Interplay between embryonic enteric neural stem cells (ENSCs) and enteric mesenchymal
cells (EMCs) in the embryonic gut is essential for normal development of the enteric
nervous system. Disruption of these interactions underlies the pathogenesis of intestinal
aganglionosis in Hirschsprung disease (HSCR). ENSC therapy has been proposed as a
possible treatment for HSCR, but whether the survival and development of postnatal-derived
ENSCs similarly rely on signals from the mesenchymal environment is unknown and has
important implications for developing protocols to expand ENSCs for cell transplantation
therapy. Enteric neural crest-derived cells (ENCDCs) and EMCs were cultured from the
small intestine of Wnt1-Rosa26-tdTomato mice. EMCs promoted the expansion of ENCDCs
9.5-fold by inducing ENSC properties, including expression of Nes, Sox10, Sox2, and
Ngfr. EMCs enhanced the neurosphere-forming ability of ENCDCs, and this persisted
after withdrawal of the EMCs. These effects were mediated by paracrine factors and
several ligands known to support neural stem cells were identified in EMCs. Using
the optimized expansion procedures, neurospheres were generated from small intestine
of the Ednrb(-/-) mouse model of HSCR. These ENSCs had similar proliferative and migratory
capacity to Ednrb(+/+) ENSCs, albeit neurospheres contained fewer neurons. ENSCs derived
from Ednrb(-/-) mice generated functional neurons with similar calcium responses to
Ednrb(+/+) ENSCs and survived after transplantation into the aganglionic colon of
Ednrb(-/-) recipients. EMCs act as supporting cells to ENSCs postnatally via an array
of synergistically acting paracrine signaling factors. These properties can be leveraged
to expand autologous ENSCs from patients with HSCR mutations for therapeutic application.
