title: The source of enteric nervous System progenitor cells present in Aganglionic gut in Hirschsprung’s disease
Creators: name: Bethell, George
Publisher: name: University of Liverpool Repository
Year: 2015

description: The source of Enteric Nervous System Progenitor Cells present in Aganglionic Gut in Hirschsprung’s disease – George Stephen Bethell The enteric nervous system develops predominantly from vagal neural crest cells which proliferate and differentiate into enteric neurons and glia whilst migrating to the distal gut. Failure of this process for unknown reasons results in Hirschsprung’s disease (HSCR) which is characterised by an absence of enteric ganglia in a variable length of distal gut. This causes life threatening bowel obstruction and requires surgical intervention to remove the affected bowel. Following surgery 10-30% of patients suffer from long term constipation or faecal incontinence which can worsen with time. Our group has previously shown that enteric nervous system progenitor cells (ENSPC) can be isolated from ganglionic HSCR gut which then differentiate into enteric neurons and glia in vitro. Most importantly, these ENSPC form clusters of cells known as neurospheres which when implanted into ex vivo aganglionic mouse gut, restore normal patterns of contractility. More recently and quite surprisingly, ENSPC have also been obtained from aganglionic HSCR gut and behave similarly to ganglionic ENSPC in vitro. The aim of this thesis is to add understanding to this finding by determining the source of ENSPC in aganglionic HSCR gut. The first results chapter focuses on the optimum medium which should be used to culture neurospheres. It was found that a horse serum based medium promotes the formation of neurospheres under adherent conditions more effectively than a more complex medium containing various growth factors. The next section tests the hypothesis that ENSPC are located within thickened nerve trunks in aganglionic gut. Using samples of aganglionic HSCR gut from patients with all variants of HSCR it was possible to correlate the presence of thickened nerve trunks and the ability to obtain ENSPC as aganglionic gut from total colonic and total intestinal HSCR doesn’t contain thickened nerve trunks. It was found that it was not possible to obtain ENSPC from aganglionic HSCR gut where thickened nerve trunks were absent. This suggests that ENSPC are associated with the thickened nerve trunks in aganglionic gut. Subsequently, experiments were aimed at determining whether aganglionic ENSPC are of neural crest lineage. Fluorescence activated cell sorting was used to obtain a sub-population of P75 positive neural crest derived cells from freshly dissociated aganglionic HSCR gut. After 6 days in culture these cells differentiated into neurons whereas this was not the case in the P75 negative sub-population. Finally immunohistochemistry was used to look at the structure of thickened nerve trunks in aganglionic gut and the expression of P75 is correlated to the possible cellular sources of aganglionic ENSPC. When combined, work in this thesis narrows down the possibilities of the cellular origin of ENSPC of which the most likely source is cells of Schwann cell lineage located within thickened nerve trunks. These findings direct further work and make the potential of in vivo ENSPC stimulation and a medical treatment for HSCR more possible.