title: Isolation, characterisation and transplantation of human enteric nervous system stem cells
Creators: name: Lindley, Richard
Publisher: name: University of Liverpool Repository
Year: 2012

description: Hirschsprung’s disease causes a life-threatening bowel obstruction in children shortly after birth, because the stem cells that will form the enteric nervous system (ENS) have failed to colonise the entire length of the gut, creating an absence of neuronal cell bodies – the aganglionic region. Although there are numerous surgical techniques to deal with this problem, many children go on to have severe problems with constipation and faecal incontinence, and some require a permanent colostomy. It was hypothesised that clinical outcomes in children with Hirschsprung’s disease could be improved by transplanting ENS stem cells into the defective bowel, as previous studies has already shown it was possible to produce such stem cells from embryonic bowel . The aim of this thesis was to develop a method of isolating such cells from the bowel of children with and without Hirschsprung’s disease, characterise these cells and investigate whether they could be successfully transplanted in an in vitro model. In the first part of this thesis, a method was developed to take samples of postnatal bowel (both small and large) and dissociate it to produce a cell suspension that will allow ENS stem (or progenitor) cells to grow in the form of neurospheres. These neurospheres are characterised in terms of growth and composition, and it is shown that they contain reproducing cells that are capable of forming neurons on subsequent differentiation. When the neurospheres are transplanted into aganglionic embryonic mouse bowel grown in tissue culture, neurons and glia from the neurosphere colonise the bowel wall. In the second part of this thesis, the neurospheres were further characterised in terms of their response to the ENS growth factors glial cell derived neurotrophic factor (GDNF) and endothelin 3 (EDN3) and this was compared to the response of neurospheres derived from embryonic and neonatal mice. It was shown that postnatal human ENS neurospheres require the addition of GDNF to produce significant neurite outgrowth. Subsequent studies then demonstrated that the human neurospheres could be dissociated and regrown in culture, exponentially increasing their numbers whilst retaining the ability to produce neurons and glia on transplantation into aganglionic embryonic hindgut. In the final part of this thesis the functional effect of neurosphere transplantation into aganglionic embryonic hindgut was investigated. Firstly it was shown that the transplanted neurons integrate closely with the recipient smooth muscle and gut pacemaker cells (the interstitial cells of Cajal). Secondly it was shown that aganglionic embryonic mouse hindgut has a higher rate of contraction than ganglionic bowel cultured in the same conditions, and that human and mouse ENS neurosphere transplantation restores the contraction frequency of aganglionic bowel to that of ganglionic bowel. Taken together the results presented in this thesis demonstrate that it is possible to generate neurospheres from postnatal human bowel samples that contain ENS stem cells which are capable of producing a restorative functional effect when transplanted into an in vitro model of Hirschsprung’s disease. This is a step toward the development of a successful stem cell treatment for this condition.