3 mg/mL. More importantly, hALB persisted for more than 4 months, consistent with long-term engraftment of human cells in the mouse liver parenchyma. Mice were sacrificed 4 months post-engraftment, and liver sections were assessed by immunostaining for a variety of human proteins (albumin, alpha-1-antitrypsine, alpha-fetoprotein). Areas of human cells were observed around central veins, and could constitute up to 15% of the mouse liver parenchyma.2 weeks post-engraftment, mice with high hALB concentration were inoculated with HCV positive sera of different genotypes (1a, 1b, 3). Serum samples were obtained at day 30, 60 and 90 post inoculation, and assessed for HCV RNA by RTgPCR. HCV RNA could be detected in the serum of every mouse at day 60 postinoculation. HCV increased up to 90 days post-infection, consistent with long-term infection of engrafted human hepatocytes in the mouse liver. Conclusion. We demonstrate here that hESCs- and hiPSCs-derived DHHs can be efficiently engrafted into the mouse liver parenchyma, and that they can be infected by HCV(+) sera of different genotypes. This approach constitutes a valuable model to study HCV infection in the context of patient's genetic background as well as in the native architecture of the liver.
Stephen Feinstone - Independent Contractor: Dynavax
The following people have nothing to disclose: Arnaud Carpentier, Abeba Tesfaye, Virginia Chu, Marian E. Major, T. Jake Liang
Discrepancies between widely used mouse models of liver progenitor cells: a comparative study between CDE- and DDC-induced chronic liver injuries
Noemi Van Hui1, Regina Español-Suñer1, Christine Sempoux1, Laurent Dallé3, Leo A. von Grunsven3, Frederic Lemaigre2, Isabelle A. Leclercg1
1Laboratory of Hepato-Gastroenterology, Institut de Recherche Expérimentale et Clinigue, Université catholigue de Louvain, Brussels, Belgium; 2Liver and Pancreas Development Unit, de Duve Institute, Université catholigue de Louvain, Brussels, Belgium; 3Liver Cell Biology Lab, Vrije Universiteit Brussel, Brussels, Belgium
Background and Aims Liver progenitor cells (LPC) are guiescent in healthy liver and are activated in chronic liver injury. We aimed to characterize and compare the LPC response observed in two widely used models of LPC activation, in terms of microenvironment, phenotype, proliferation and differentiation. Methods Mice received an ethionine-supplemented choline-deficient diet (CDE) or 3, 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet to induce liver injury. LPC phenotype was investigated by immunohistochemistry using markers such as K19, E-cadherin and Osteopontin (〇PN) while Sirius Red, Laminin, a-SMA and F4/80 were used for defining the micro-environment. To follow the fate of LPC, we performed lineage tracing experiments using mice that express tamoxifen-inducible Cre recombinase under control of osteopontin (OPN) regulatory region (OPNiCreERT2; Rosa26RYFP mice) exposed to CDE or DDC diet followed by 2 weeks of recovery. Results The CDE diet targets specifically hepatocytes and activates a compartment of small cells that give rise to elongated transit-amplifying cells expanding from portal tracts towards central veins. Myofibroblast activation and extracellular matrix (ECM) deposition precedes this cell expansion, and a laminin-rich basement membrane sustains those LPC. In the DDC model, accumulation of (proto)porphyrin obstructs the hepatobiliary system resulting in highly proliferative cells forming bile duct-like structures delineated by a thin layer of Laminin. Those duct-like structures localize within the portal mesenchyme. In both CDE and DDC models, LPC similarly co-express K19, S〇X9 and 〇PN. DDC pseudo-biliary cells exhibited a strong membrane E cadherin staining compared to the subtle expression in CDE invading cells, supporting strong intercellular cohesion. DDC-induced proliferation of the ductular cells was accompanied by a dense inflammatory infiltrate in the portal mesenchyme. Portal inflammation is not seen in CDE livers, rather Kupffer cells were intermingled with invading LPC within the parenchyma. Cell tracking experiments revealed that LPC are able to generate functional hepatocytes (forming biliary canaliculi, expressing hepato-specific enzymes and accumulating glycogen) during the recovery period after CDE and not after DDC exposure. Conclusions While the LPC induced during CDE diet have a more undifferentiated and migration-supporting phenotype, they are able to differentiate into hepatocytes. In contrast, the accumulating cells observed in the DDC portal areas resemble dysmorphic cholangiocytes participating to the restoration of the bile duct(ule)s.
The following people have nothing to disclose: Noemi Van Hul, Regina EspanolSuner, Christine Sempoux, Laurent Dolle, Leo A. van Grunsven, Frederic Lemaigre, Isabelle A. Leclercg
Human Fibrolamellar Hepatocellular Carcinomas: Evidence for their Derivation from Biliary Tree Stem Cell Subpopulations
Tsunekazu Oikawa1, Eliane Wauthier1,Andrea Reyna-Neyra2, Nancy Carrasco2, Ron Levine3, Yunfang Wang4, Vincenzo Cordinale5, Guido Carpino6, Domenico Alvaro5, Eugenio Gaudio6, Lola M. Reid1
1Department of Cell Biology and Physiology, UNC Lineberger Comprehensive Cancer Center of the University of North Carolina School of Medicine, Chapel Hill, NC; 2Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT; 3Yale University School of Medicine, New Haven, CT; 4The Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing, China; 5Division of Gastroenterology, Department of Scienze e Biotecnologie Medico-Chirugiche, Fondazione Eleonora Lorillord Spencer Cenci, Polo Ponfino, Italy; 6Deporfmenf of Human Anatomy, Sapienzo University of Rome, Rome, Italy
Human fibrolamellar hepatocellular carcinomas (hFL-HCCs) are cancers not recognized prior to ∼1960. For reasons unknown, hFL-HCCs have increased in freguency world-wide and now constitute ∼5% of all liver cancers. Egually disturbing, hFL-HCCs present in children, teenagers and young adults without evidence of hepatitis viruses, fibrosis or any other condition known relevant to other forms of liver cancers. No treatments, other than surgery, have been found effective, and surgery is not useful for metastatic tumors. An hFL-HCC transplantable tumor line has been established in immuno-compromised murine hosts with cells culture-selected in Kubota's Medium (KM), designed for endodermal stem cells. Transplantation in KM supplemented with hyaluronans, hepatocyte growth factor (HGF), and vascular endothelial cell growth factor (VEGF) resulted in nodular tumors comprised of >65% host mesenchymal cells if injected subcutaneously and >90% if injected intraperitoneally. Xenografted tumors were established in primary cultures in KM on plastic or hyaluronans. The phenotypic traits of hFL-HCCs, both in vivo and in vitro, match closely those of a normal biliary tree stem cell (hBTSC) subpopulation negative for epithelial cell adhesion molecule, EpCAM, and being precursors to both liver and pancreas. They strongly express endodermal stem cell markers (PDX1, SOX9, SOX17, LGR5), pluripotency genes (NANOG, SOX2, OCT4, SALL4, BMI1, TROP-2), multidrug resistance genes (MDR1, ABCG2), matrix and membrane receptors (CD44, laminin, E-cadherin, syndecan-1, VCAM, VEGF-R2), hepatic markers (CK7,18,19, HepPar-1), sonic hedgehog, sodium iodide symporter (NIS), and CD68. The cells are positive for NGN3 and weakly so for MUC6, markers of intermediate stages to pancreatic islets. They are consistently negative for albumin, alpha-fetoprotein, CD13, CD31, CD34, CD45, and CD146. The hFL-HCCs are distinct from the normal hBTSCs in forming “catena”，floating chains of cells bound tightly via lateral adhesion mechanisms (e. g. cadherins), in having tunneling nanotubes and in overproduction of matrix-degrading enzymes. We hypothesize that hFL-HCCs are malignant transformants of hBTSC subpopulations. The hFL-HCC's phenotypic traits are predictive of resistance to chemotherapies but vulnerability to multiple candidate therapies including radioactive I131, inhibitors of heparanse and other matrix-degrading enzymes, antagonists to EGF, HGF or VEGF, and/or treatment with differentiation factors prior to attempts at chemotherapy. This is the first and only model of hFL-HCCs ever established, offering opportunities for studies on tumor biology and/or strategies for treatments.
Lola M. Reid - Consulting: PhoenixSongs Biologicals; Grant/Research Support: Vesta Therapeutics, NIH, The Hamner Institute
The following people have nothing to disclose: Tsunekazu Oikawa, Eliane Wauthier, Andrea Teyna-Neyma, Nancy Carrasco, Ron Levine, Yunfang Wang, Vincenzo Cardinale, Guido Carpino, Domenico Alvaro, Eugenio Gaudio
Insulin is reguired for the differentiation and proliferation of hepatocyte progenitor cells in vitro: A novel role for insulin receptor substrate 2
Luke A. Noon1,2, Alicia Martinez-Romero1, Jose E. O'Connor4, Anne Corlu3, Pascale Bouillé5, Christiane Guillouzco3, Deborah J. Burks1,2
1Centro de Investigación Principe Felipe, Valencia, Spain; 2CIBER de Diabetes y Enfermedades Metabólicas (CIBERDEM), Valencia, Spain; 3lnserm, UMR 991, CHU Pontchaillou, F-35033' Rennes, France; 4Department of Biochemistry, University of Valencia,, Valencia, Spain; 5Vectalys, Canal Biotech, Toulouse, France
Background: Insulin/IGF1 play an important role in the control of liver growth and metabolism. Insulin is also a key component of protocols used to differentiate pluripotent stem cells to hepatocyte-like cells in vitro, however the precise role of this pathway in the de novo differentiation of hepatocytes remains to be elucidated. HepaRG cells differentiate from bipotent “hepatoblast-like” cells to cholangiocytes and hepatocytes in vitro and thus are a novel tool for the study of human hepatogenesis. Methods: We assessed how supplemented insulin influenced HepaRG differentiation, proliferation and hepatocyte maturation using a novel Apolipoprotein A2-GFP hepatocyte reporter system. Lentiviral shRNA was used to knockdown key components of the insulin signaling pathway and the effect on hepatocyte gene expression was analyzed by immunostaining and Western blot. Results: Omitting insulin (0.88uM) reversibly blocked hepatocyte differentiation, as did stable knockdown of insulin receptor-β (IRβ) and both insulin receptor substrate (IRS)1 and IRS2. In the early stages of differentiation insulin drove differentiation in a proliferation independent manner, via phosphatidylinositol 3-kinase signaling. However insulin was also reguired for the later proliferation of differentiating hepatocytes expressing Apolipoprotein A2. We show that IRS2 expression in precursor cells enhanced insulin sensitivity, proliferation and survival, thereby promoting hepatogenesis. Interestingly, IRS2 expression was downregulated as hepatocytes matured and expressed Cyp3A4. This correlated with reduced proliferation. Enforced IRS2 expression in the presence of insulin led to dramatically increased numbers of immature hepatocytes but impaired maturation as well as aberrant lipid accumulation/steatosis suggesting dampening of insulin signaling was reguired for normal hepatocyte maturation. Conclusion: Insulin, promotes HepaRG-hepatocyte differentiation and proliferation, however maintained high levels of signaling through this pathway impair maturation and lead to aberrant lipid accumulation. These results may facilitate refinement of current strategies designed to produce mature cells from various progenitor sources in vitro.
The following people have nothing to disclose: Luke A. Noon, Alicia MartinezRomero, Jose E. O'Connor, Anne Comlu, Pascale Bouillé, Christiane Guillouzco, Deborah J. Burks
Mesenchymal Stem Cells Transplantation in Patients with End Stage Liver Disease improve Hepatic fibrosis
Abdelrahman Zekri1, Hosny M. Salama2, Abeer Bahnassy3, Shereen M. Al Alim2, Ola Ahmed1, Mai Lotfy1, Eman Medhat2, Rasha Ahmed2,Sherief Muso2
1Virology ond immunology Unit, Cancer Biology, National Cancer Institute, Cairo, Egypt; 2Hepatology and Tropical Medicine, El-Kasr Al-Aini School of Medicine, Cairo, Egypt; 3PathoIogy Department' National Cancer Institute, Cairo, Egypt
Aim: To assess the utility of autologous mesenchymal stem cells (MSCs) peripheral vein infusion as a possible therapeutic modality and to confirm the supportive role of the stem cell (SC) treatment for patients with end-stage liver diseases Methods: Forty patients with post-HCV end-stage liver diseases were randomized into 2 groups. Group 1, comprising 20 patients and they have received granulocyte colony stimulating factor (GCSF) for 5 days followed by autologous MSC peripheral veins infusion. Group 2, comprising 20 patients and they have received regular liver supportive treatment and have served as a control group. Results: In the infused group (Gr. I), There was near normalization of liver enzymes and improvement in liver synthetic function (S. Albumin, Prothrombin time and concentration) in 54%. There was significant changes in albumin (p=0.000), bilirubin (p=0.002), INR (p=0.017), prothrombin conc. (p=0.029), AST (p=0.156) and ALT levels (p=0.029). Also, in Gr. I, there was stabilization of the clinical and biochemical status in 13% of cases. None of the patients in the control group (Gr. II) showed any significant improvement. Hepatic fibrosis was assessed in the treated group by detection of procollagen Ill C peptide level (PIIICP) (9.4 ± 4.2) and procollagen Ill N peptide level (PIIINP) (440 ± 189) (Pre treatment Value) in the patients' serum. It was reported a decrease in the level of PIIICP (8.1 ± 2.6) and PIIINP (388 ± 102) after stem cell therapy (three months treatment Value) but they have not reached the significant value (6 months treatment Value) (p=0.7), however there was a significant correlation coefficient after three months between the serum level of the PIIINP and prothrombin concentration (p=-0.5) and between the serum level of the PIIICP and ascites (p=0.550) Conclusion: Our data showed that autologous mesenchymal stem cells infusion into the peripheral veins was effective and showed the same result as intrahepatic infusion from our previous studies (Salama et al, 2011) and confirmed the supportive role of mesenchymal stem cell treatment for end-stage liver disease with satisfactory tolerability and beneficial effects on liver synthetic functions and hepatic fibrosis. We have also observed in this study that the serum albumin has been improved within the first two weeks and prothrombin concentration improvement was delayed for almost one month. According to the data obtained from this study in combination with our previous studies; we recommend patients infusion with pure CD133 and CD34 at first then followed by infusion of in vitro differentiated MSC within one week followed by another infusion session after 3 months.
The following people have nothing to disclose: Abdelrahman Zekri, Hosny M. Salama, Abeer Bahnassy, Shereen M. Al Alim, Ola Ahmed, Mai Lotfy, Eman Medhat, Rasha Ahmed, Sherief Musa
Mesenchymal stem cells induce the generation of a novel tolerogenic dendritic cells through miR-378 and IL-6/STAT3/SOCS1/TLR4 signaling network
Guo-Ying Wang, Yi-nan Deng, Yong Zou, Minru Li, Qi Zhang, GuiHua Chen
Liver Transplantation Center, the Third Affiliated HospitaI, Sun Yat-sen University, Organ Tansplantation Research Institute, Guangzhou, China
Mesenchymal stem cells (MSC) display a striking immunoregulatory property. This property has been used in several clinical settings; particularly, MSC infusion could resolve severe, acute graft-vs-host disease. Most of the data suggest that this property involves secretion of specific cytokines and mechanisms mediated by cell-cell contact. In addition, MSC are also likely to modulate the differentiation and function of dendritic cells (DC). However, the underlying mechanisms are still poorly understood. In this study, we found that human MSC from umbilical cord (huc-MSC) induced immature dendritic cells (iDC) to differentiate into a novel tolerogenic DC subset (MSC-DC) with a stable phenotype and function when cocultured. MSC-DC display the low immunogenicity and immune tolerance by triggering a T helper type 2-polarizing program and down-regulating the pro-inflammatory factor production. Further study demonstrates that huc-MSC induce the tolerogenic MSC-DC generation through the IL-6/STAT3/SOCS1/TLR4 signaling network. Huc-MSC induced the higher expression of SOCS1 in MSC-DC, which were activated by secreting a larger number of IL-6 through the JAK-STAT pathway, repressing toll like receptor 4 (TLR4) signaling pathway, and ultimately inducing the generation of novel tolerogenic dendritic cells. Moreover, Huc-MSC could increase phophorylation of Akt, but inhibit phophorylation of IRF3. We also observed that amount of microRNAs changed when cocultured. We found that miR-378 was an important factor in the generation of novel tolerogenic dendritic cells by targeting STAM2. These results indicate that microRNAs could play essential roles in the production of the tolerogenic MSC-DC. Taken together, our data proposed a new molecular mechanism of MSC in regulating tolerogenic DC production and promote the clinical application of MSC in new and broader immune applications, including treatment of allograft rejection and graft-vs-host disease in organ transplantation and autoimmune liver diseases.
The following people have nothing to disclose: Guo-Ying Wang, Yi-nan Deng, Yong Zou, Minru Li, Qi Zhang, Gui-Hua Chen
Precise Replication of Human Fetal Liver Development on Decellularized Liver Matrix
Pedro M. Baptista, Dipen Vyas, Emma Moran, Anthony Atala, Shay Soker
Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC
Bioengineering of a fully functional tissue reguires precise recapitulate normal tissue development. Specifically for the liver, one may use bipotent human liver progenitor cells (hFLCs) capable of differentiation into hepatocytes and cholangiocytes. The goal of the current study was to develop a system that would efficiently recapitulate embryonic development of hepatic parenchymal tissue and bile ducts, using decellularized liver extracellular matrix (ECM) as scaffolds. Bioengineered liver organoids were generated by seeding freshly isolated hFLC through the vena cava and portal vein of the intact liver scaffold. These seeded scaffolds remained in a perfusion bioreactor up to two weeks. In parallel, hFLCs were seeded on decellularized liver ECM discs (300 μm thick, 8 mm diameter) and were cultured for 3 weeks in hepatic differentiation medium. Confocal microscopy was used to determine the extent of progenitor cell differentiation into hepatocytes and cholangiocyies in disk organoids and whole liver scaffold. Urea, albumin and drug metabolism were guantified as paramaters of liver function. hFLCs seeded on liver ECM discs differentiated into hepatocytes and cholangiocytes. The cells showed predominant albumin expression along with loss of AFP expression at 3 weeks. The cells also expressed other mature hepatocyte markers like HNF-4α, α-1AT and CYP450 1A2, 2A and 3A. The cells in the ductular structures expressed bile duct specific markers like CK19, SOX9, EpCAM, ASBT, β-catenin and the presence of primary cilia, thus demonstrating differentiation towards cholangiocyte lineage along with maintaining apicobasal polarity. Similarly, hFLC seeded in whole liver scaffolds showed progressive tissue formation and organization with clusters of hepatocytes expressing albumin, AFP, CYP450 3A and 2A, E-cad, Hep-1 and EpCAM, and several long ductular structures staining positive for biliary markers CK19, EpCAM, ASBT spawning for 200-400μm in length. Urea and albumin secretion was higher in the whole bioengineered liver and liver disc organoids compared to control hFLCs cultured in petri dishes. Several metabolites of diazepam and 7-ethoxycoumarin were also detected by LC-MS/MS, showing broad CYP450 activity in both culture systems. Our results demonstrate the efficient generation of bioengineered human liver tissue with hFLC that recapitulates stepwise development of hepatocyte and bile duct formation. Altogether, this study demonstrates the potential of this technology to study and mimic human liver development. These models provide novel approaches for liver bioengineering, drug discovery and toxicology, and ultimately for the treatment of liver disease.
The following people have nothing to disclose: Pedro M. Baptista, Dipen Vyas, Emma Moran, Anthony Atala, Shay Soker
FTY720 increases the hepatic retention of purified haematopoietic stem cells in chronic liver injury resulting in an enhanced anti-fibrotic action
Andrew King1,Diarmaid D. Houlihan1,Dean P. Kavanagh2, Abhilok Garg1, Shankar Suresh1,Henry Sumption1, Jon Frampton3, David H. Adams1,Philip N. Newsome1
1Centre for Liver Research and NIHR Liver Biomedical Research Unit, University of Birmingham, Birmingham, United Kingdom; 2Centre for Cardiovascular Sciences, University of Birmingham, Birmingham, United Kingdom; 3School of immunity and Infection, University of Birmingham, Birmingham, United Kingdom
There is considerable interest in the use of bone marrow(BM) cells in liver cirrhosis, however the role of purified haematopoietic stem cells(HSC) and use of repeated infusions have not been studied. We also set out to determine whether increased retention of HSC within the injured liver by modulating their response to Sphingosine 1-phosphate(S1P) would augment their anti-fibrotic effect. Liver injury was induced in BoyJ(CD45.1) mice by twice weekly ip injection of carbon tetrachloride(1 mg/kg) for ten weeks, whilst purified FACS-sorted HSC(c-kit+ sca1 + lineage-) isolated from BM of C57BL6(CD45.2) mice were infused via tail vein at weeks 7,8,9. Tracking of DiR labelled HSC was performed using the IVIS Spectrum imaging system and cell numbers guantified by flow cytometry. The partial S1P receptor agonist FTY720(1mg/kg) was administered by ip injection. Repeated infusions of HSC resulted in a significant reduction in liver scarring as assessed by: picrosirius red(PSR) staining(48.8% reduction vs control, p<0.001), hepatic hydroxyproline content(436 vs 313mg/g 丨iver, p<0.01), αSMA immunostaining(7.0 vs 2.4% staining, p<0.001), as well as increased serum albumin(3.1 vs 4.0g/dl, p<0.001). In separate BM transplantation studies liver injury was seen to result in a 4.4 fold increase in the number of BM-derived HSC in the liver(vs controls, p<0.001). Increased hepatic S1P levels in liver injury resulted in a reduced S1P gradient between liver and lymph, and was a result of increased hepatic sphingosine kinase 1 expression. FTY720 reduced HSC migration to S1P and resulted in a 1.7 fold increase in BM-derived HSC accumulating in the liver(vs no FTY720, p<0.01)and a 1.9 fold increase in the number of infused HSC in the liver 4 days after infusion(vs no FTY720, p<0.01). Intravital microscopy demonstrated this was not due to increased hepatic recruitment of HSC. Repeated administration of FTY720 during infusions of HSC resulted in a further reduction in hepatic fibrosis compared with HSC infusions alone(PSR 21.7% reduction, p<0.05; αSMA 25% reduction, p<0.05). The antifibrotic effect of HSC was also seen with infusions of lymphoid progenitors lacking myeloid potential. Infused cells(CD45.2+) were not detected in livers 7 days after infusion, although there were increased numbers of recipient(CD45.1+) neutrophils and macrophages(2.2 and 1v fold increase vs control, p<0. 01) in the liver following HSC infusion. Our data demonstrate the potent anti-fibrotic action of repeated infusions of purified HSC, which is mediated by recruitment of endogenous cells. Moreover, we demonstrate that increasing hepatic retention of HSC with FTY720 augments their antifibrotic action.
Philip N. Newsome - Grant/Research Support: Novo Nordisk
The following people have nothing to disclose: Andrew King, Diarmaid D. Houlihan, Dean P. Kavanagh, Abhilok Garg, Shankar Suresh, Henry Sumption, Jon Frampton, David H. Adams
The Role of Pleiotrophin Signaling in the Hepatic Stem Cell Niche
Anikia Tucker1, Gregory A. Michelotti2, Steve S. Choi2,3, Guanhua Xie2, Gamze Karaca2, Marzena Swiderska-Syn2, Leandi Kruger2, Mariana V Machado2, Katherine S. Garman2,3, Anna Mae Diehl2
1School of Medicine, Duke University, Durham, NC; 2Division of Gastroenterology, Duke University Medical Center, Durham, NC; 3Section of Gastroenterology, Durham Veterans Affairs Medical Center, Durham, NC
Background: To better treat liver disease we must decipher mechanisms controlling progenitor fate. Pleiotrophin (PTN) regulates hematopoietic stem cells. PTN knockout mice have poor liver regeneration after partial hepatectomy and hepatic PTN expression increases in cirrhosis and liver cancer suggesting PTN may regulate liver progenitors. Aim: To localize PTN in guiescent/injured liver and determine whether hedgehog (Hh) signaling modulates PTN expression. Methods: We used immunohistochemistry to localize GFP expression in liver from PTN-GFP reporter mice. Expression of PTN and its receptor, protein tyrosine phosphatase receptor type Z (PTPRZ1) was assessed by PCR in isolated hepatocytes, hepatic stellate cells (HSC), liver sinusoidal endothelial cells (LSEC) and Kupffer cells from eight healthy adult wild-type mice. PTN and PTPRZ1 were also assessed in the clonally derived mouse cholangiocyte cell line 603B. Cells were activated in culture for up to seven days. Three injury models were used in wild type mice: CCl4, high fat diet with and without CCl4, and bile duct ligation. To assess PTN expression when Hh was blocked, cultured HSC cells were treated with DMSO (control) or 0.4μM-2.0μM of the hedgehog inhibitor GDC-0449. Adult a-smaCreERT2/floxed Smoothened double transgenic mice underwent BDL (n=8 mice/group) or partial hepatectomy to evaluate PTN response after liver injury in animals with abolished HSC Hh signaling. Results: In healthy livers, PTN expression was highest in LSEC and HSC and PTPRZ1 expression was highest in HSC. Healthy adult PTN-GFP mice expressed GFP in stromal cells in periportal areas, a putative progenitor niche. Serial sections suggest co-expression of PTN with desmin, supporting HSC expression of PTN. After activation in culture PTN expression fell in LSEC but increased in activated myofibroblastic (MF)-HSC suggesting MF-HSC are the major PīN source in liver injury. PTN expression also increased in in vivo mouse models of acute and chronic liver injury. Treating MF-HSC cultures with Hh inhibitor decreased PTN expression. Treating a-smaCreERT2/floxed Smoothened mice with tamoxifen to block MF-HSC Hh signaling was associated with decreased PTN expression after bile duct ligation and partial hepatectomy. Conclusion: HSC express PīN and increase expression during activation. PTN expression increases in liver injury. Since PTN expression is decreased when Hh signaling is blocked, Hh signaling modulates PTN expression during HSC activation and liver injury in vivo. Our results suggest a novel liver repair mechanism involving Hhdependent HSC PTN production. PTN may show promise for staging or treatment of human liver disease.
Anna Mae Diehl - Consulting: Bristol Myers Sguibb, Synergy, GlaxoSmithKline, Norgine; Grant/Research Support: GlaxoSmithKline
The following people have nothing to disclose: Anikia Tucker, Gregory A. Michelotti, Steve S. Choi, Guanhua Xie, Gamze Karaca, Marzena Swiderska-Syn, Leandi Kruger, Mariana V. Machado, Katherine S. Garman
Oxygen gradient on a chip for optimizing the differentiation of human pluripotent stem cells into hepatic cells
Giovanni G. Giobbe1,2, Federica Tonon2,4, Alessandro Zambon2,3, Federico Michielin2,5, Nicola Elvossore2,3, Annarosa Floreoni1
1Surgery, Oncology and Gastroenterology, University of Padova, Padova, italy; 2lndustrial Engineering, University of Padova, Padova, Italy; 3Venetian Institue of Molecular Medicine, Padova, italy; 4lndustrial Engineering and Information Technology, University of Trieste, Trieste,Italy; 5The Scripps Research Institute, La Jolla, CA
Developing new strategies for mimicking early organogenesis and deriving functional hepatocytes from human pluripotent stem cells (hPSCs) has a high scientific relevance and therapeutic potential. The role of oxygen tension as a key regulatory mechanism in hepatic differentiation has not yet been well described. Aims: a) to recapitulate early in vitro organogenesis in physiological conditions and efficiently derive mature hepatic cells from hPSCs under a stable oxygen gradient. b) to integrate the specific lineages into a microfluidic platform to obtain a functional liver tissue on a chip. Methods: Human embryonic stem cells (hESCs line HES2, NSCB, Madison WI) and human induced pluripotent stem cells (hiPSCs line ADHF#1, iCEMS, Kyoto University) were expanded and differentiated on matrigel-coated channels. We developed a multi-stage microfluidic technology to derive mature cells from pluripotent cells. This technology was implemented with generation of 1 mm (diameter of hepatic lobule) stable oxygen gradient. Obtained cells have been characterized both with hepatic markers (AFP, CK 18 −19, ALB, CYP3A) and functional tests (proliferation, glycogen storage, indocyanine green uptake, albumin secretion). Results: We developed a microfluidic technology to generate a stable 〇2 gradient and culture and differentiate human pluripotent cells. We efficiently differentiated both hESCs and hiPSCs. Two hepatic lineages were obtained: hepatocyte- and cholangiocyte-like cells showing high CYP3A expression, ICG uptake, glycogen storage and albumin secretion over a 14-day period. This technology allowed to accurately control hPSC expansion and fate toward early endoderm commitment, hepatic development and functional maturation on a chip. Compared to conventional culture, microfluidic oxygen-defined platform allowed shortening of the time reguired for differentiation and enhanced functional activity. The proportion between hepatocyte- and cholangiocyte-like cells was 3: 1. In particular, we obtained 75% of cells with glycogen storage capacity, whereas the number of CYP3A-positive cells resulted in 60% of the total, with 20% increase compared to standard hepatocytes differentiation. Albumin production was about 40% higher than standard conditions. Conclusions: The engineerization of hPSC differentiation into hepatic lineages under defined oxygen gradient will allow us to further understand the mechanisms involved in tissue development. Moreover, mature hepatic cells fully integrated on a chip could be directly used for temporaldefined toxicological assays and drug screening.
The following people have nothing to disclose: Giovanni G. Giobbe, Federica Tonon, Alessandro Zambon, Federica Michielin, Nicola Elvassore, Annarosa Floreani
Liver progenitor cells as potential target of TLR4 oncogenic signaling to generate tumor-initiating stem-like cells
Chio-Lin Chen1, Hidekazu Tsukamoto2,3, Jian-Chang Liu1,Linda S. Sher4, Lydia M. Petrovic4, Keigo Machida1,2
1Molecular Micmobi ology and Immunology, University of Southern California Keck School of Medicine' Los Angeles' CA; 2Southe; n California Research Center for ALPD and Cirrhosis, Los Angeles, CA; 3Pathology, University of Southern California Keck School of Medicine, Los Angeles, CA; 4Surgery, University of Southern California Keck School of Medicine, Los Angeles, CA
We have identified the TLR4-NANOG oncogenic pathway in the genesis of CD133+CD49f+ tumor initiating stem cell-like cells (TICs) and liver tumor in alcohol-fed HCV Ns5aTg mice and our most recent finding extends this notion to other HCC models including DEN/Pb-treated or Spnb2+/- mice. Although ectopic TLR4 expression is presumed to have occurred primarily in hepatocytes in these models, the evidence also suggests liver progenitor cells (LPCs) may be the source of TLR4+ TICs. [Aim] The present study investigated whether and why hepatocytes (HC) vs. liver progenitor cells (LPCs) are the primary target of the oncogenic TLR4 pathway. [Methods and Results] Firstly, we expressed a constitutive active (ca) mutant of TLR4 (CD4hTlr4) in vivo by creating the compound mice carrying pLAP-tTA which expresses Tet-off tTA under HC/HB-specific LAP promoter and a Tet-response element (TRE) upstream of caTlr4. The mice were withdrawn from Doxycycline (Dox) at 2 months old and subjected to 1) partial hepatectomy (PH) that primarily stimulates HC proliferation or 2) PH plus 2-AAFadministration that inhibits HC proliferation and induces a LPC population. After 12 months, PH mice developed liver tumor in 15% (x/y) of the mice while PH+2-AAF mice developed more aggressive multinodular tumors in 60% (a/b) with lung metastasis in 15%. Secondly, we have generated caTLR4 Tg mice using a-fetoprotein promoter (Afp-tTA: Tet(TRE)-caTIr4) to target LPCs. Dox was withdrawn either at the beginning of the last trimester of gestation (E14) or at 2 months after birth (P60). After 12 months, 58% (7/12) of the E14-withdrawaI mice developed liver tumors while only 9% (1/9) of P60-withdrawal mice had tumor, confirming the LPS/HB are the primary target of the TLR4 oncogenic pathway. Thirdly, we directly tested if expression of caTLR4 by a lentiviral vector induces Nanog in mature mouse HC, E12.5 HB, and PIL4 cells (p53 deficient HB cell line) in vitro. Nanog mRNA induction and colony formation in soft agar were detected in caTLR4-transduced HB and PIL4 cells but not in HC. These differential effects were associated with demethylation of a Nanog distal enhancer in HB compared to HC, particularly at an E2F1 binding site (nt −5113/-5106) shown to be critical for E2F1-NFĸB cooperation for optimal Nanog transcription. Genome-wide NANOG-binding site analysis (ChlP-seq) in TICs demonstrates that NANOG regulates oxidative phosphorylation in mitochondria as a master regulator of mitochondria biogenesis. [Conclusion] These results support the notion that LPC/HB are the primary target of TLR4 oncogenic pathway because of epigenetic de-repression of Nanog involving DNA hypomethylation.
Hidekazu Tsukamoto - Consulting: Shionogi & Co., S. P. Pharmaceutics; Grant/Research Support: The Toray Co.
The following people have nothing to disclose: Chia-Lin Chen, Jian-Chang Liu, Linda S. Sher, Lydia M. Petrovic, Keigo Machida
The Role of Keap1/Nrf2 in Modulating Redox Status of Hepatic Progenitor Cells
Soona Shin1,2, Naman Upadhyay1,2, Klaus H. Kaestner1,2
1Genetics, University of Pennsylvania, Philadelphia, PA; 2Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania, Philadelphia, PA
Background: Transplantation of hepatic progenitor cells (HPCs) is considered to be promising alternative to organ transplantation for the treatment of liver diseases. Understanding the role of redox status in HPCs is reguired for optimizing their expansion and differentiation. Nrf2 is a transcription factor that regulates cellular defenses against oxidative stress. Keap1 is the cytoplasmic binding partner of Nrf2, and suppression of Keap1 expression induces nuclear translocation of Nrf2 and expression of downstream antioxidant genes. Therefore, we investigated the role of the Keap1/Nrf2 signaling pathway in protecting HPCs from oxidative stress-induced cell death. Methods: We isolated Foxl1-expressing HPCs from FoxI1-Cre; RosaYFP/YFP mice and expanded clonal HPC cell lines in culture (1). We utilized lentiviral shRNAs to modulate expression of Keap1 and Nrf2 in HPCs. Results: ShRNA targeting of Keap1 led to dramatic induction of Nrf2 downstream antioxidant target genes. Conversely, shRNA targeting of Nrf2 led to suppression of these genes. In addition, HPCs transduced with Keap1-targeting shRNA were more resistant to menadioneinduced oxidative stress compared to HPCs transduced with control shRNA, while HPCs transduced with Nrf2-targeting shRNA were more susceptible to oxidative stress-induced cell death. We also confirmed transduction of HPCs with Keap1-targeting shRNA and Nrf2-targeting shRNA does not affect the ability of HPCs to proliferate and differentiate into hepatocytes. Conclusion: Our results indicate that targeting Keap1/Nrf2 signaling is a feasible strategy to protect HPCs from oxidative stress. Reference: 1. Shin S, Walton G, Aoki R, Brondell K, Schug J, Fox A, Smirnova O, Dorrell C, Erker L, Chu AS, Wells RG, Grompe M, Greenbaum LE, Kaestner KH, “FoxI1-Cremarked adult hepatic progenitors have clonogenic and bilineage differentiation potential, ” Genes & Development, Vol.25(11), pp.1185-1192, 2013.
The following people have nothing to disclose: Soona Shin, Naman Upadhyay, Klaus H. Kaestner
Human parthenogenetic stem cell-derived hepatocyte transplantation controls serum bilirubin in Gunn rats
Alina Ostrowska, Larisa Agapova, Tiffany Chu, Trudy ChristiansenWeber, Ruslan Semechkin
International Stem Cell Corporation, Carlsbad, CA
Background: Extensive studies indicate that pluripotent stem cells are a highly promising alternative source of histocompatibie cells for cell replacement therapy. Hepatocyte-like cells (HLCs) derived from human parthenogenetic stem cells (hpSCs) might be transplanted to treat a wide array of metabolic liver diseases including CN1 (Crigler-Najjar syndrome type I). CN1 is the paradigm of inherited liver-based metabolic disorders in that the host liver is lacking one hepatic enzyme - UGT1A1, which is essential for the conjugation and excretion of bilirubin. To obtain proof that differentiation has been achieved, following the preliminary evaluation in vitro, we tested hepatocyte-like cells in vivo using an animal model of CN1: Gunn rats which accumulate toxic plasma levels of unconjugated bilirubin. Methods: Highly enriched populations of definitive endoderm were generated from hpSCs in a novel 3D-differentiation system and induced to differentiate towards HLCs. Cells were characterized using RT-gPCR, immunohistochemistry and FACS analysis for hepatocyte-specific markers, drug metabolism assays to determine the activity of CYP450s, and a luminescent method for measuring UGT activity. Production of liver-specific proteins was measured by guantitative ELISA. To evaluate engraftment and functional repopulation in vivo, CFSE-labeled hpHCs were injected (10x106 per animal) into the spleen of 4-6 week old Gunn rats. Blood serum samples of tested animals were evaluated for indirect bilirubin levels 4, 8 and 19 weeks post-transplantation. Liver tissue samples were embedded in OCT compound and snap frozen, for cryosectioning. Results: CFSElabeled HLCs transferred into the spleen were shown to migrate to the liver. Multiple engrafted cells were observed in the periportal regions of the liver lobules and formed morphologically distinct aggregates. The overall liver structure appeared undamaged without signs of inflammation, fibrosis or tumor. Significant decrease and long-term stabilization of bilirubin levels was seen in the serum of tested animals in comparison with shamtreated controls. Although by week 19 serum indirect bilirubin was not normalized in transplanted animals, it resulted in an average 70% reduction compared with pretreatment levels. Conclusion: This pre-clinical study describes important supportive evidence of the potential efficacy and safety of hpSCderived hepatocytes which might constitute an easily available source of a large number of transplantable cells for regenerative treatments of CN1. In the long-term, experience with HLCs transplantation for CN1 can be used to develop therapeutic strategies for more common inherited liver diseases.
Alina Ostrowska - Employment: International Stem Cell Corporation
Larisa Agapova - Stock Shareholder: ISCO
Tiffany Chu - Employment: International Stem Cell Corporation
Trudy Christiansen-Weber - Employment: International Stem Cell Corportation; Stock Shareholder: International Stem Cell Corporation
Ruslan Semechkin - Employment: International Stem Cell Corporation; Stock Shareholder: International Stem Cell Corporation
Interleukin-17 favors progenitor cell proliferation and differentiation during liver regeneration
Adrien Guillot, Nabila Hamdaoui, Sophie Lotersztajn, Fouad Lafdil
INSERM U955, UPEC University of Paris, Créteil, France
Introduction. After moderate injury, the liver displays high regenerative capacity from remaining and healthy hepatocytes. However, when the proliferative properties of residual hepatocytes are altered, liver regeneration is driven by the proliferation and differentiation of liver progenitor cells (LPCs) named oval cells in rodents. In human, LPC proliferation is also freguently observed, in cirrhosis, hepatitis B infection, alcoholic or non-alcoholic steatohepatitis and referred to as ductular reaction. Previous studies suggested an important role of T lymphocyte immune response on LPCs accumulation in regenerating livers. Indeed, T helper 1 (Th1) lymphocytes promote LPC development via IFN-g production. Interestingly, IL-27 has been described as an IFN-g-like cytokine, but its role in LPC-mediated liver regeneration remains unknown. Furthermore, recent studies identified Th17 lymphocytes producing IL-17 and IL-22, in several chronic liver diseases. While the pro-mitogenic role of IL-22 on LPCs has been shown, the implication of IL-17 has not been yet studied. Thus, we proposed to determine the involvement of IL-17 and IL-27 on LPC proliferation and differentiation in liver regeneration. Materials and methods. Wild-type (WT) and IL-17-deficient (IL-17-/-) mice were subjected to a model of liver regeneration from LPCs induced by a choline deficient, and ethionine supplemented diet (CDE). In vivo, the immune response was analyzed by guantitative RT-PCR. LPC accumulation was assessed by immunohistochemistry using an anti-cytokeratin 19 (CK19) antibody and by qRT-PCR. In vitro, IL-17 effects on murine macrophage (RAW) phenotype were analyzed by gRT-PCR. Murine oval cell (BMOL) proliferation and differentiation were studied by proliferation assay (MTS) and gRT-PCR, respectively. Results. After 3 weeks of CDE diet, IL-17/- mice displayed less liver injury as compared to WT mice. IL17-deficiency was associated with reduced CK19+ LPCs, and with weaker induction of LPC activation marker expressions (afoeto-protein, M2-PK, Cx43) when compared with WT mice. In addition, the lack of IL-17 led to a reduction of both macrophage recruitment (F4/80, MCP-1) and pro-inflammatory cytokine expression (TNF-a IL-6) including IL-27. Interestingly, in vitro, IL-17 induced macrophage IL-27 expression. While IL-17 stimulated LPC proliferation, IL-27 treatment led to increased biliary cell (Cx43, CK7, CK19) and hepatocyte (Alb, Cx32, HFN4-a) marker expressions. Conclusion. Our results revealed that IL-17 directly favors oval cell proliferation, and indirectly enhances their differentiation by inducing macrophage IL-27 production during liver regeneration.
The following people have nothing to disclose: Adrien Guillot, Nabila Hamdaoui, Sophie Lotersztajn, Fouad Lafdil
Repopulation of mouse liver with human hepatocytes from core biopsies: a new approach to modeling human liver diseases
Branden Tarlow1, Willscott E. Naugler2, Susan L. Orloff4, Annelise Haft3, Markus Grompe3;
1Cell and Developmental Biology, Oregon Health & Sciences University, Portland, OR; 2 Medicine/Gas-troenterology, Oregon Health & Sciences University, Portland, OR; 3Pediatrics, Oregon Health & Sciences University, Portland, OR; 4Abdominal Organ Transplant, Portland VA Medical Center, Portland, OR
Background: A shortcoming of existing transgenic mouse models of cirrhosis is that they only partially recapitulate the features of human liver disease. Modeling chronic liver disease with human tissue, especially at early stages, may allow for better understanding of the pathophysiology of diseases like non-alcoholic steatohepatitis (NASH). Patient-specific xenograft models may highlight factors driving the variability in disease progression and aid in the selection of therapies that are likely to modify disease pathophysiology in particular patients. Methods: Previously, our group and others have used the Fah-/- Rag2-/-Il2ry-/- (FRG) mouse, a model of tyrosinemia, type I, to propagate and study normal human hepatocytes from large surgical wedge resections. Here, 32mm × 16-gauge core needle biopsies were collected from human liver explants or surgically resected tissue with patient consent and intuitional review board approval. No donor tissues were obtained from executed prisoners or other institutionalized persons. Tissue was digested with an EDTA and collagenase-based digestion protocol in a shaking water bath. Viable hepatocytes were identified by trypan blue exclusion and attachment to tissue culture plates. Hepatocytes were transplanted via the portal vein into FRG mice. Mice were treated postoperatively with antibiotics and cycled on the drug NTBC (nitisinone) to allow selective expansion human hepatocytes. All experimental animal procedures were conducted with the approval and oversight of the OHSU Institutional Animal Care and Use Committee. Results: We show that hepatocytes can be isolated from core needle biopsy tissue of human liver tissue, resulting in liver humanization. Approximately 30,000 - 80,000 live human hepatocytes were isolated per biopsy from diseased liver. Portal vein cell infusion was well tolerated in immune-compromised mice with greater than 80% perisurgical survival. Human serum albumin was detected by human specific ELISA 6-10 weeks after transplantation of human hepatocytes. Engrafted mice showed an increase in human serum albumin over time, indicating progressive liver humanization. Immunohistochemical stains confirm the presence of engrafted human hepatocytes as noted by positive Fah staining, which is absent in Fah-/- host mice. Conclusion: Human hepatocytes from small clinically available tissue samples can be engrafted into the livers of mice for further study and analysis. Modeling chronic liver disease from percutaneous biopsy tissue may improve the understanding of disease pathophysiology.
Markus Grompe - Board Membership: Yecuris Corp.; Consulting: Yecuris Corp.; Stock Shareholder: Yecuris Corp.
The following people have nothing to disclose: Branden Tarlow, Willscott E. Naugler, Susan L. Orloff, Annelise Haft
Identification of liver cancer progenitors whose malignant progression depends on autocrine IL-6 signaling
Debanjan Dhar1, Hayato Nakagawa1,3, Hisanobu Ogata1, Yuhong Jiang1, Ekihiro Seki2, Shabnam Shalapour1, Michael Karin1;
1DEPARTMENT OF PHARMACOLOGY, UCSD, La Jolla, CA;2Department of Medicine, UCSD, La Jolla, CA; 3Department of Gastroenterology, University of Tokyo, Tokyo, Japan
Background: Hepatocellular carcinoma (HCC) is believed to evolve from premalignant lesions in chronically damaged livers. However, it was never established that premalignant lesions actually contain tumor progenitors that give rise to cancer. Results: We have isolated and characterized the HCC progenitor cells (HcPC) from two different mouse HCC models: (1) Hepatocarcinogen DEN treated WT mice and (2) Hepatocyte specific deletion of TAK1 (MAP3K) mice that develops spontaneous HCC. We have characterized the HcPC based on several cell surface markers and activated signaling pathways and found that the cells resembling HcPC reside within dysplastic lesions that appear several months before macroscopic HCC nodules. Although cancer stem cells have been isolated from several well-developed tumors, we were able to isolate HcPC long before the tumors are visible. Hepatocyte preparations by collagenase digestion of DEN-exposed and Tak1 deficient livers (long before actual tumors appear) contain rare collagenase resistant cell aggregates that are enriched in HcPC. Unlike fully malignant HCC, HcPC give rise to cancer only when introduced into a liver undergoing chronic damage and compensatory proliferation such as that of Mup-uPA mice (where the liver undergoes chronic low grade damage due to hepatocyte specific expression of Plasminogen Activator) or that of wild-type mice treated with retrorsine and carbon tetrachloride (CCl4). Furthermore, we have identified that DEN-induced premalignant lesions and HcPC exhibit autocrine IL-6 production that is critical for tumorigenic progression. Knockdown of IL-6 in HCC derived cell line as well as freshly isolated HcPC reduced their tumorigenicity when transplanted into Mup-uPA liver. Also, HcPC isolated from IL-6ko liver had reduced tumorigenicity compared to WT-HcPC. Unlike early hepatocarcinogenesis that depends on paracrine IL-6 production by inflammatory cells, HcPC had acquired autocrine IL-6 signaling that stimulates their in vivo growth and malignant progression. The autocrine IL-6 production is activated in part due to the upregulation of LIN28, an RNA binding protein that prevents processing of microRNA Let-7 that is known to downregulate IL-6. We found that Let-7 miRNA along with other miRNAs that target IL-6 are also down-regulated. We further show that the autocrine IL-6/LIN28 loop is also activated in human pre-malignant lesions (needle biopsies of HCV-infected livers that contain dysplastic lesions). Conclusions: We successfully isolated and characterized HcPC from tumor bound livers and identified that HcPC acquire the ability to produce their own IL-6 that is critical for their malignant progression.
The following people have nothing to disclose: Debanjan Dhar, Hayato Nakagawa, Hisanobu Ogata, Yuhong Jiang, Ekihiro Seki, Shabnam Shalapour, Michael Karin