Stem cells and progenitors


55

Nrf2 Deficiency Causes Transient but Massive Hepatocyte Dedifferentiation during Liver Regeneration

Yuhong Zou, Joonyong Lee, Shashank M. Nambiar, Min Hu, Qi Bao, Guoli Dai

Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN

Nuclear factor erythroid 2-related factor 2 (Nrf2), a basic leucine zipper transcription factor, is a central regulator of cellular defense responses against xenobiotic insults, oxidative stress, and inflammation. Several studies indicate important roles for Nrf2 in modulating liver injury and repair. To gain insights into how Nrf2 participates in regulating liver regeneration, wild-type and Nrf2-null mice were subjected to 2/3 partial hepatectomy (PH) and were sacrificed at multiple time points after surgery for various analyses. Nrf2-null mice exhibited sluggish liver regrowth, although the lost liver mass was eventually restored 7 days after PH. Nrf2 deficiency did not affect the number of hepatocytes entering the cell cycle, but delayed hepatocyte mitosis due to excessive accumulation of Cyclin A2 protein and reduced activity of Wee1/Cdc2/Cyclin B1 pathway in regenerating livers. Both strains of mice showed an overall trend of progressively increased hepatocyte hypertrophy during the entire course of liver regeneration. However, at 60 h after PH, hepatocytes lacking Nrf2 had markedly reduced cell sizes and condensed nuclei, accompanied by severe liver regrowth retardation. Moreover, DNA microarray analysis revealed three hepatic progenitor marker genes which were dramatically upregulated at the time point due to Nrf2 absence. They are CD133, tumor necrosis factor-like weak inducer of apoptosis (TWEAK) receptor (Fn14), and trefoil factor family 3 (TFF3). Verification at protein level by western blotting showed that hepatic CD133, Fn14, and TFF3 proteins were drastically activated at 60 h after PH in Nrf2-null, but not wild-type, mice during the entire course of liver regeneration. Immunohistochemically, almost all hepatocytes lacking Nrf2 expressed CD133 evenly in their cytosol, Fn14 on their surface, and TFF3 cytosolically concentrated as aggregates. Thus, the vast majority of hepatocytes deficient in Nrf2 exhibited a phenotype of CD133+/Fn14+/TFF3+. Reduction in cell size and activation of the three hepatic progenitor markers indicate that Nrf2-null hepatocytes were undergoing transient but massive dedifferentiation. Taken together, we demonstrate that Nrf2 is required for timely M phase entry of replicating hepatocytes by ensuring proper regulation of Cyclin A2 and Wee1/Cdc2/Cyclin B1 pathway. Most remarkably, we, for the first time, discovered a phenomenon of massive hepatocyte dedifferentiation in vivo in a pathological condition (Nrf2 absence and liver injury). The finding indicates a role for Nrf2 in maintaining hepatocyte identity during liver regeneration.

Disclosures:

The following people have nothing to disclose: Yuhong Zou, Joonyong Lee, Shashank M. Nambiar, Min Hu, Qi Bao, Guoli Dai

56

lncRNA MALAT1 inhibits Smad2/3 signaling in hepatic cells

Jinqiang Zhang, Chang Han, Kyoungsub Song, Tong Wu

Pathology and Laboratory Medicine, Tulane Univeraity School of Medicine, New Orlenas, LA

Long non-coding RNA (lncRNA) is defined as RNA longer than 200 bases that does not encode mRNA, rRNA or tRNA. In this study we carried out RNA immunoprecipitation assays and identified lncRNA MALAT1 association with pSmad2/3 in transformed human hepatocytes (Hep3B and SK-Hep1). qRTPCR analysis showed that MALAT1 was enriched by antipSmad2/3 antibodies approximately 15 times more than that by IgG control. Combinational FISH and immunofluorescence staining revealed co-localization of MALAT1 and pSmad2/3 in nuclear speckles. Sequence alignment analysis and immunoprecipitation assays showed that pSmad2/3 bound to MALAT1 through SETD2 (a protein termed SET domain containing 2). Knockdown of SETD2 by siRNA dramatically decreased the binding between pSmad2/3 and MALAT1 as determined by RNA Immunoprecipitation assay. Furthermore, knockdown of MALAT1 by shRNA or antisense oligonucleotide increased Smad2/3 activity, as reflected by enhanced Smad2/3 DNA binding and luciferase reporter activity. Accordingly, knockdown of MALAT1 potentiated TGF-β-mediated inhibition of Hep3B cell proliferation. Knockdown of MALAT1 was found to decrease the binding of pSmad2/3 to PPM1A (Protein Phosphatase 1A, a Smad2/3 phosphatase that terminates pSmad2/3 activity). Using iPS cell to hepatocyte-like cell differentiation model, we found that knockdown of MALAT1 in iPS cells by antisense oligonucleotide enhance Activin A- or TGFβinduced hepatocyte differentiation, characterized by increased expression of α-Fetoprotein, Albumin, Glucose6-phosphatase, cytokeratin18 et al. Therefore, MALAT1 (together with SETD2) might act as a scaffold to facility the interaction between pSmad2/3 and PPM1A, which leads to dephosphorylation of pSmad2/3 and inhibition of TGFβ/Samd2/3 signaling.

Disclosures:

The following people have nothing to disclose: Jinqiang Zhang, Chang Han, Kyoungsub Song, Tong Wu

57

Regression of cirrhosis: The maturation sequence of buds arising from hepatocyte progenitor cells

Ashley E. Stueck, lan R.. Wanless

Department of Pathology, Dalhousie University, Halifax, NS, Canada

Cirrhosis can regress if the primary disease is controlled. Regression requires infill of collapsed regions by division of mature hepatocytes or generation of new cells from hepatocyte progenitor cells (HPC). New hepatocytes have been identified in buds associated with CK19+ ductular structures (Wanless 2000, Falkowski 2003). These hepatocytes express EpCAM (Yoon 2011). Glutamine synthetase (GS) is also expressed in these hepatocytes and in the cholangiocytes of budding ducts (Fleming 2013). The purpose of this study is to define the natural history of buds with a morphologic “bud maturation sequence” and immaturity index (IMI). Methods. 35 large liver specimens with cirrhosis or controls from archives of QEII-HSC were stained with EpCAM, CK19, CD34, GS and Ki-67.Specimens were stratified by etiology (biliary, non-biliary) and Laennec stage. Buds were defined as clusters of hepatocytes within septa. Five levels of bud maturation (0-4) were defined by progressive enlargement and decreasing CK19+ to CK19- ratio (cholangiocytes and hepatocytes, respectively): in brief, level-1 buds contain CK19+ cells adjacent to all hepatocytes, while level-4 buds are clusters of >10 hepatocytes without intervening CK19+ cells. Immunohistochemical expression (% of positive hepatocytes or sinusoids) was correlated with bud level. A novel IMI was defined as the % hepatocyte area with EpCAM+ hepatocytes. Results. Buds evolve sequentially with activation of ducts (to GS+), generation of hepatocytes within ducts, replication to form clusters of hepatocytes, followed by merging of buds together or with established parenchyma. As buds enlarge and mature from level-1 to level-4, expression declines for GS (98 to 46%, p<0.01), EpCAM (75 to 8%, p<0.01), and CD34 (100 to 19%, p<0.0001). Buds are more numerous in severe cirrhosis (p<0.0007) and in non-biliary disease (p<0.0002). IMI varied from 0 to 69% (e. g. 4C=31% vs 4a/B=6%, p<0.0008; non-biliary=30% vs biliary=2%, p=0.0065). Ki-67 data (not shown) indicates that proliferation shifts from cholangiocytes/HPC to hepatocytes as buds mature. Vascular maturation occurs in parallel: sinusoidal endothelial cells are CD34+ in immature buds and are largely CD34- in mature buds where hepatic vein drainage appears to be developing. Conclusions: The bud maturation sequence and IMI provide useful markers for the study of regression of cirrhosis. The high IMI values indicate that a large percentage of parenchyma in regressed cirrhosis may be derived from HPC as buds. Vascularization of buds, especially hepatic vein drainage, may be a limiting factor controlling bud development. Low bud number in biliary disease may reflect bile duct injury.

Disclosures:

The following people have nothing to disclose: Ashley E. Stueck, Ian R. Wanless

58

Epigenetic Reprogramming Modulates Malignant Properties of Human Liver Cancer Cells

Chiara Raggi, Valentina M. Factor, Seo Daekwan, Agnes Holczbauer, Jens U. Marquardt, Snorri S. Thorgeirsson

Laboratory of Experimental Carcinogenesis, NCI / NIH, Bethesda, MD

Background and Aims: Modulation of cellular fate in solid tumors is defined to a large extent by DNMT1-regulated epigenetic machinery and cellular and non-cellular constituents in the tumor-initiating cells (TICs) niche. Current study examines the significance of the DNMT1-cellular interactions in reprogramming of TICs properties. Methods: Seven HCC cell lines were plated in 2D culture at various cell densities and exposed to a transient nontoxic dose of a DNMT1-inhibitor Zebularine (ZEB). After a 3-day treatment, cells were cultured in 3D non-adherent condition in ZEB- and serum-free media to generate primary spheres (G1) which were then passaged through generation G5.Differences in long-term self-renewal, gene expression, tumorigenicity and metastatic potential of G1-G5 spheres were examined. Results: Transient exposure to ZEB produced the differential cell density-depended responses in 5/7 tested HCC cell lines. In cells grown at low density (LD), ZEB caused a remarkable increase in G1 sphere formation. This effect persisted through G5.In striking contrast, untreated LD cells failed to form primary spheres while the sphere forming potential of high density (HD) and HD ZEB-treated (HDZ) cells rapidly decreased over the first 3 generations. Likewise DNMT1 depletion by shRNA promoted acquisition of self-renewal potential in LD cells. The increase in sphere forming potential of LDZ cells strongly correlated with a stable overexpression of cancer stem cell-related markers and key genes involved in self-renewal and epithelial-mesenchymal transition. Moreover, when dissociated LDZ, HD and HDZ spheres were injected subcutaneously into NO D/SCID mice, LDZ cells generated tumors more rapidly and were more metastatic. Both gene reactivation and tumorigenic-ity progressively increased from G1 to G4.Tumors derived from G1-G4 LDZs were also increasingly more vascular. Global transcriptome analysis of LDZ spheres at G1-G4 confirmed that a LDZ signature was enriched in genes associated with oncogenic signaling pathways and could predict clinical outcome of liver cancer patients. Conclusion: DNMT1 inhibition combined with cellular context-dependent cues results in reprogramming of hepatic TICs which persists long after the drug removal and affects their fate. These findings may provide a new venue for therapeutic strategy in HCC patients.

Disclosures:

The following people have nothing to disclose: Chiara Raggi, Valentina M. Factor, Seo Daekwan, Agnes Holczbauer, Jens U. Marquardt, Snorri S. Thorgeirsson

59

Myofibroblastic Cells Function as Progenitors to Regenerate Murine Livers after Partial Hepatectomy

Marzena Swiderska-Syn, Wing-Kin Syn, Guanhua Xie, Leandi Kruger, Anna Mae Diehl

Division of Gastroenterology, Duke University Medical Center, Durham, NC

Background: Smoothened (SMO), a membrane co-receptor of the Hedgehog (Hh) pathway, is a master regulator of liver epithelial repair. We recently showed that SMO signaling promotes mesenchymal-to-epithelial transitions in hepatic stellate cell (HSC)-derived myofibroblasts (MF). HSC-derived MF also exhibit features of liver progenitors. We reported that treating mice with a systemic pharmacologic inhibitor of SMO impairs liver regeneration after partial hepatectomy (PH). Here we investigate the specific roles of SMO+ MF in adult liver regeneration by evaluating the effects of conditional SMO deletion in aSMA+ cells (i. e., MF) after PH. Methods: αSMA-CreERT2xSMO /flox mice (n–55) were treated with vehicle (Veh, Control) or tamoxifen (TMX, to delete SMO), and then sacrificed at 24, 48, 72, and 96 hrs after PH. Regenerating livers were analyzed for proliferation, progenitors, and fibrosis by qRT-PCR, and quantitative IHC. Results were normalized to liver segments resected at PH (time 0). For tracing progeny of aSMA(+) cells, aSMA-Cre-ERT2xRO SA-Stop-flox-YFP mice (n=40) were treated with Veh or TMX; livers were stained for YFP protein, and hepatocytes isolated 48 and 72 hrs post-PH were analysed for YFP fluorescence by FACS. Results: In VehαSMA-SMO mice, PH increased expression of Gli1 and Gli2 (Hh target genes), stimulated accumulation of liver progenitors (Sox 9, AFP, K19, Lgr5(+) cells, mRNA & protein), and induced proliferation of hepatocytes and cholangiocytes (all p < 0.05 vs time 0). In contrast, TMX-αSMA-SMO mice exhibited loss of whole liver SMO expression, repression of Gli1 and Gli2 to pre-PH levels, >5-fold reduced accumulation of progenitors, 7-fold inhibition of hepatocyte and cholangiocyte proliferation, and decreased recovery of liver weight/body weight (all p<0.05). Loss of SMO in αSMA+ cells also attenuated the fibrogenic response to PH (αSMA, Desmin, Vimentin, OPN, MMP9), while upregulating E-Cadherin and quiescent HSC markers (GFAP, PPAR٧) (p<0.05). Lineage tracing using TMX-αSMA-YFP mice showed accumulation of YFP+ cholangiocytes and hepatocytes 48 and 72 hrs post PH. FACS of primary hepatocytes confirmed that up to 25% were derived from αSMA-YFP+ cells. Conclusion: Loss of SMO in αSMA+ cells abrogates Hh activity, reduces liver progenitors, inhibits liver cell proliferation, attenuates fibrogenesis, and suppresses liver regeneration after PH. At least 25% of hepatocytes in regenerating livers derive from αSMA+ cells, confirming that αSMA-SMO + cells become liver progenitors to help regenerate the liver after PH.

Disclosures:

Anna Mae Diehl - Consulting: Bristol Myers Squibb, Synergy, GlaxoSmithKline, Norgine; Grant/Research Support: GlaxoSmithKline

The following people have nothing to disclose: Marzena Swiderska-Syn, WingKin Syn, Guanhua Xie, Leandi Kruger

60

Hepatic Stellate Cells Are the Dominant Source of Myofibroblasts Across all Types of Chronic Liver Injury but do not Contribute to the Generation of Hepatocytes

Ingmar Mederacke, Christine C. Hsu, Juliane Tröger, Peter Huebener, Dianne H. Dapito, Pradere Jean-Philippe, Robert Schwabe

Medicine, Columbia University, New York, NY

BACKGROUND: The cellular sources of hepatic myofibroblasts and their relative contribution to liver fibrosis remain elusive due to the lack of cell fate tracking studies. Although several candidates, including hepatic stellate cells (HSCs), portal fibroblasts, and fibrocytes differentiate into myofibroblasts in vitro, none of these cells have been proven to contribute to the myofibroblast pool in the fibrotic liver. Moreover, it is believed that their contribution varies significantly between toxic and biliary liver fibrosis, and that some cells such as HSCs not only give rise to myofibroblasts but also to newly generated hepatocytes. AIM: Employing genetic cell fate tracking, we seek to determine the relative contribution of HSCs to the myofibroblast pool in different types of liver fibrosis, and to investigate whether HSCs are a source of newly generated hepatocytes. METHODS: We generated a novel BAC-transgenic mouse, in which Cre expression is driven by the murine lecithin retinol acyltransferase promoter (LratCre). For cell fate tracking, LratCre-expressing mice were bred with 3 different Cre reporter mice. To assess the contribution of HSCs to the collagen-producing myofibroblast pool, triple transgenic mice, expressing LratCre, Cre reporter and a Col-GFP reporter, were generated. Liver injury and fibrosis were induced by CCl4, bile duct ligation (BDL), MCDE diet, DDC diet and by Mdr2 knockout (Mdr2ko). RESULTS: LratCre labeled 99% of HSCs in liver sections, in culture and by FACS analysis (p<0.0001), without marking a significant percentage of other hepatic cell populations including hepatocytes, macrophages, or endothelial cells. Using αSMA staining or Col-GFP reporter expression, we observed that >95% of myofibroblasts were derived from LratCre-marked HSCs in toxic liver fibrosis. Surprisingly, even in biliary fibrosis induced by BDL, Mdr2ko or DDC diet, we found >80% of Col-GFP+ and aSMA+myofibroblasts to be HSC-derived. The functional contribution of HSCs was further confirmed by HSC depletion studies with Cre-inducible diphtheria toxin receptor-mediated ablation, which significantly reduced the number of myofibroblasts (80%) in the fibrotic liver. LratCre-marked HSCs did not contribute to the generation of HNF4α-expressing hepatocytes after BDL, CCl4 treatment, DDC or MCDE diet or by Mdr2ko. Finally, bone marrow (BM) transplantation studies revealed that LratCre-marked HSCs are not BM-derived in normal liver, and BDL- or CCl4-induced liver fibrosis. CONCLUSION: HSCs are the dominant and liver-resident source of myofibroblasts across different types of liver fibrosis, and do not contribute to the generation of newly formed hepatocytes.

Disclosures:

The following people have nothing to disclose: Ingmar Mederacke, Christine C. Hsu, Juliane Tröger, Peter Huebener, Dianne H. Dapito, Pradere Jean-Philippe, Robert Schwabe

Ancillary