Investigating reprogramming factors and neural conditions to convert human dental pulp stem cells into neural stem cells
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(Thesis)
Date
2020
Authors
Gancheva, Maria Roumenova
Editors
Advisors
Koblar, Simon
Kremer, Karlea
Gronthos, Stan
Thomas, Paul
Kremer, Karlea
Gronthos, Stan
Thomas, Paul
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Thesis
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Abstract
Human dental pulp stem cells (DPSC), harvested from the pulp tissue of molar teeth, are a heterogeneous population of multipotent adult stem cells that possess neurogenic potential. They exert positive effects in the injured and diseased brain via multiple molecular and cellular mechanisms, and are potential candidates for cell-based therapies for treatment of neurological diseases, including stroke. Previous research of human DPSC in a stroke model demonstrated their ability to improve neurobehavioural function, which was mediated by paracrine effects but not cell replacement. In the adult brain, there are neurogenic niches in which reside neural stem cells (NSC). These NSC have a role in repairing the brain, however their capacity is limited under harsh conditions. Cellular reprogramming has been used to convert more accessible cell types, such as fibroblasts, into NSC for potential therapeutic use. Induced NSC have demonstrated positive outcomes in stroke models. DPSC may be a suitable cell type from which to obtain NSC, due to their neural crest origin. This may enhance their neurogenic capacity, including their ability to replace lost neural cells. The aim of this thesis was to investigate the ability to reprogram human DPSC into NSC. Multiple transcription factors were selected for reprogramming and over-expressed via lentiviral transduction. Cells were cultured in neural conditions, and analysed for expression of neural markers and stem cell properties (self-renewal and multi-lineage differentiation). RNA-sequencing was performed to investigate the transcriptome-wide effects of reprogramming, and used to examine differentially expressed genes. In vivo analysis was performed in a developmental avian model to examine the ability of the cells to respond to endogenous signals and differentiate. DPSC over-expressing the transcription factor OCT4 when cultured using a multi-step neural inductive protocol, underwent morphological changes, displaying a neural-like phenotype. They showed increased expression levels of neural markers in response to the environmental conditions and decreased levels of the mature neuronal marker in response to the transcription factor, suggesting that the cells were directed to an early neural state. The reprogrammed cells had a greater capacity for sphere formation, which indicated a greater number of stem cells present. They also displayed enhanced neuronal differentiation but not glial differentiation. Transcriptomic analysis showed significant differences to the controls, with an increased expression in genes associated with neural and neuronal function and components, and similarities with NSC. In vivo analysis showed the cells’ ability to survive in the developing central nervous system, localising to established endogenous axonal processes and differentiating into neuronal-like cells, as well as their capacity to induce neuroplasticity. These results suggest that DPSC may be reprogrammed along the neural lineage. Enhanced neural properties were observed in the OCT4 over-expressing, neural induced human DPSC, with data suggesting they resemble neuronal progenitor cells. Future research with these cells will aim to examine their response in a stroke model, to determine if they enhance functional recovery and whether cell replacement is a mechanism of action. An efficient and reliable reprogramming method could provide an alternative source of NSC for use in cell-based therapies for neurological diseases.
School/Discipline
Adelaide Medical School
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2021
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This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals