Assessing Human Embryonic Stem Cell-Derived Dopaminergic Neuron Progenitor Transplants Using Non-invasive Imaging Techniques



Mousavinejad, Masoumeh
(2021) Assessing Human Embryonic Stem Cell-Derived Dopaminergic Neuron Progenitor Transplants Using Non-invasive Imaging Techniques. PhD thesis, University of Liverpool.

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Abstract

Parkinson’s disease (PD) is a neurodegenerative disease that results, in part, from the progressive loss of dopaminergic (DA) neurons in the Substantia Nigra pars compacta (SNpc). Several groups have shown that human Pluripotent Stem Cell (hPSC)- derived dopaminergic neuron Progenitor Cells (DAPCs) can generate mature DA neurons and improve motor function following intrastriatal transplantation in animal models of PD. This has now evolved to the point that the first in human hPSC-based DA neural transplants are being undertaken or are being planned in patients with PD. However, prior to undertaking larger-scale clinical studies, animal experiments are needed to adequately assess the safety of the therapies. Key safety concerns with such therapies for PD and other Central Nervous System (CNS) disorders include the risk that the implanted cells could proliferate and form space-occupying masses and/or migrate to off-target sites within the CNS and/or induce major neuroinflammation. In addition to considering the potential risks, it is also important to monitor the long-term viability and differentiation capacity of implanted cells, as to be effective, they must differentiate into the appropriate phenotype and persist in the brain. Monitoring viability and biodistribution of the DAPCs over time requires non-invasive imaging techniques. In this project, a bimodal imaging strategy based on Bioluminescence (BLI) and Magnetic Resonance Imaging (MRI) to monitor the safety of the human Embryonic Stem Cell (hESC)-based therapy in immunocompromised nude rats has been investigated. BLI is the preferred technique for monitoring viability and proliferation in vivo, but spatial resolution is poor, meaning that it cannot be used to assess intracranial biodistribution. However, this can be overcome by labelling cells with Iron Oxide Particles (IOPs) so that they can be imaged using MRI, a technique that provides very high spatial resolution. RC17 hESCs were transduced with bicistronic Luciferase-ZsGreen lentiviral particles and directed to differentiate to DAPCs. Expression of DAPC markers was assessed to confirm the success of the differentiation. Furthermore, a group of RC17 hESCs were differentiated into DAPCs and labelled using Micron-sized Particles of Iron Oxide (MPIOs) to be visualized using MRI. DAPCs expressing Luciferase-ZsGreen or labelled with MPIOs were transplanted in the striatum of nude rats (n = 6 per group). DAPCs were tracked in vivo using BLI and MR imaging modalities. Transgene silencing in differentiating DAPCs accompanied with signal attenuation due to animal growth, rendered the BLI undetectable by week 2 post intrastriatal transplantation. However, MR imaging of MPIO-labelled DAPCs showed that transplanted cells remained at the site of injection for over 120 days. Post-mortem histological analysis of DAPC transplants demonstrated that labelling with either Luciferase-ZsGreen or MPIOs did not affect the ability of cells to differentiate into mature dopaminergic neurons. Importantly, labelled cells did not elicit increased glial reactivity compared to non-labelled cells.

Item Type: Thesis (PhD)
Divisions: Faculty of Health and Life Sciences
Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 09 Sep 2021 15:35
Last Modified: 11 Feb 2022 08:13
DOI: 10.17638/03131530
Supervisors:
  • Murray, Patricia
  • Poptani, Harish
  • Plagge, Antonius
URI: https://livrepository.liverpool.ac.uk/id/eprint/3131530