title: Primary Human Hepatocytes Repopulate Livers of Mice After In Vitro Culturing and Lentiviral-Mediated Gene Transfer.
abstract: Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo.
authors.affiliation: 1 Department for General, Visceral, Thoracic, and Vascular Surgery, University Medical Center Bonn , Bonn, Germany .
authors.forename: Jeanette
authors.initials: J
authors.lastname: Bierwolf
authors.affiliation: 2 Department of Internal Medicine, University Medical Center Hamburg-Eppendorf , Hamburg, Germany .
authors.forename: Tassilo
authors.initials: T
authors.lastname: Volz
authors.affiliation: 2 Department of Internal Medicine, University Medical Center Hamburg-Eppendorf , Hamburg, Germany .|3 Department of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf , Hamburg, Germany .
authors.forename: Marc
authors.initials: M
authors.lastname: Lütgehetmann
authors.affiliation: 2 Department of Internal Medicine, University Medical Center Hamburg-Eppendorf , Hamburg, Germany .
authors.forename: Lena
authors.initials: L
authors.lastname: Allweiss
authors.affiliation: 4 Department of Stem Cell Transplantation, Research Department Cell and Gene Therapy, University Medical Center Hamburg-Eppendorf , Hamburg, Germany .
authors.forename: Kristoffer
authors.initials: K
authors.lastname: Riecken
authors.affiliation: 2 Department of Internal Medicine, University Medical Center Hamburg-Eppendorf , Hamburg, Germany .
authors.forename: Michael
authors.initials: M
authors.lastname: Warlich
authors.affiliation: 4 Department of Stem Cell Transplantation, Research Department Cell and Gene Therapy, University Medical Center Hamburg-Eppendorf , Hamburg, Germany .
authors.forename: Boris
authors.initials: B
authors.lastname: Fehse
authors.affiliation: 1 Department for General, Visceral, Thoracic, and Vascular Surgery, University Medical Center Bonn , Bonn, Germany .
authors.forename: Joerg C
authors.initials: JC
authors.lastname: Kalff
authors.affiliation: 2 Department of Internal Medicine, University Medical Center Hamburg-Eppendorf , Hamburg, Germany .|5 German Center for Infection Research , Hamburg-Lübeck-Borstel Partner Site, Hamburg, Germany .
authors.forename: Maura
authors.initials: M
authors.lastname: Dandri
authors.affiliation: 1 Department for General, Visceral, Thoracic, and Vascular Surgery, University Medical Center Bonn , Bonn, Germany .
authors.forename: Joerg-Matthias
authors.initials: JM
authors.lastname: Pollok
mesh_terms: D000818:Animals; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism / Q000637:transplantation; D064593:Heterografts; D006801:Humans; D016086:Lentivirus; D008099:Liver / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D008822:Mice, Transgenic; D014161:Transduction, Genetic; D018183:Transplantation Chimera / Q000235:genetics / Q000378:metabolism*
chemical_list: D049452:Green Fluorescent Proteins