• Hepatocyte transplantation, a step forward?

      Ott, Michael; Castell, Jose V; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Elsevier, 2019-06-01)
    • Homologous recombination mediates stable Fah gene integration and phenotypic correction in tyrosinaemia mouse-model.

      Junge, Norman; Yuan, Qinggong; Vu, Thu Huong; Krooss, Simon; Bednarski, Christien; Balakrishnan, Asha; Cathomen, Toni; Manns, Michael P; Baumann, Ulrich; Sharma, Amar Deep; et al. (2018-02-27)
      To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout (Fah-/-)mice by homologous-recombination-mediated targeted addition of theFahgene.
    • MicroRNA-125b-5p mimic inhibits acute liver failure.

      Yang, Dakai; Yuan, Qinggong; Balakrishnan, Asha; Bantel, Heike; Klusmann, Jan-Henning; Manns, Michael P; Ott, Michael; Cantz, Tobias; Sharma, Amar Deep; Twincore Centre of Experimental and Clinical Infection Research; a joint venture between the Hannover Medical School and the Helmholtz Centre for Infection Research, Hannover 30625, Germany. (2016)
      The lack of broad-spectrum anti-acute liver failure (ALF) therapeutic agents contributes to ALF-related mortality. MicroRNAs (miRNAs) are suggested to be potent serum biomarkers for ALF, but their functional and therapeutic relevance in ALF are unclear. Here we show an unbiased approach, using two complementary miRNA screens, to identify miRNAs that can attenuate ALF. We identify miR-125b-5p as a regulator of cell death that attenuates paracetamol-induced and FAS-induced toxicity in mouse and human hepatocytes. Importantly, administration of miR-125b-5p mimic in mouse liver prevents injury and improves survival in models of ALF. Functional studies show that miR-125b-5p ameliorates ALF by directly regulating kelch-like ECH-associated protein 1, in turn elevating expression of nuclear factor-E2-related factor 2, a known regulator in ALF. Collectively, our findings establish miR-125b-5p as an important regulator of paracetamol-induced and FAS-induced cell death. Thus, miR-125b-5p mimic may serve as a broad-spectrum therapeutic attenuator of cell death during ALF.
    • Determinants of tetanus, pneumococcal and influenza vaccination in the elderly: a representative cross-sectional study on knowledge, attitude and practice (KAP).

      Klett-Tammen, Carolina J; Krause, Gerard; Seefeld, Linda; Ott, Jördis J (2016)
      Severity and incidence of vaccine-preventable infections with influenza viruses, s. pneumoniae and c. tetani increase with age. Furthermore, vaccine coverage in the elderly is often insufficient. The aim of this study is to identify socio-economic and knowledge-, attitude- and practice- (KAP)-related determinants of vaccination against influenza, pneumococcal disease and tetanus in the older German population.
    • Concise review: cell therapies for hereditary metabolic liver diseases-concepts, clinical results, and future developments.

      Cantz, Tobias; Sharma, Amar Deep; Ott, Michael; TWINCORE, Centre for Experimental and Clinical Infection Research, Feodor-Lynen Str. 7, 30625, Hannover, Germany. (2015-04)
      The concept of cell-based therapies for inherited metabolic liver diseases has been introduced for now more than 40 years in animal experiments, but controlled clinical data in humans are still not available. In the era of dynamic developments in stem cell science, the "right" cell for transplantation is considered as an important key for successful treatment. Do we aim to transplant mature hepatocytes or do we consider the liver as a stem/progenitor-driven organ and replenish the diseased liver with genetically normal stem/progenitor cells? Although conflicting results from cell tracing and transplantation experiments have recently emerged about the existence and role of stem/progenitor cells in the liver, their overall contribution to parenchymal cell homeostasis and tissue repair is limited. Accordingly, engraftment and repopulation efficacies of extrahepatic and liver-derived stem/progenitor cell types are considered to be lower compared to mature hepatocytes. On the basis of these results, we will discuss the current clinical cell transplantation programs for inherited metabolic liver diseases and future developments in liver cell therapy. Stem Cells 2015;33:1055-1062.
    • E-N-cadherin heterodimers define novel adherens junctions connecting endoderm-derived cells.

      Straub, Beate K; Rickelt, Steffen; Zimbelmann, Ralf; Grund, Christine; Kuhn, Caecilia; Iken, Marcus; Ott, Michael; Schirmacher, Peter; Franke, Werner W (2011-11-28)
      Intercellular junctions play a pivotal role in tissue development and function and also in tumorigenesis. In epithelial cells, decrease or loss of E-cadherin, the hallmark molecule of adherens junctions (AJs), and increase of N-cadherin are widely thought to promote carcinoma progression and metastasis. In this paper, we show that this "cadherin switch" hypothesis does not hold for diverse endoderm-derived cells and cells of tumors derived from them. We show that the cadherins in a major portion of AJs in these cells can be chemically cross-linked in E-N heterodimers. We also show that cells possessing E-N heterodimer AJs can form semistable hemihomotypic AJs with purely N-cadherin-based AJs of mesenchymally derived cells, including stroma cells. We conclude that these heterodimers are the major AJ constituents of several endoderm-derived tissues and tumors and that the prevailing concept of antagonistic roles of these two cadherins in developmental and tumor biology has to be reconsidered.
    • The temporal and hierarchical control of transcription factors-induced liver to pancreas transdifferentiation.

      Berneman-Zeitouni, Dana; Molakandov, Kfir; Elgart, Marina; Mor, Eytan; Fornoni, Alessia; Domínguez, Miriam Ramírez; Kerr-Conte, Julie; Ott, Michael; Meivar-Levy, Irit; Ferber, Sarah; et al. (2014)
      Lineage-specific transcription factors (TFs) display instructive roles in directly reprogramming adult cells into alternate developmental fates, in a process known as transdifferentiation. The present study analyses the hypothesis that despite being fast, transdifferentiation does not occur in one step but is rather a consecutive and hierarchical process. Using ectopic expression of Pdx1 in human liver cells, we demonstrate that while glucagon and somatostatin expression initiates within a day, insulin gene expression becomes evident only 2-3 days later. To both increase transdifferentiation efficiency and analyze whether the process indeed display consecutive and hierarchical characteristics, adult human liver cells were treated by three pancreatic transcription factors, Pdx1, Pax4 and Mafa (3pTFs) that control distinct hierarchical stages of pancreatic development in the embryo. Ectopic expression of the 3pTFs in human liver cells, increased the transdifferentiation yield, manifested by 300% increase in the number of insulin positive cells, compared to each of the ectopic factors alone. However, only when the 3pTFs were sequentially supplemented one day apart from each other in a direct hierarchical manner, the transdifferentiated cells displayed increased mature β-cell-like characteristics. Ectopic expression of Pdx1 followed by Pax4 on the 2(nd) day and concluded by Mafa on the 3(rd) day resulted in increased yield of transdifferentiation that was associated by increased glucose regulated c-peptide secretion. By contrast, concerted or sequential administration of the ectopic 3pTFs in an indirect hierarchical mode resulted in the generation of insulin and somatostatin co-producing cells and diminished glucose regulated processed insulin secretion. In conclusion transcription factors induced liver to pancreas transdifferentiation is a progressive and hierarchical process. It is reasonable to assume that this characteristic is general to wide ranges of tissues. Therefore, our findings could facilitate the development of cell replacement therapy modalities for many degenerative diseases including diabetes.
    • Ectopic expression of murine CD47 minimizes macrophage rejection of human hepatocyte xenografts in immunodeficient mice.

      Waern, Johan M; Yuan, Qinggong; Rüdrich, Urda; Becker, Pablo D; Schulze, Kai; Strick-Marchand, Helene; Huntington, Nicholas D; Zacher, Behrend J; Wursthorn, Karsten; Disanto, James P; et al. (2012-10)
      Macrophages play an important role in the rejection of xenogeneic cells and therefore represent a major obstacle to generating chimeric mice with human xenografts that are useful tools for basic and preclinical medical research. The signal inhibitory regulatory protein α (SIRPα) receptor is a negative regulator of macrophage phagocytic activity and interacts in a species-specific fashion with its ligand CD47. Furthermore, SIRPα polymorphism in laboratory mouse strains significantly affects the extent of human CD47-mediated toleration of human xenotransplants. Aiming to minimize macrophage activity and thus optimize human cell engraftment in immunodeficient mice, we lentivirally transduced murine CD47 (Cd47) into human liver cells. Human HepG2 liver cells expressing Cd47 were less frequently contacted and phagocytosed by murine RAW264.7 macrophages in vitro than their Cd47-negative counterparts. For the generation of human-mouse chimeric livers in immunodeficient BALB-ΔRAG/γ(c) -uPA (urokinase-type plasminogen activator) mice, freshly thawed cryopreserved human hepatocytes were transduced with a lentiviral expression vector for Cd47 using a refined in vitro transduction protocol immediately before transplantation. In vivo, Cd47-positive human primary hepatocytes were selectively retained following engraftment in immunodeficient mice, leading to at least a doubling of liver repopulation efficiencies. Conclusion: We conclude that ectopic expression of murine Cd47 in human hepatocytes selectively favors engraftment upon transplantation into mice, a finding that should have a profound impact on the generation of robust humanized small animal models. Moreover, dominance of ectopically expressed murine Cd47 over endogenous human CD47 should also widen the spectrum of immunodeficient mouse strains suitable for humanization. (HEPATOLOGY 2012).
    • Regional transient portal ischemia and irradiation as preparative regimen for hepatocyte transplantation.

      Koenig, S; Yuan, Q; Krause, P; Christiansen, H; Rave-Fraenk, M; Kafert-Kasting, S; Kriegbaum, H; Schneider, A; Ott, M; Meyburg, J; et al. (2011)
      Hepatocyte transplantation is regarded as a promising option to correct hereditary metabolic liver disease. This study describes a novel method involving regional transient portal ischemia (RTPI) in combination with hepatic irradiation (IR) as a preparative regimen for hepatocyte transplantation. The right lobules of rat livers (45% of liver mass) were subjected to RTPI of 30-120 min. Liver specimens and serum samples were analyzed for transaminase levels, DNA damage, apoptosis, and proliferation. Repopulation experiments involved livers of dipeptidylpeptidase IV (DPPIV)-deficient rats preconditioned with RTPI (60-90 min) either with or without prior partial hepatic IR (25 Gy). After reperfusion intervals of 1 and 24 h, 12 million wild-type (DPPIV positive) hepatocytes were transplanted into recipient livers via the spleen. RTPI of 60-90 min caused limited hepatic injury through necrosis and induced a distinct regenerative response in the host liver. Twelve weeks following transplantation, small clusters of donor hepatocytes were detected within the portal areas. Quantitative analysis revealed limited engraftment of 0.79% to 2.95%, whereas control animals (sham OP) exhibited 4.16% (determined as relative activity of DPPIV when compared to wild-type liver). Repopulation was significantly enhanced (21.43%) when IR was performed prior to RTPI, optimum preconditioning settings being 90 min of ischemia and 1 h of reperfusion before transplantation. We demonstrate that RTPI alone is disadvantageous to donor cell engraftment, whereas the combination of IR with RTPI comprises an effective preparative regimen for liver repopulation. The method described clearly has potential for clinical application.
    • Generation of healthy mice from gene-corrected disease-specific induced pluripotent stem cells.

      Wu, Guangming; Liu, Na; Rittelmeyer, Ina; Sharma, Amar Deep; Sgodda, Malte; Zaehres, Holm; Bleidissel, Martina; Greber, Boris; Gentile, Luca; Han, Dong Wook; et al. (2011-07)
      Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH⁻/⁻ mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH⁻/⁻-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH⁻/⁻ iPS cell lines, we aggregated FAH⁻/⁻-iPS cells with tetraploid embryos and obtained entirely FAH⁻/⁻-iPS cell-derived mice that were viable and exhibited the phenotype of the founding FAH⁻/⁻ mice. Then, we transduced FAH cDNA into the FAH⁻/⁻-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell-derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.
    • Induction of a mature hepatocyte phenotype in adult liver derived progenitor cells by ectopic expression of transcription factors.

      Iacob, Razvan; Rüdrich, Urda; Rothe, Michael; Kirsch, Sarah; Maasoumy, Benjamin; Narain, Nidhi; Verfaillie, Catherine M; Sancho-Bru, Pau; Iken, Marcus; Popescu, Irinel; et al. (2011-05)
      By ectopic expression of a distinct combination of transcription factors we aimed to induce a mature hepatocyte phenotype in an adult liver derived progenitor cell population (ALDPC).