Investigating the Interaction of the Enteric Nervous System With the Extrinsic Nervous System and the Immune System in Children and Development of Cell Therapies for Hirschsprung's Disease
Conditions: Hirschsprung Disease
Sponsor: Alder Hey Children's NHS Foundation Trust

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Investigating the interaction of the enteric nervous system with the extrinsic 
nervous system and the immune system in children and development of cell 
therapies for Hirschsprung’s Disease 
HAWAII 
Hirschsprung’s Advances; Working towards Autologous tIssue therapIes 
 
 
 
1. Investigators 
Chief Investigator: 
Miss Rachel Harwood 
Department of Paediatric Surgery 
Alder Hey Children’s NHS Foundation Trust 
East Prescot Road 
Liverpool 
 
Co-investigators: 
Professor Simon Kenny 
Miss Sarah Almond 
Dr Bettina Wilm 
Dr Megan Smith 
 
 
 

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2. Background 
The gastrointestinal (GI) tract is responsible for many functions essential to life such as digestion, 
absorption and excretion, all of which are under the control of its own intrinsic nervous system, the 
enteric nervous system (ENS) (1). The ENS is part of the autonomic peripheral nervous system and 
provides the intrinsic innervation of the bowel - the submucosal and myenteric plexuses are the two 
networks that comprise the ENS and consist of bundles of enteric neurons and glia – the enteric 
ganglia that are connected by nerve fibres. The ENS is essential for the autonomic regulation of 
gastric motility and secretion (2). 
Hirschsprung’s disease (HSCR) is a gut motility disorder, affecting 1 in 5000 people worldwide (3). HSCR 
is characterised by the absence of ganglia and nerve fibres of the ENS to various extents of the bowel: 
80% of cases affect the distal colon and internal anal sphincter, but 10% of cases have total colonic 
aganglionosis and 1% total intestinal aganglionosis (4). Between the ganglionic and aganglionic bowel 
there is a transition zone. The transition zone is abnormally innervated due to a reduced number of 
enteric ganglia (3). 
Initial presentation of neonates with HSCR typically includes distal intestinal obstruction, with 
symptoms including abdominal distention or vomiting and/or failure to pass meconium within the first 
48hrs of life.  Children with HSCR can also have difficulty feeding, severe constipation, and symptoms 
of HSCR- associated enterocolitis (HAEC) (5, 6).  
Due to its lack of ENS, the aganglionic region of HSCR bowel is tonically contracted (3). This results in 
constipation and without treatment is fatal. The current gold-standard treatment for HSCR is a surgical 
pull-through procedure in which the aganglionic region and TZ is resected in an attempt to restore 
normal function.  
Our group has previously demonstrated the presence of enteric nervous system progenitor cells 
(ENSPCs) in the aganglionic bowel of children with HSCR. ENSPCs have both a multipotent nature and 
proliferative capacity (7). There has been a significant amount of research into the feasibility of using 
ENSPCs to reduce or correct bowel dysmotility in mouse and rat models of ENS neuropathies (8-10). 
ENSPCs have commonly been cultured as neurospheres (9, 11-19). Neurospheres are free-floating 
cultures of neural stem cells that also contain neurons and glia and in the case of enteric neurospheres, 
ENSPCs (11, 12, 15-17). 
ENSPCs cultured in neurospheres have the potential to be used as an autologous therapy for HSCR 
which would avoid the risk of immune rejection and can be isolated from embryonic and postnatal 
mouse, rat and human tissue (9, 11, 15, 17, 20). Enteric neurospheres have been shown to contain 
cells expressing various progenitor, neuronal and glial markers.  In our lab, Almond et al. (11), 
demonstrated that both embryonic mouse caecum derived neurospheres and postnatal human 
colonic neurospheres expressed p75, Tuj1, S100, the proliferation marker BrdU and the neuronal 
subtype markers NOS and VIP showing that neurosphere cells were capable of proliferation and 
differentiation. This work was further explored in our group by Lindley and Wilkinson (15, 17, 20) and 
neurospheres were cultured from tissue from the aganglionic bowel of patients with HSCR. This 
demonstration(17), indicates the potential for autologous therapies using the patients’ own 
aganglionic bowel due to the presence of progenitor cells that were capable of differentiation into 
different neuronal subtypes in vitro. 
The transplantation of enteric neurospheres to aganglionic bowel has provided insight into the ability 
of such cells to rescue a model with a HSCR phenotype (9, 11, 15, 17). Using ex vivo models of 

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aganglionic tissue, multiple groups have demonstrated that transplanted neurospheres can integrate 
into tissue and differentiate into enteric glia and electrophysiologically active neurons (19). 
Recently, we have found that there is an interaction in the transition zone of children with HSCR 
between the intrinsic ENS and the extrinsic nerves of the aganglionic bowel (21). As the transition zone 
is also resected during surgery for HSCR, this could provide a source of ENSPCs to use for autologous 
transplantation. However, transition zone neurospheres have not yet been characterised. We plan to 
include transition zone neurospheres in our investigation to determine whether these cells may be 
useful for transplantation. This forms part of our next set of experiments to investigate future 
therapies for HSCR. 
The clinical outcomes of children with Hirschsprung’s Disease have been poorly reported. Recently a 
core outcome set has been developed to help to standardise how children’s outcomes are reported 
and to encourage reporting of patient’s experiences as well as clinician’s experiences. After pull-
through 30% of patients suffering constipation or incontinence, with clear ‘constipation’ and ‘rapid 
transit’ phenotypes being evident (22). This may be caused by abnormal motility of the ganglionic 
region or by the fact that the aganglionic internal anal sphincter remains after surgery (22). Future 
therapies for HSCR aim to reduce long term morbidities associated with HSCR (23) but a key part of 
this includes drawing together children’s lived experience with the findings within the laboratory. This 
group is currently undertaking a study of the long-term outcomes of children and adults with 
Hirschsprung’s Disease (REC: 18/NNW/0608) and this study offers an opportunity to combine clinician 
and patient reported data with immunohistochemical data. 
3. Research question 
Can enteric nervous system neural progenitor cells (ENSPCs) isolated from the bowel of children with 
and without Hirschsprung’s disease be modulated in vivo or transplanted as mature neural cells to 
restore function to the abnormal diseased bowel? 
4. Aims 
The aims of this study are to answer the following questions: 
1. How are the nerve progenitor cells different in the different sections of the bowel of children with 
Hirschsprung’s Disease (ganglionic, aganglionic and transition zone) and do these differ to the nerve 
progenitor cells of children without Hirschsprung’s Disease? 
2. Do neurospheres cultured from different parts of the bowel of children with Hirschsprung’s Disease 
have the same composition of cells within them? 
3. Can the differentiation potential of the progenitor cells within neurospheres be manipulated via 
signalling pathways such as NOTCH, serotonin, endothelin 3 and Sonic Hedgehog. 
4. Can cultured neurospheres from ganglionic, transition zone and aganglionic bowel integrate and 
persist in aganglionic bowel? 
5. Do cultured neurospheres from ganglionic, transition zone and aganglionic bowel restore function 
to aganglionic bowel? 
6. Are there differences in the immune profile of the ganglionic, transition and aganglionic sections of 
the bowel of children with Hirschsprung’s Disease 
7. Does ganglionic bowel of children with Hirschsprung’s Disease have different properties which 
relate to their clinical experience of living with the disease? 

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5. Rationale and significance 
The current gold-standard treatment for HSCR is to surgically remove the affected bowel to restore 
normal bowel function and enable passage of stool. However, this often has unsatisfactory outcomes: 
after surgery ~30% of children have constipation, incontinence or recurrent episodes of enterocolitis 
and 10% will need a permanent stoma (6). These outcomes are associated with psychological 
difficulties and significantly impact the quality of life of children and their families. Thus, there is an 
urgent need to improve the treatment of babies and children with HSCR. 
The proposed work is a logical continuation of our previous studies (Research Ethics Committee 
(04/09/RE “Isolation of Human Enteric Nervous System Stem Cells” and 10/69/RE “Enteric nervous 
system stem cell behaviour in mature muscle models”).  We have previously demonstrated that we 
can isolate and culture human ENSPCs and that these cells can be transplanted into aganglionic bowel 
to restore contractility (17). However, there are still outstanding questions on how these cells can be 
optimised for successful transplantation before this work can be translated to clinical trials: 
1) What are the characteristics of the ENSPCs that can be transplanted and go on to proliferate 
and differentiate.   
2) Safety: Do all these cells differentiate? Are the stem cells adequately controlled within 
microenvironmental niches that prevent uncontrolled growth? 
3) What is the ideal method of transplantation? This may be whole neurospheres or single cells, 
with and without pharmacological agents to aid the differentiation of the ENSPCs. 
4) Can the progenitor cells be modulated in vivo to avoid transplantation using medication? 
 
This project is designed to begin to answer these questions using established protocols from our lab 
as well as incorporating new approaches such as single cell transcriptomics. Firstly, we aim to compare 
the progenitor populations in ganglionic, transition zone and aganglionosis bowel from children with 
HSCR as well as control ganglionic bowel. We will do this by examining full thickness bowel tissue 
sections and neurospheres with histology and single cell transcriptomic analysis. While recent 
publications have used single cell analysis of neuronal and glial cell populations (24, 25), it has not 
been carried out in progenitor populations or in HSCR samples.  
Secondly, we will identify and target signalling pathways such as NOTCH, serotonin and sonic 
hedgehog to try and improve the potential of progenitor cells in neurospheres to differentiate when 
transplanted. Finally, we will optimise the method used for transplantation of neurospheres using 
explants of aganglionic bowel to see if we can use transplanted cells to restore function. This may 
involve transplanting dissociated or intact neurospheres with and without signalling pathway 
manipulation. 
The lived experience of children with Hirschsprung’s Disease is very variable, even when the length of 
bowel affected by Hirschsprung’s Disease is the same(26). This heterogeneity may be due to the effect 
of differing genetic backgrounds or differences in the development of the ENS in the ganglionic part 
of bowel children with HSCR. These differences may reflect differences in the progenitor population 
and could affect the success of transplantation. Investigation of whether there are visible differences 
is a key step towards understanding why children have different experiences and will help to 
understand the translational impact of this work. 
In this project we build on many years of HSCR research undertaken by our team. Our long-term goal 
is to use the patients’ own neural stem/progenitor cell population to innervate the aganglionic bowel 
of infants with HSCR, thus avoiding the need for life-changing surgery and improving outcomes. To 
reach the point of clinical translation, a robust understanding of the developmental origin and the 

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properties of neural progenitor cells is essential. We have shown that neurospheres derived from 
bowel cell cultures contain ENSPCs that could be used as therapies (17). Therefore, it is necessary to 
identify any therapeutic cell population within HSCR bowel and subsequently neurospheres, and to 
determine the impact of external signalling pathways on the cultured cells. Furthermore, 
understanding the molecular mechanisms that control proliferation is a prerequisite for safe 
progenitor cell therapies. Linking these findings with clinical investigations, including whole genome 
sequencing, and outcomes is a key part in determining the potential of autologous therapies for 
translation to clinical practice. 
6. Research design 
This will be a prospective study. 
7. Demographics 
Infants and children undergoing intestinal resection or stoma formation.  Samples will be taken from 
bowel resected for clinical necessity.  No additional tissue will be removed. 
8. Research methods 
Inclusion criteria 
Any child undergoing therapeutic intestinal resection. Specifically, patients in which it is not necessary 
for all resected tissue to be sent for histopathological analysis. No additional tissue will be removed 
for research purposes. 
Exclusion criteria  
None 
Handling of human tissue samples  
Human tissue samples will be stored at the Institute in the Park, Alder Hey Children’s Hospital, 
Liverpool and Stem Cell laboratory, Department of Molecular Physiology and Cell Signalling, Nuffield 
Wing, Crown Street, University of Liverpool.  Samples will be stored and disposed of according to the 
regulations set down in the Human Tissue Act (HTA) and under the remit of the University of Liverpool 
HTA governance officers. 
Of the following investigations of the tissue – not every tissue will undergo each evaluation but a 
selection of investigations will be used depending on the study question being answered. 
Isolation of ENSC and culture of neurospheres. 
The sample of bowel will be mechanically and chemically disrupted in order to produce a dissociated 
cell suspension which is then cultured to promote the formation of neurospheres (aggregates of stem 
cells, neurons and glia) using optimised cell culture and culture media. This will be performed as per 
our previous studies(11, 15, 17). Neurospheres will be used for subsequent analysis using histology 
and other techniques described below. 
Histology 
As previously demonstrated, we will analyse the characteristics of the bowel tissue by looking at key 
markers from tissue sections and neurosphere sections. Using established techniques tissue and 
neurospheres will be stained using antibodies for immunofluorescence and immunohistochemistry 
staining such as Haematoxylin and Eosin (17, 21, 27). Such antibodies used may include those to 

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identify proliferating cells, neuronal and glial cells and progenitor cells. The modified thymidine 
analogue EdU and apoptosis assays will be used to assess proliferation and apoptosis within 
neurospheres in culture. This will allow us to match progenitor status with proliferative behaviour and 
identify aberrant responses. The stained tissue and neurospheres will be imaged using various 
microscopy techniques including but not limited to confocal and light microscopy. 
Single cell analysis 
Following dissociation of tissue to single cells from patient tissue we would prepare for a recovery of 
10,000 cells and 20,000 reads per cell. The samples will be run on a NovaSeq 6000 using an S2 flow 
cell for an estimated output of 3.3 billion reads, at the Centre for Genomic Research (CGR, University 
of Liverpool). We will use the Chromium X fixed RNA workflow which allows collection and storage of 
samples, followed by library preparation and sequencing of all collected samples in one day. The single 
cell whole-transcriptome sequencing data will be analysed using the computing program R and cells 
will be clustered according to their principle components using cell cluster analysis 
software/programmes CellRanger and Seurat. Neuronal progenitor cells will be identified and 
transcription factors will be interrogated for significant differences using Ingenuity Pathway Analysis. 
Specifically, we will be looking at the molecular profiles of individual cells to discriminate between 
different types of cells.  
qPCR analysis 
RNA will be extracted from tissue preparations encompassing the myenteric plexus from patient tissue 
samples to confirm expression of significantly upregulated markers using quantitative RT-PCR (qRT-
PCR). Specimens will be analysed for the presence and quantity of markers such as SOX10, p75, Tuj, 
GFAP, calretinin and GLUT1 using established primers but also those identified in scRNA-Seq; β -actin 
will be used as a reference gene and each measurement will be performed in triplicate. This analysis 
will confirm any novel transcript expression detected by scRNA-Seq and differences in expression 
levels between bowel samples. To include spatial analysis of significant markers we will perform the 
in situ hybridisation technique RNAscope. 
Cell sorting 
Tissue preparations encompassing the myenteric plexus will be dissociated described and single cell 
solutions labelled for flow cytometry using standardised methods established within our laboratory 
on a FACSCanto II flow cytometers within the University of Liverpool Flow cytometry facility. We will 
quantify cells expressing neuronal, glial and progenitor cell markers, including those assessed by qRT-
PCR as well as novel markers determined by scRNA-Seq. Our analysis will provide quantification of 
numbers of cell types and relative expression levels by signal intensity. 
Stimulation of key signalling pathways in neurospheres 
The role of the following key signalling pathways will be assessed for their capacity to modulate 
progenitor cells within cultured neurospheres: 
a. Notch signalling – using chemical inhibition (DAPT) and siRNA knockdown of RBPkJ 
b. Serotonin signalling – using pharmacological serotonin receptor agonists and antagonists 
c. Sonic hedgehog signalling – pharmacological inhibition of the sonic hedgehog pathway 
d. Pathways identified in the scRNA-Seq analysis will be included in this work stream. 
Transplantation of cells 

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Cells will be dissociated from neurospheres and then labelled with a fluorescent protein such as GFP 
using lentiviral transfection to allow the tracking of the migration of these cells and their progeny post 
transplantation.  Following purification (removal of viral particles) a single cell suspension will be 
produced and microinjected into bowel explants. The transplanted model will then be cultured and 
assessment of the degree of colonisation made at set time points.  This assessment will be made by 
various microscopy techniques and immunofluorescent analysis as described above. Single cells as 
well as intact neurospheres may be transplanted to compare the optimal transplantation technique.  
Linking with clinical outcomes 
Patients recruited for this study who have Hirschsprung’s Disease are also eligible for the ALOHA study 
(REC: 18/NNW/0608) which is being run by the same study team in the same institution. Linkage of 
the clinical outcomes data with the laboratory-based data described above is expected to shed novel 
light onto the findings and is an important step towards clinical translation of this work. 
Statistical analysis 
All quantitative results will be analysed using appropriate statistical measures. For complex statistics 
the Biostatistics Department at the University of Liverpool will be consulted. 
9. Outcome measures 
The milestones for outcome will be: 
1) Successful optimisation of a scRNAseq protocol using dissociated human bowel cells. 
2) Identification and description of the populations of progenitor cells which are present within the 
dissociated cells from different regions of children with HSCR and control patient groups. 
3) Determination of the mechanisms and signalling pathways responsible for ENSPC proliferation and 
differentiation in neurospheres. 
4) Manipulation of neurospheres using targeted agonists and antagonists for key pathways. 
5) Transplantation of cells into explant tissue ex vivo and characterising of cell integration. 
9. Study end-point 
It is anticipated that achieving these study outcomes including refining protocols and gaining 
sufficient data within each workstream will require 150 Hirschsprung’s specimens and 300 control 
specimens.  
11. Dissemination 
The results will be widely disseminated by presentations at national and international 
gastroenterology, paediatric surgical and developmental biology meetings, and by peer-reviewed 
publication in appropriate gastroenterology or developmental biology journals. The research group 
hold regular patient and family meetings where research findings are fed back to participating families. 
The group’s close links with Hirschsprung’s charities, particularly CHAMPS, means that research finds 
are also fed back through the group’s channels. 
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