lecture 18: Hirschsprung’s Disease

Question 1

What is Hirschsprung disease?

Answer 1

• 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

Question 2

What is the history of Hirschspring disease?

Answer 2

• 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

Question 3

What is the organisation of the ENS?

Answer 3

• 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

Question 4

Is Hirschsprung a genetic disease?

Answer 4

• 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

Question 5

What are some genes that have been shown to contribute in mice when homozygously inactivated?

Answer 5

• 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 **
• ET_B
  
  • 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.

Question 6

In what way is Hirschsprung Disease a quantitative problem?

Answer 6

• 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

Question 7

How does Hirschsprung Disease occur?

Answer 7

• 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

Question 8

How is Hirschsprung disease treated now?

Answer 8

• 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
• cost
  
  • excess of $A20,000/yr/patient (Tony Catto-Smith, Head, Gastroenterology, RCH)

Question 9

A different treatment for Hirschsprung disease?

Answer 9

• 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

Question 10

What is the potential for stem/progenitor cell therapy?

Answer 10

• 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

Question 11

Is there anything in our favour?

Answer 11

• 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

Question 12

Can ENS form from progenitor cells in post-natal gut?

Answer 12

• 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

Question 13

What is cell therapy for Hirschsprung Disease (and other enteric neuropathies)?

Answer 13

• 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)

Question 14

What is the aim of this research?

Answer 14

• 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

Question 15

What are proof of principle studies in animal models?

Answer 15

• mouse and rat models of Hirschsprung disease exist - same gene defects, same megacolon phenotype

Question 16

What stem/progenitor cells to use?

Answer 16

• 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

Question 17

Can mouse neural stem/progenitor cells form ENS in normal post-natal mouse gut?

Answer 17

• 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

Question 18

Can embryonic neural stem/progenitor cells form ENS in normal post-natal gut?

Answer 18

yes

Question 19

Can embryonic neural stem/progenitor cells form ENS in aganglionic (“Hirschsprung”) post-natal gut?

Answer 19

yes

Question 20

Can post-natal neural stem/progenitor cells form ENS in normal post-natal gut?

Answer 20

yes

Question 21

Can this cell therapy produce functional improvement in Hirschsprung models?

Answer 21

???

• transplanted progenitors generate functional enteric neurons in the postnatal colon, Ryo Hotta, et al.

Question 22

But what about a human model?

Answer 22

• great if it works in a mouse but one of the aims of the research is to determine whether human enteric neural stem/progenitor cells (post-natal, autologous) can be obtained and used
• aims of research:
  
  • to determine whether human enteric neural stem/progenitor cells (post-natal, autologous) can be obtained
  • to determine if they can colonise the post-natal colon TISSUES from Hirschsprung Disease patients
  • to determine whether the grafted cells migrate, proliferate and differentiate into cells (neurons, glia) of the appropriate phenotype in PATIENT’S COLON TISSUE IN CULTURE
  • to determine if these can restore SOME MUSCULAR function

Question 23

Can ENS cells be obtained from patients?

Answer 23

• 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

Question 24

Can ENS cells be combined with Hirschsprung gut from patients?

Answer 24

• 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

Question 25

What are future aims of this research?

Answer 25

• we can get teh cells and it appears they will do “the right thing” in the colon
• what are the crucial challenges?
  
  • how do we get enough ENS somatic precursor cells?
    
    • devise new techniques to culture autologous ENS somatic precursor cells in vitro to enable proliferation on a large scale
    • human iPS cells from patients (i.e. autologous), proliferate in vitro, then switch them to become ENS somatic precursor cells (we don’t really know what the switches are, yet!)
  • how do you get the ENS somatic precursor cells into the colon wall?
    
    • have ENS somatic precursor cells growing on a biodegradeable membrane to “wrap” the colon, after first opening up the serosa – the “skin” of the colon. Rely on cells migrating inside the colon
    • there’s no b