Investigation of neuronal dysfunction in Parkinson's disease using advanced physiological induced pluripotent stem cells
The study of Parkinson’s Disease (PD) pathogenesis has been greatly facilitated by the revolutionary induced pluripotent stem cells (iPSC) technology which permits modelling of PD using human neurons and with human-specific genetics. However, current iPSC-derived models are still largely dependent o...
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| Format: | Thesis |
| Language: | English |
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2022
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| _version_ | 1811140380597420032 |
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| author | Do, B |
| author2 | Wade-Martins, R |
| author_facet | Wade-Martins, R Do, B |
| author_sort | Do, B |
| collection | OXFORD |
| description | The study of Parkinson’s Disease (PD) pathogenesis has been greatly facilitated by the revolutionary induced pluripotent stem cells (iPSC) technology which permits modelling of PD using human neurons and with human-specific genetics. However, current iPSC-derived models are still largely dependent on adherent monoculture of a single cell type with limited capacity to capture the complex neuronal microenvironment and the resulting plethora of cell-cell interactions evident in vivo. Here, I described attempts at establishing three different strategies of advanced culturing of iPSC-derived neurons, namely i) the striatal presynaptic microcircuit consisting of iPSC-derived medium spiny neurons (MSN), cortical and dopaminergic neurons , ii) human midbrain-like organoids (hMLO) and iii) the fluid-walled microfluidic-based corticostriatal pathway. The striatal microcircuit recapitulated the directed connectivity of the presynaptic landscape of MSN wherein both cortical and dopaminergic axons converged on the MSN. Electrophysiological recordings of the MSN of the striatal presynaptic microcircuit delineated the pro-maturational roles of cortical and dopaminergic inputs on MSN physiology, as well as the pathological effect of DaN harbouring PD-related GBA N370S mutation on inducing early and transient immature and hyperexcitable-like phenotype of the connected healthy MSNs. On the other hand, characterisation of the hMLO suggested expression of DaN of floor-plate origin surrounded by diverse subsets of neuronal and neural cells. However, hMLO derived from PD patients carrying the triplication of SNCA or the N370S GBA mutation lacked phenotypic DaN degeneration or synucleinopathy. Lastly, I recreated the corticostriatal pathway on the novel fluid-walled microfluidic platform. This provided the proof-of-concept of the possibility to recapitulating any neuronal circuits of interest in an open-access fashion free of the obstructive barrier apparent in conventional microfluidic approach, rendering it suitable for direct physical manipulation such as directed axotomy. Live monitoring of axon growth post directed axotomy suggested the positive effect of permissive environment such as that induced by exogenously supplied brain-derived trophic factor (BDNF) or endogenous post-synaptic target (i.e. MSN) on cortical axon regeneration. Taken together, this work offers frameworks of novel advanced culture strategies that could be employed to improve neuronal maturation in in vitro modelling as well as to facilitate studies of network-dependent biological phenomena. |
| first_indexed | 2024-03-07T07:55:01Z |
| format | Thesis |
| id | oxford-uuid:c37fdb12-3875-4de7-9338-56b33e0c65ca |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-09-25T04:21:04Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | oxford-uuid:c37fdb12-3875-4de7-9338-56b33e0c65ca2024-08-05T09:14:41ZInvestigation of neuronal dysfunction in Parkinson's disease using advanced physiological induced pluripotent stem cellsThesishttp://purl.org/coar/resource_type/c_db06uuid:c37fdb12-3875-4de7-9338-56b33e0c65caPhysiologyneuroscienceEnglishHyrax Deposit2022Do, BWade-Martins, RBengoa-Vergniory, NMarquez Gomez, RMagill, PThe study of Parkinson’s Disease (PD) pathogenesis has been greatly facilitated by the revolutionary induced pluripotent stem cells (iPSC) technology which permits modelling of PD using human neurons and with human-specific genetics. However, current iPSC-derived models are still largely dependent on adherent monoculture of a single cell type with limited capacity to capture the complex neuronal microenvironment and the resulting plethora of cell-cell interactions evident in vivo. Here, I described attempts at establishing three different strategies of advanced culturing of iPSC-derived neurons, namely i) the striatal presynaptic microcircuit consisting of iPSC-derived medium spiny neurons (MSN), cortical and dopaminergic neurons , ii) human midbrain-like organoids (hMLO) and iii) the fluid-walled microfluidic-based corticostriatal pathway. The striatal microcircuit recapitulated the directed connectivity of the presynaptic landscape of MSN wherein both cortical and dopaminergic axons converged on the MSN. Electrophysiological recordings of the MSN of the striatal presynaptic microcircuit delineated the pro-maturational roles of cortical and dopaminergic inputs on MSN physiology, as well as the pathological effect of DaN harbouring PD-related GBA N370S mutation on inducing early and transient immature and hyperexcitable-like phenotype of the connected healthy MSNs. On the other hand, characterisation of the hMLO suggested expression of DaN of floor-plate origin surrounded by diverse subsets of neuronal and neural cells. However, hMLO derived from PD patients carrying the triplication of SNCA or the N370S GBA mutation lacked phenotypic DaN degeneration or synucleinopathy. Lastly, I recreated the corticostriatal pathway on the novel fluid-walled microfluidic platform. This provided the proof-of-concept of the possibility to recapitulating any neuronal circuits of interest in an open-access fashion free of the obstructive barrier apparent in conventional microfluidic approach, rendering it suitable for direct physical manipulation such as directed axotomy. Live monitoring of axon growth post directed axotomy suggested the positive effect of permissive environment such as that induced by exogenously supplied brain-derived trophic factor (BDNF) or endogenous post-synaptic target (i.e. MSN) on cortical axon regeneration. Taken together, this work offers frameworks of novel advanced culture strategies that could be employed to improve neuronal maturation in in vitro modelling as well as to facilitate studies of network-dependent biological phenomena. |
| spellingShingle | Physiology neuroscience Do, B Investigation of neuronal dysfunction in Parkinson's disease using advanced physiological induced pluripotent stem cells |
| title | Investigation of neuronal dysfunction in Parkinson's disease using advanced physiological induced pluripotent stem cells |
| title_full | Investigation of neuronal dysfunction in Parkinson's disease using advanced physiological induced pluripotent stem cells |
| title_fullStr | Investigation of neuronal dysfunction in Parkinson's disease using advanced physiological induced pluripotent stem cells |
| title_full_unstemmed | Investigation of neuronal dysfunction in Parkinson's disease using advanced physiological induced pluripotent stem cells |
| title_short | Investigation of neuronal dysfunction in Parkinson's disease using advanced physiological induced pluripotent stem cells |
| title_sort | investigation of neuronal dysfunction in parkinson s disease using advanced physiological induced pluripotent stem cells |
| topic | Physiology neuroscience |
| work_keys_str_mv | AT dob investigationofneuronaldysfunctioninparkinsonsdiseaseusingadvancedphysiologicalinducedpluripotentstemcells |