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Title: The effect of hypoxia on the differentiation of dopaminergic neurons from human pluripotent stem cells
Author: Badger, J. L.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2013
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Parkinson’s disease (PD) is a debilitating neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Human pluripotent stem cells (hPSCs) are capable of generating any cell from the body, including dopaminergic neurons, so are an ideal source of material for a cell replacement therapy for PD. Hypoxia is a cell culture tool used to recapitulate the oxygen tension of the in vivo environment. Atmospheric oxygen is approximately 20 % O2, which is significantly greater than the in vivo environment of hPSCs and dopaminergic neurons. Studies in human foetal neural progenitor cells (NPCs) demonstrate that hypoxia increases the yield of dopaminergic neurons. Therefore, this thesis examines the ability of hypoxia to enhance the yield of dopaminergic neurons from hPSCs and hPSC-derived NPCs. The work of this thesis begins by validating the hypoxic (2 % O2) model and the use of mouse embryonic fibroblasts as a substrate for hPSCs, and then progresses to generate a scalable protocol for the production of dopaminergic neurons from hPSCs. The protocol uses a free floating method to generate a greater number of NPCs per cm2, with the basic protocol producing 16.67 % FOXA2/TH dual positive cells. Methods to increase the yield of the protocol through the use of hypoxia and lentiviral vectors are then assessed. The final chapter assesses how hypoxia might affect canonical Wnt signalling, which is heavily involved in neural development and differentiation. The key findings from this thesis are that an LMX1A-encoding lentivirus causes a reduction in FOXA2 in differentiated neurons, whilst canonical Wnt agonist CHIR99021 causes a decrease in FOXA2 and LMX1A in expanded and differentiated neurospheres, compromising the yield of dopaminergic neurons. However, hypoxia is able to improve the yield of FOXA2/TH dual positive neurons from normoxic mature neurospheres to 23.27 %, which is comparable to recently published monolayer differentiation techniques.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available