Transcript - Catalyst

Regenerative Therapy for
Hirschsprung’s Disease
Harry Flaster, MD
• Transplantation of native or induced pluripotent
stem cells has exciting potential to treat
Hirschsprung’s disease
• Among the many possible “cell-based”
approaches to the treatment of Hirschsprung’s is
the use of skin-derived precursors
• This approach has its limitations
• Therefore, treatment for Hirschsprung’s disease
will remain operative
• But, this is an exciting area of research, and HD is
a great disease to study
Hirschsprung’s Disease
• Most common neural crest cell pathology in
humans affecting 1:5000 newborns
• Result of incomplete colonization of bowel with
enteric neural crest cells, resulting in peristalsis,
causing chronic constipation
• Treatment for HD is a “pull through” procedure,
where by aganglionic portions of bowel are
resected and then anastomosed to healthy bowel
while preserving sphincter function.
Neural Crest Cells
• Biologic Rebels: “they break all the rules”
Enteric Nervous System
• ENS is derived from neural crest cells originating in the
vagal neural crest of the developing embryo
• Neural crest cells delaminate from the vagal neural
crest, then congregate in the caudal brachial arches,
then enter the anterior foregut during the 4th week of
• The cells then proliferate rapidly, forming a migratory
“wave” that reaches the distal hindgut by week 7.5, but
it is not until week 10 that the cells coalesce into what
will eventually become the myenteric plexus
• Then, a few days later, there is a second centripetal
wave inward, forming the submucosal plexus.
Enteric Neural Crest Migration
Other Neural Crest Associated Defects
• Mutation in cKit  migration defects
• Waadenburg’s Pax3 mutation
• Di George Syndrome: 22q11 deletion: disrupts
neural crest migration into pharyngeal arches
• Skin-derived precursor cells (SKPs) are somatic
cells located in the bulge of hair follicles
• Have similar properties to neural crest cells
Figure 1: SKPS express marker of embryonic neural
crest and differentiate into peripheral neurons and
Schwann cells
Materials and Methods
• Foreskin tissue was collected from 5 patients, aged 2
months to 12 years old, who had undergone
• Used mice with the Ret knockout mutation
• Prepared both human and mice tissue were prepared
in a similar fashion in order to isolate the SKPs and
then grow them in culture
• Both the mice and human SKPs were labeled with GFP
using recombinant adenovirus in culture
• The Ret knockout embyronic guts were excised and five
GFP-labeled mouse and human SKP spheres were
mixed with basement membrane extract than
transplanted to the hindgut
Ex vivo gut explants
• The mouse guts were then cultured for an
additional three days, then fixed, stained, and
• The experiments were repeated three times
• Successfully isolated SKPs and grew SKP
spheres in culture
SKPs express pluripotent transcription
• “Stemness” genes, transcription factors none
to be capable of inducing pluripotency in
fibroblasts, were expressed in SKPs, including
Sox2, Klf4, and c-Myc (Oct-4 missing)
• SKPs also express neural crest cell makers, but,
surprisingly, not Ret
Figure 2
Figure 3
Figure 4
Problems with this specific study
• The SKPs express neuronal markers and look like
neurons. Do they work like neurons?
• “Cell migration occurs over distances between 0.60
mm and 1.03 mm and the average migration distance
is 0.78 mm away from the site of transplantation.”
• Even in HD limited to the sigmoid colon, both the
population of transplanted enteric nervous system
pluripotent cells and the distances required for them to
travel would be vast
• However, there is the potential for a more limited
transplantation to the anastomotic region after a pullthrough
Challenges to this field of research
• Migration distances
• Potential for neoplasm (germ cell tumor)
• Will the transplanted cells restore
• Are the transplanted cells sufficient? What
other factors must be manipulated?
To solve multi-factorial problems, it takes a multi-factorial approach
Works Cited
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Thank You Ped Surg at SCH