O1115: Enteric Progenitors Can Be Induced to Migrate at Different Developmental Stages in an Embryonic Gut Explant Model of Hirschsprung’s Disease

Background: Hirschsprung’s disease is a congenital neuropathy of the intestine. It remains unknown if neural progenitors of Hirschsprung’s intestine, at different developmental stages, can be induced to migrate distally in the intestine. Amniotic fluid stem cells (AFSC) have been proven to enhance cell migration through a paracrine effect, and to secrete growth factors that possess chemoattractive capacity, such as glia cell derived neurotrophic factor (GDNF). We hypothesize that administration of AFSC condition media (AFSC-CM) or GDNF can promote migration of enteric progenitors in both early migratory stage (E11.5) and post-migratory stage (E14.5).

Methods: Ednrb knockout mice (Ednrb-/-) were studied as an animal model of Hirschsprung’s disease, with transgenic expression of Sox10-Venus to allow tracing of enteric progenitors. Following ethical approval (#47780), embryonic guts of wildtype and Ednrb-/- embryos at E11.5 and E14.5 with Sox10-Venus expression were isolated and embedded into Matrigel domes. Explants were cultured in control media (E11.5: wildtype n=8, Ednrb-/- n=3, E14.5: wildtype n=3, Ednrb-/- n=3), media conditioned by AFSC for 24 hours (E11.5: wildtype n=8, Ednrb-/- n=3, E14.5: wildtype n=3, Ednrb-/- n=3), or media supplemented with GDNF (100ng/mL) (E11.5: wildtype n=7, Ednrb-/- n=3, E14.5: wildtype n=4, Ednrb-/- n=4). Explants were maintained for 48 hours, followed by live imaging to track cell migration.

Results: Prior to AFSC-CM or GDNF administration, a delayed migration of Sox10+ cells was observed in Ednrb-/- embryonic guts from both E11.5 and E14.5 (Fig. A, D). Forty-eight hours after administration of either AFSC-CM or GDNF to E11.5 explants, outward migration of Sox10+ cells was observed in both wildtype and Ednrb-/- embryonic gut explants (Fig. B), with quantification of neurite extension length confirming significant increased migration compared to explants cultured in control media (Fig. C). However, in E14.5 explants, only GDNF treatment demonstrated an outward migration of Sox10+ cells in wildtype and Ednrb-/- explants, while AFSC-CM treatment did not induce migration of enteric progenitors (Fig. E). This was also confirmed by quantification of neurite extension length (Fig. F).

Conclusion: Administration of AFSC-CM or GDNF both significantly enhanced Sox10+ enteric progenitor migration in wildtype and Ednrb-/- embryonic gut explants at early migratory stage E11.5, while only GDNF can significantly induce migration at post-migratory stage E14.5. These results suggest that enteric progenitors can be promoted to migrate in both early and post-migratory stage, but early migratory progenitors are more plastic in response to a wider range of chemoattractants. These findings open the way to the development of a novel prenatal treatment for Hirschsprung’s disease.