Primary Human Hepatocytes Repopulate Livers of Mice AfterIn VitroCulturing 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.

Multi-color RGB marking enables clonality assessment of liver tumors in a murine xenograft model

“…To visualize RGB-marked cells, we performed fluorescence microscopy of liver sections and generated overlays for the three basic colors red, green and blue detected in three different channels as described [ 14 ]. Immunohistochemistry was carried out using the following primary antibodies for 1h incubation: anti-green fluorescent protein (GFP) (1:300; Invitrogen, Karlsruhe, Germany) [ 50 ], anti-human cytokeratin 18 (1:100; Antibodies-Online, Aachen, Germany) [ 51 ], and anti-human albumin (1:100; DAKO, Hamburg, Germany). Visualization was performed with DAKO REAL EnVision Kit (DAKO) according to the manufacturer’s instruction.…”

Strategies to capitalize on cell spheroid therapeutic potential for tissue repair and disease modeling

“…Though liver microarchitecture is highly complex, primary hepatocytes formed into spheroids will spontaneously organize into functional liver architecture 102 – 104 . This is of particular interest for pharmacology and toxicology in vitro studies, but there are clear regenerative applications.…”

“…This is of particular interest for pharmacology and toxicology in vitro studies, but there are clear regenerative applications. Following intrasplenic injection, genetically transduced human hepatocyte spheroids were successfully engrafted in uPA/SCID mouse livers, a primary model of viral hepatitis 104 . When delivered intraportally, ASC spheroids localized in the liver, increased regeneration in a liver failure model and added an immunomodulatory effect through secretions of PGE 2 105 .…”

Novel vectors and approaches for gene therapy in liver diseases

“…Transduction efficiency has been improved with the use of lentiviral vectors achieving in vitro transduction levels close to 90% of the cells. 120,121 Hepatocyte transplantation, however, remains relatively inefficient and variable, likely due to poor engraftment and limited persistence of engrafted hepatocytes if there is not a proliferative advantage. Recently, liver-directed ex vivo gene therapy using a lentiviral vector to integrate a corrected Fah gene was shown to cure liver disease in a pig model of HT1 thanks to the selective advantage of modified hepatocytes.…”