Enteric nervous system progenitor cells from aganglionic gut for Hirschsprung's therapies

Each year, approximately 170 babies in the UK are born with Hirschsprung's disease (HSCR). Affected babies develop an intestinal blockage, which can be life threatening, due to missing nerve cells in the lower part of their intestine (called 'aganglionic' intestine). Currently, the only treatment is surgical removal of the affected segment of the intestine. However, after surgery up to 75% of children with HSCR suffer from constipation or incontinence, and 10% of children require a permanent artificial opening of the intestine. Thus, there is a need to improve the treatment of babies and children with HSCR.

We have isolated stem cells from normal segments of intestine, cultured them in a sterile dish and have shown that these cells can become nerve cells which are able to regulate bowel activity under laboratory conditions. Surprisingly, we have recently demonstrated that similar stem cells can also be found in the aganglionic segments of intestine of children with HSCR, which lack nerve cells. This novel finding opens up exciting new questions for treatment options: Is it possible to transplant the patient's own stem cells, after converting them in to nerve cells in the laboratory? Can the stem cells, while still present in the aganglionic segments of intestine, be stimulated to become new nerve cells? These possibilities could improve surgical outcomes or avoid surgery altogether.

Based on these findings, the overarching aim of this project is to develop new approaches to treat patients with Hirschsprung's disease.

As our previous observations showed that the stem cells present in the aganglionic intestine also give rise to nerves similar to those from normal gut, our current work is focussed on establishing to what extent the stem cells from the aganglionic intestine are the same as those from normal gut. It is also important to understand whether the stem cells isolated from aganglionic intestine can be controlled to make sure that they do not form tumours after they have been stimulated. Therefore, the main focus of the proposed project is to determine the mechanisms which regulate the behaviour of these new stem cells. This information is necessary before we can consider any use of the stem cells in therapies for patients.

In this project we will isolate stem cells in the laboratory from tissue samples obtained at surgery from children with and without HSCR. Once the stem cells are isolated, they are cultured under laboratory conditions. The next step will be to assess whether the same mechanisms that regulate the behaviour of stem cells in normal bowel, also control the change of stem cells derived from aganglionic HSCR bowel in to functioning nerves in culture. Once we know what these mechanisms are, we will be able to use specific drugs to regulate the behaviour of the HSCR stem cells in order to convert them into the nerves necessary to restore normal function to the bowel.

The information gained from this project is an essential prerequisite for clinical trials to test the efficacy of the stem cells isolated from aganglionic bowel to be used in stem cell therapies.

Many children with Hirschsprung's disease face a lifetime of psychological, social and physical difficulties affecting them and their families. Improved outcomes from the proposed treatment have the potential to permanently change the lives of children and reduce the demands on health and social services.

The aim of this project is to determine mechanisms that regulate the behaviour of progenitor cells from the aganglionic gut of HSCR patients.

Objectives:
1.To determine the expression and role of key molecules of the Notch signalling pathway in aganglionic HSCR gut and in derived ENSPC.
2.Characterise and compare differentiation and proliferation responses to Notch, 5-HT and EDN3 signalling in ENSPC derived from aganglionic HSCR gut with those from ganglionic gut.
3.To analyse the effect of Notch, 5-HT and EDN3 signalling on restoring functionality in aganglionic gut explants ex vivo.

Methods:
-Collection and isolation of cells from human ganglionic and aganglionic colon samples during surgical procedures (HSCR patients and controls).
-Culture of ENSPCs as neurospheres in primary cultures.
-Lentiviral labelling of neurosphere cells for constitutive expression of GFP to enable cell tracking.
-Analysis of components of the Notch signalling pathway in ENSPC prior to isolation and during culture, by observation of expression and subcellular localization of Notch receptors and activation-signalling intermediary RBPkJ, using immunohistology with confocal microscopy.
-Assessment of the roles of:
(a)Notch signalling - using chemical inhibition (DAPT) and siRNA knockdown of RBPkJ.
(b)5-HT signalling - using pharmacological 5-HT4 receptor agonists, e.g. Mosapride citrate or Prucalopride and antagonists, e.g. SB-207266 or GR113808.
(c)EDNRB /EDN3 signalling - using EDN3 and pharmacological EDNRB antagonists e.g. BQ-788 or A192621.
-Assessment of neuronal differentiation and functionality in gut explants using inverted phase contrast microscopy to assess contractility and Diamtrak software to measure frequency and amplitude of contractions after transplantation of aganglionic gut derived neurospheres into cultured aganglionic embryonic bowel explants with or without prior manipulation of neuronal differentiation using the siRNA and pharmacological approaches.

Despite surgical advances, postoperative outcomes for children with Hirschsprung's disease remain poor. Approximately 10-30% of children suffer from long-term problems with constipation, enterocolitis or incontinence and 10% of children require a permanent colostomy. These outcomes have a significant impact on childrens' psychological, social and physical wellbeing including demands on their family and health and social services.

Children with HSCR would directly benefit from a novel therapeutic agent which could be used to induce neurogenesis in the aganglionic segment of bowel, particularly if it could be applied to the affected bowel topically. Alternatively, injection of autologous aganglionic progenitor cells or their derivates into the aganglionic internal anal sphincter, which remains in situ after surgery, may also improve the outcomes of these children. However, before considering the clinical applications of enteric nervous system progenitor cells or any therapeutic agent, information regarding the safety and efficacy of treatment is paramount. This project serves as a stepping stone toward potential novel future treatments by assessing the mechanisms which control aganglionic progenitor cells, in order to identify any potential therapeutic agents, and is an essential prerequisite to clinical trials.

We have strong links with the charity CHAMPS appeal (Curing Hirschsprung's and Making Positive Steps) who have supported our research at the University of Liverpool and are very interested in any scientific advancements arising from their financial support which may benefit all children and adults with HSCR. This research will not only advance our scientific knowledge regarding the aetiology of HSCR and behaviour of aganglionic progenitor cells, it also has the potential to permanently transform the lives of children with Hirschsprung's disease.

A recently published White Paper, authored by 30 members of the enteric nervous system (ENS) basic science and clinical field, sets out opinions on efforts to establish novel stem cell therapies for enteric neuropathies of the gastrointestinal tract (DOI:10.1016/j.ydbio.2016.04.001.). Based on the current literature and international experience, the paper includes clearly set out methods and approaches to identify, isolate, purify, expand and optimise enteric nervous system stem cells and progenitors, transplant them in to the bowel, and assess transplant success as a way to restore gut function. The aim of the White Paper is to define standard methodologies that can be adapted to provide the necessary safety, regulatory and good manufacturing practice protocols required for eventual clinical application and our future work will be in accordance with these proposals. Creating a forum for discussion and communication of results with these international groups is essential when considering animal models prior to human application of therapy in order to combine expertise.

Our previous research has been cited in the White Paper and our current research fits in to the remit of the development of novel stem cell based therapies for the treatment of enteric neuropathies. By focussing on the development of a therapeutic agent to induce neurogenesis in situ as an alternative to cell replacement therapy, our current research has the potential to overcome some of the challenges associated with autologous stem cell therapy, such as the optimal quantity and method of transplantation. Therefore, the results will likely have a significant impact on other international research groups involved in preclinical studies aimed at developing cell replacement therapies. Taking into account the quantity of stem cells required to repopulate the aganglionic segment including the process of stem cell isolation and culture, it is likely that a therapeutic agent would be more cost efficient as a therapy overall.