lecture 18: Hirschsprung’s Disease Flashcards
1
Q
What is Hirschsprung disease?
A
- very distended stomach
- last part of colon is very constricted
- megacolon: balloons out and makes the abdomen swell
- congenital megacolon
- an absence of enteric nervous system
- intractable constipation
- 1/5000 births (males more than females)
- lack of peristalsis
- lack of regional:distal intestine
- length affected variable; most descending and sigmoid colon, can be all the colon
- world’s simplest birth defect
- longitudinal muscle and circular muscle
- lack of ganglion cells (neurons)
- thick bundles of extrinsic nerves
- untreated it will be fatal
2
Q
What is the history of Hirschspring disease?
A
- 1691 - Ruysch (Holland)-first description of megacolon disease
- Harold Hirschsprung (1830-1916) Danish physician and paediatrician
- 1886: published the monograph describing in detail the disease that bears his name
- he misidentified the megacolon as the origin of the problem
- 1901: Tittel
- absence of ganglion cells
- children with many forms of constipation were included as “Hirschsprung disease”, confusion about the cause continued until late 1940s
- 1932: A Kuntz
- showed ENS derived from hindbrain neural crest in chicken – clinical significance could not be grasped
- 1946: Ehrenpreis
- realised the colon was distended because of the lack of ganglion cells
- Orvar Swenson (1909-2012)
- US based surgeon
- 1946-48 – focussed on the correct site, the constricted segment of colon
- performed first effect surgical treatment, colostomy above above the constriction
- 1950s – improved diagnostic and surgical techniques
- CL Yntema and WS Hammond
- 1954: published experimental embryology paper showing in chickens the ENS comes from the vagal or hind brain neural crest
3
Q
What is the organisation of the ENS?
A
- relatively simple
- small ganglia (variable # neurons + glia) in two layers (plexuses)
- neurite bundles between local ganglia, and to nearby gut tissues
- replicated countless times → ENS network; self-organising, no grand plan
4
Q
Is Hirschsprung a genetic disease?
A
- yes
- mutations in many genes predispose to hirschsprung disease
- genetic complexity and variability → simple disease phenotype
- many modifier genes: affect penetrance/severity
- up to 70
- when mutated won’t give hirschsprung but will affect severity when you have it
- penetrance– discordancy even in MZ twins
- possibility of environmental influences
5
Q
What are some genes that have been shown to contribute in mice when homozygously inactivated?
A
-
RET
- 10q11.2
- absence of neurons from small and large intestine
-
GDNF
- 5p12-13.1
- absence of neurons from small and large intestine
-
GFRa1
- 10q25
- **absence of neurons from small and large intestine **
-
ETB
- 13q22
- **absence of neurons from distal-most large intestine **
-
ET-3
- **20q13.2-13.3 **
- **absence of neurons from distal-most large intestine **
-
ECE-1
- 1p36.1
- **absence of neurons from distal-most large intestine **
-
Sox10
- 22q13
- **absence of neurons from entire gastrointestinal tract **
- SIP1
- 2q22
- absence of neurons from distal-most large intestine
- PAX3
- 2q37
- absence of neurons from small and large intestine
- Phox2b
- 4p12
- absence of neurons from entire GIT
- IHH
- 2q33 to q35
- absence of neurons from parts of the small int. and colon
- SHH
- 7q36
- ectopic neurons within mucosa
- HOX11L1
- 2p13.1
- ENS hyperplasia in colon and hypoplasia in small int.
6
Q
In what way is Hirschsprung Disease a quantitative problem?
A
- the ENS is about 95% normal
- the trouble is, instead of 100% of the gut having 95% of normal neuronal density, which would be no functional problem
- 95% of the gut has 100% neuron density, and 5% of the gut as 0% neuron density
- that 5% is the problem
7
Q
How does Hirschsprung Disease occur?
A
- the ENS arises from emigrés from the CNS–neural crest cells
- nearly all come from hind brain in early development
- about 2-3 weeks post fertilisation in humans migrate out of hindbrain a short distance to a bit of the nearby gut that we call the foregut which in later stages will end up as oesophagus, stomach and duodenum
- in the following four weeks, this population expands, spreads and colonises the rest of the gut getting down to the anal end by ~7 weeks
- but in HD, for some reason or another these do not migrate to the end
- hence why always distal, and why varying lengths
8
Q
How is Hirschsprung disease treated now?
A
- surgical treatment for HD and postoperative problems
- acute
- anastomosis failure; leakage (1.5~8%), stricture (3~35%)
- enterocolitis (~40%)
- chronic
- motility disorders
- constipation; 27% of patients are maintained on a stool softener
- incontinence; 44% of patients suffer from faecal soiling
- motility disorders
- cost
- excess of $A20,000/yr/patient (Tony Catto-Smith, Head, Gastroenterology, RCH)
9
Q
A different treatment for Hirschsprung disease?
A
- could Hirschsprung disease be relieved by adding in new nerve cells to the colon, instead of cutting out colon without nerve cells?
- new nerve cells would have to be derived from stem/progenitor cells
- potential of cell therapy to treat pediatric motility disorders: Ryo Hotta, Dipa Natarajan, Nikhil Thapar
10
Q
What is the potential for stem/progenitor cell therapy?
A
- problem arises before 7 weeks gestation
- diagnosed ~2 weeks post-natally
- so is emplacement of a new ENS possible?
- stem/progenitor cells must be available and of appropriate developmental capability (neural crest stem cells?)
- the cells must be able to be expanded in vitro before application
- stem/progenitor cells must be able to be introduced into the aneural gut
- stem/progenitor cells preferably not subject to immune attack (autologous?)
- conditions in the post-natal gut must be appropriate for these cells (much more mature and larger than when ENS is generated)
- response must include coverage of gut musculature (migration), proliferation, neuronal and glial differentiation, connectivity, integration with host ENS
11
Q
Is there anything in our favour?
A
- most of ENS in Hirschsprung patients is OK; so no a priori reason why ENS cannot be formed even from patient’s cells
- ENS is self-organising with local rules – no requirement for a grand plan
- ENS does not need to be “perfect” to function adequately – highly redundant and compensatory (like CNS)
- somatic NC stem/progenitor cells may be harvested from various tissues (skin and hair, gut, dentine, etc)
- tonnes of papers
- NC stem/progenitor cells may be created from human ES cells and iPS cells
12
Q
Can ENS form from progenitor cells in post-natal gut?
A
- the plasticity of the defecation reflex pathway in the enteric nervous system of guinea pigs
- in vivo experiments were performed on guinea pigs…
- resection of a 2cm segment of distal colon, with anastomosis of the exposed ends
- the R-IAS reflex relaxations recovered and some bundles of fine nerve fibres were able to be seen interconnecting the oral and anal ends of the myenteric plexus
- also surprisingly, new neurons were found to have generated from neural stem cells at the anastomotic ends
- these new neurons had constructed mature enteric neural networks including ganglionic-like structures eight weeks after surgery
13
Q
What is cell therapy for Hirschsprung Disease (and other enteric neuropathies)?
A
- the basic idea is that we get some kind of stem or progenitor cell of a neural type, preferably a neural crest type
- from somewhere - pluripotent cells (ES or iPS cells), maybe CNS neural stem cells in foetal brain, or enteric gut neural crest stem cells (foetal or post-natl or adult bowel)
- grow in cultures to enormous numbers and somehow or other stick them back into the wall of the intestine that lacks these cells
- hopefully by some magic will work
- (perhaps still sounds farfetched)
14
Q
What is the aim of this research?
A
- to determine whether enteric neural stem/progenitor cells (post-natal, autologous) can colonise the post-natal colon of Hirschsprung Disease patients
- to determine whether the grafted cells migrate, proliferate and differentiate into cells (neurons, glia) of the appropriate phenotype
- to determine if these can restore function sufficiently that bowel-removal surgery is not necessary
15
Q
What are proof of principle studies in animal models?
A
- mouse and rat models of Hirschsprung disease exist - same gene defects, same megacolon phenotype
16
Q
What stem/progenitor cells to use?
A
- donor cells: genetically labelled – fluorescent GFP – neural crest-derived cells from the gut of embryonic mice
- generated by Hideki Enomoto
- experiments by Heather Young (anatomy) lab
- embryonic or post-natal colon
- separate all gut cells
- ENS cells are green
- select the green cells with FACS
- centrifuge the green cells to form a clump
- grow the clumped cells (neurospheres)
- divide the neurospheres to make more neurospheres
17
Q
Can mouse neural stem/progenitor cells form ENS in normal post-natal mouse gut?
A
- transplantation of neural stem/progenitor cells into the colon of post-natal mice
- post-natal mouse anaesthetised
- distal colon surgically exposed
- 1-3 neurospheres surgically implanted into wall of colon → neurosphere inserted in slit made in colon wall
- colon replaced in body cavity
- wound closed and mouse recovers
- extensive migration of graft-derived cells within the post-natal colon (normal AND hirschsprung)
- graft-derived (green) neurons fire action potentials and connect with other neurons
23
Q
Can ENS cells be obtained from patients?
A
- all human ENS cells can be identified by specific antibodies - example HNK-1
- the target molecule of the HNK-1 antibody is on the other surface of the ENS cells (there are other antibodies with this useful property, e.g. p75)
- this means that HNK-1 or p75 can be used as a “hook” to “catch” living ENS cells
- colon with ENS is dissociated into a cell suspension (trypsin enzymes etc)
- cell suspension is labelled with HNK-1 (or p75) coupled to a fluorescent reporter
- only the ENS cells are labelled with fluorescent reporter; the other cells (muscle, fibroblasts etc) are not labelled
- the Fluorescence Activated Cell Sorter (FACS) is a machine that can separate the fluorescent cells from the rest
- cultured (adherent) for more than 1 day
- FACS profile of dissociated p75-labelled human patient colon cells
- p75 draws these cells out
- determine if they are neural crest cells by looking for other markers
24
Q
Can ENS cells be combined with Hirschsprung gut from patients?
A
- distal end (no ENS) of bowel resected from patients is cut into 2mm pieces
- neurosphere inserted in pouch created between muscles layers
- after 8 days extensive spread of cells into gut muscle
- implantation site identified by MTR+ cells
- broad distribution of MTR+ ENS cells around this implantation site = migration
- HU+ and TUJ1+ (neuronal) cells, S100b + glial cells = differentiation
- this means that what happened in the mouse model is also happening in vitro in human models
- don’t know if there is any restoration of function
- maybe appropriate phenotype
