Wilkinson, David 
ORCID: 0000-0001-5165-8230
Origin,
behaviour
and
control
of
enteric
nervous
system
progenitor
cells
in
Hirschsprung’s
disease.
PhD thesis, University of Liverpool.
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Abstract
The enteric nervous system (ENS) arises mainly from cells exiting the vagal neural crest, entering the bowel and migrating caudally. A failure in this migratory process is thought to result in the clinical entity Hirschsprung’s disease (HSCR), which is typically characterised by the absence of enteric ganglia in the colon and rectum (aganglionosis). Untreated HSCR may present with life-‐threatening bowel obstruction in the first few days of life. However, despite recent surgical advances children and adults still suffer significant life-‐long post-‐ operative morbidity. ENS progenitor cells (ENSPC) have been shown to persist in the postnatal bowel, thereby stimulating research into the potential of manipulating or transplanting these cells to improve long-‐term outcomes. Early results have been promising, ENSPC have been isolated from children with HSCR and clonally expanded in cultured neurospheres, after which they have been transplanted into aganglionic embryonic mouse gut ex-‐vivo and shown to restore a normal pattern of contractility. However, questions still remain about the functional potential and the safety implications of transplanting these cells back into humans. Therefore, this thesis is aimed at improving our understanding of the properties of these cells and the mechanisms controlling their behaviour when they are brought into culture and after subsequent transplantation. The first part of this thesis focuses on the proliferation of ENSPC within the in-‐vitro environment of cultured mouse and human neurospheres. The main findings were that cells undergoing proliferation are predominantly found at the neurosphere periphery rather than being evenly distributed throughout the neurosphere. Subsequently, post-‐ mitotic peripheral cells were found to be distributed throughout the neurosphere. Furthermore, using embryonic and postnatal mouse tissue it was demonstrated that the proportion of cells undergoing of proliferation decreased with increasing maturity of the source tissue. The second part of the thesis identifies the requirement for Notch signalling pathway in the self-‐renewal of undifferentiated human ENSPC. Inhibition of the Notch signalling pathway with both chemical inhibitors and siRNA knockdown resulted in decreased ENSPC proliferation together with increased neuronal differentiation. Manipulation of this pathway may therefore improve both the effectiveness and safety of any future cell-‐based therapy. The next section of the thesis describes the development of an ex-‐vivo human colonic smooth muscle model to investigate the behaviour of ENSPC in an environment closer to that of the human colon. The chapter describes the development and maintenance of human colonic smooth muscle cells in long-‐term culture and demonstrates contractility, thus providing a model in which ENSPC behaviour can be investigated in a more physiologically relevant environment. During the routine culture of human aganglionic smooth muscle preliminary observations were made consistent with the presence of cells that could give rise to neurospheres, despite the absence of an ENS in this region of the bowel. The final part of this thesis confirms the presence of ENSPC within the aganglionic bowel and characterises their behaviour. These progenitors are demonstrated to differentiate into mature ENS specific neuronal phenotypes, and importantly have the capability to restore a normal pattern of contractility in the embryonic aganglionic gut model. Taken together the work in this thesis furthers our understanding of the control and behaviour of human ENSPC. Furthermore, demonstration of the existence of ENSPC in the bowel of children with HSCR raises important questions regarding both the aetiology HSCR and the potential to utilise the cells in future autologous cell-‐based therapies.
| Item Type: | Thesis (PhD) |
|---|---|
| Additional Information: | Date: 2014-06 (completed) |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 26 Mar 2015 15:38 |
| Last Modified: | 17 Dec 2022 01:07 |
| DOI: | 10.17638/02009128 |
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/2009128 |
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