Regenerative Therapy for Hirschsprung’s Disease Harry Flaster, MD

Summary 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 4 th week of gestation 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

Abstract Skin-derived precursor cells (SKPs) are somatic cells located in the bulge of hair follicles

SKPs 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 circumcision 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 photographed The experiments were repeated three times

Results Successfully isolated SKPs and grew SKP spheres in culture

SKPs express pluripotent transcription factors “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

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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 pull- through

Challenges to this field of research Migration distances Potential for neoplasm (germ cell tumor) Will the transplanted cells restore functionality? Are the transplanted cells sufficient? What other factors must be manipulated?

To solve multi-factorial problems, it takes a multi-factorial approach

Works Cited

Thank You Ped Surg at SCH