Human embryonic stem cell-derived neural crest stem cells and Hirschsprung disea

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Hirschsprung disease, or aganglionic megacolon, is a congenital defect that affects 1 out of 5,000 live births and is characterized by a failure to form enteric nervous system (ENS) in a variable length of the hindgut [1]. This potentially fatal condition results in an inability to coordinate peristaltic movements of the bowel and is most commonly caused by mutations that reduce signaling through the glial cell line-derived neurotrophic factor (GDNF) or endothelin-3 (EDN3) signaling pathways [2]. Both the GDNF receptor, Ret, and the EDN3 receptor Endothelin receptor B (EDNRB) are expressed by the neural crest stem cells (NCSCs) that give rise to the ENS ADDIN EN.CITE [3]. These signaling pathways interact to regulate the proliferation and migration of NCSCs and other neural crest progenitors that colonize the gut, though questions remain about whether the primary role of EDN3 signaling is to inhibit premature differentiation or to promote migration ADDIN EN.CITE [3-10]. Neural crest cells never migrate into the aganglionic portion of the gut in animals affected by Ret or Ednrb deficiency ADDIN EN.CITE [3,6]. These observations raise the possibility of improving the treatment of Hirschsprung disease by combining traditional surgical approaches with cell therapy in which NCSCs are transplanted directly into the aganglionic portion of the gut to generate enteric ganglia by bypassing the migration/proliferation defects ADDIN EN.CITE [3,6,11,12]. Consistent with this possibility, we and others have shown that NCSCs isolated from the fetal rodent gut can engraft and form enteric neurons after transplantation into the aganglionic region of the gut from rodent models of Hirschsprung disease ADDIN EN.CITE [6,11-13]. Nonetheless, before such a therapy can be contemplated for patients it will be necessary to obtain human NCSCs in quantities adequate for clinical use. Since human fetal tissue is very limited and of inconsistent quality for clinical use [14], it would be ideal to derive NCSCs with enteric characteristics from human embryonic stem (hES) cells. Having extensively characterized mouse and rat enteric NCSCs, we propose to optimize culture conditions to derive human NCSCs with similar properties from hES cells. We will inject these human NCSCs into the aganglionic hindgut of Ednrb mutant rats to test the ability of these cells to form neurons and glia in vivo. These studies will test whether NCSCs with specific regional characteristics can be derived from hES cells and whether these cells engraft in the gut of an animal model of Hirschsprung disease.