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Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

Brunel University London

Gene delivery by integrating vectors has the potential to cause genotoxicity in the host by insertional mutagenesis (IM). Previously, the risk of IM by replication incompetent retroviral vectors was believed to be small. However, the recent observation of leukaemic events due to gamma retroviral vector insertion and activation of the LMO-2 proto-oncogene in patients enrolled in the French and British gene therapy trials for X-SCID demonstrates the need to understand vector associated genotoxicity in greater detail. These findings have led to the development of in vitro, ex vivo, and in vivo assays designed to predict genotoxic risk and to further our mechanistic understanding of this process at the molecular level. In vitro assays include transformation of murine haematopoietic stem cells by integrating retroviral (RV) or lentiviral (LV) vectors and measurement of cell survival resulting from transformation due to integration mainly into the Evi1 oncogene. Ex vivo assays involve harvesting haematopoietic stem cells from mice followed by gene transfer and re-infusion of RV or LV infected cells to reconstitute the immune system. Insertional mutagenesis is then determined by analysis of clonally dominant populations of cells. The latter model has also been made highly sensitive using cells from mice predisposed to oncogenesis by lack of the P53 and Rb pathways. Our investigations on fetal gene therapy discovered a high incidence of liver tumour development that appears to be associated with vector insertions into cancer-related genes. Many genes involved in growth and differentiation are actively transcribed in early developmental and are therefore in an open chromatin configuration, which favours provirus insertion. Some of these genes are known oncogenes or anti-oncogenes and are not usually active during adulthood. We found that in utero injection of primate HIV-1, HR'SIN-cPPT-S-FIX-W does not result in oncogenesis as opposed to administration of non-primate equine infectious anaemia virus (EIAV), SMART 2 lentivirus vectors and, most recently, the non-primate pLIONhAATGFP (FIV) vector, which both give rise to high frequency hepatocellular carcinoma. The peculiar integration pattern into cancer-related genes observed in this model makes the fetal mouse a sensitive tool, not only to investigate long-term vector-mediated gene expression, but also vector safety in an in vivo system with minimal immunological interference. The identification of distinct differences in genotoxic outcome between the applied vector systems i.e. EIAV or FIV vectors versus HIV may indicate a particular biosafety profile of the HIV-1-based vector, which renders it potentially suitable for safe prenatal gene therapy.

Monitoring for potential adverse effects of prenatal gene therapy: genotoxicity analysis in vitro and on small animal models ex vivo and in vivo.

Recent advances in the isolation of tissue-resident adult stem cells and the identification of inductive factors that efficiently direct differentiation of human pluripotent stem cells along specific lineages have facilitated the development of high-fidelity modelling of several tissues  Many of the novel approaches have employed self-organizing three-dimensional (3D) culturing of organoids, which offer several advantages over conventional two-dimensional platforms. Organoid technologies hold great promise for modelling diseases and predicting the outcome of drug responses  Here, we outline the historical background and some of the recent advances in the field of three-dimensional organoids. We also highlight some of the current limitations of these systems and discuss potential avenues to further benefit biological research using three-dimensional modelling technologies.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Three-dimensional cell culture: from evolution to revolution.

Retroviral and lentiviral vectors often use the envelope G protein from the vesicular stomatitis virus Indiana strain (VSVind.G). However, lentivector producer cell lines that stably express VSVind.G have not been reported, presumably because of its cytotoxicity, preventing simple scale-up of vector production. Interestingly, we showed that VSVind.G and other vesiculovirus G from the VSV New Jersey strain (VSVnj), Cocal virus (COCV), and Piry virus (PIRYV) could be constitutively expressed and supported lentivector production for up to 10 weeks. All G-enveloped particles were robust, allowing concentration and freeze-thawing. COCV.G and PIRYV.G were resistant to complement inactivation, and, using chimeras between VSVind.G and COCV.G, the determinant for complement inactivation of VSVind.G was mapped to amino acid residues 136-370. Clonal packaging cell lines using COCV.G could be generated; however, during attempts to establish LV producer cells, vector superinfection was observed following the introduction of a lentivector genome. This could be prevented by culturing the cells with the antiviral drug nevirapine. As an alternative countermeasure, we demonstrated that functional lentivectors could be reconstituted by admixing supernatant from stable cells producing unenveloped virus with supernatant containing envelopes harvested from cells stably expressing VSVind.G, COCV.G, or PIRYV.G.

Michael Themis

Division of Biosciences,
Department of Life Sciences,
College of Health and Life Sciences,
Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences

Brunel University London

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

Michael Themis

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Biosciences,Department of Life Sciences,College of Health and Life Sciences,Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences

Brunel University London

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

Division of Biosciences,
Department of Life Sciences,
College of Health and Life Sciences,
Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences