Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.806784
Title: Integration of imaging to enable iPSC-derived hepatocyte tracking in vivo
Author: Ashmore-Harris, Candice
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2020
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Abstract:
Primary hepatocyte transplantation (HTx) has been demonstrated as a safe cell therapy for liver disease patients which can act as an alternative or bridging therapy to orthotopic liver transplant (OLT), however wider application is circumvented by poor cell engraftment due to limitations in hepatocyte quality and transplantation strategies. Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (hiPSC) are considered a promising alternative cell therapy but also require optimisation of transplantation conditions and comprehensive safety assessments. Whole-body in vivo imaging would be a highly beneficial means to assess transplanted HLC engraftment non-invasively allowing both short and long-term monitoring of cell survival and biodistribution. To realistically achieve long-term monitoring, a reporter gene-based cell tracking approach matched with highly sensitive 3D tomography is required. The human sodium iodide symporter (hNIS) can serve as such a reporter and can utilise radiotracers already available for clinical use to enable positron emission tomography (PET) or single photon emission computed tomography (SPECT)-afforded whole body in vivo cell tracking. Radionuclide-fluorescence dual-mode reporters have previously been shown to streamline preclinical research through facilitating traceable cell generation as well as downstream ex vivo validation. In this thesis two distinct approaches were explored to yield expression of the dual-mode radionuclide-fluorescence hNIS-mGFP reporter in HLCs. Firstly, a lentiviral transduction approach was employed to engineer hiPSC-derived HLCs during differentiation. This strategy resulted in the successful production of NIS-expressing HLCs in two hiPSC lines, as well as in primary foetal hepatocyte controls. These were functionally characterised in vitro relative to wild-type HLCs to assess reporter impact on the hepatic phenotype downstream. NIS-expressing HLCs were transplanted intrahepatically, either pre-labelled with a radiotracer in vitro or unlabelled, and radionuclide tomography (SPECT/CT) was used to track their fate over time in mice. Retention of their hepatic phenotype in vivo was assessed by histology and serum albumin ELISA. A second approach used direct gene editing of hiPSCs with transcription activator-like effector nucleases (TALEN) to incorporate the hNIS-mGFP reporter into the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbour locus of a cGMP compliant hiPSC line, so that all subsequent progeny expressed NIS. NIS-expressing hiPSCs, hepatic progenitor cells and mature HLCs were phenotyped to assess the impact of the reporter on pluripotency and differentiation capacity. Retention of reporter expression was also confirmed throughout differentiation. Subsequently, NIS-hiPSCs were used to produce hNIS-mGFP+ liver bud organoids by differentiation and co-culture of hepatic endoderm, endothelial and septum transverse mesenchyme cells. Liver buds were transplanted into healthy and liver injured mice, and tracked using PET. These studies are the first to explore the capacity of NIS-afforded tracking of hiPSC-derived HLCs in vivo and highlight the potential for this tracking strategy to answer wider questions in the HLC field, including their use as a tool to robustly assess whether HLCs could act as an alternative to HTx.
Supervisor: Fruhwirth, Gilbert Otto Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.806784  DOI: Not available
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