Potential use of skin-derived precursors (SKPs) in establishing a cell-based treatment model for Hirschsprung's disease.

BACKGROUND: Hirschsprung's (HSCR) disease is characterized by absence of ganglia in the distant bowel. Skin-derived precursor cells (SKPs) are somatic stem cells located in the bulge of hair follicles with high neural plasticity. In this study, we elucidated the therapeutic potential of SKPs for replenishing absent ganglia in HSCR bowel. METHODS: SKPs were isolated from mouse or human skin and cultured in neural differentiation medium to generate various types of neural cells. Expression of stem cell and neural differentiation markers were monitored by reverse-transcription polymerase chain reaction and immunocytochemistry, respectively. Engraftment and differentiation potentials of SKPs were further assessed using ex vivo gut culture with Ret(k/k) aganglionic gut. RESULTS: Expression studies revealed that SKPs express a panel of neural crest markers and three key stemness factors (Klf4, c-Myc and Sox2), which may account for the multipotency of these cells. Subsequent differentiation assays directly demonstrated that both mouse and human SKPs retain high differentiation capacities to form enteric neurons, and glia. Importantly, with ex vivo gut explants assay, we further showed that SKPs colonize and differentiate in the Ret(k/k) aganglionic hindgut explants. CONCLUSION: Our data suggest that SKPs may represent an alternative source of stem cells for the study of cell-based therapy for HSCR.

Hedgehog/Notch-induced premature gliogenesis represents a new disease mechanism for Hirschsprung disease in mice and humans.

Hirschsprung (HSCR) disease is a complex genetic disorder attributed to a failure of the enteric neural crest cells (ENCCs) to form ganglia in the hindgut. Hedgehog and Notch are implicated in mediating proliferation and differentiation of ENCCs. Nevertheless, how these signaling molecules may interact to mediate gut colonization by ENCCs and contribute to a primary etiology for HSCR are not known. Here, we report our pathway-based epistasis analysis of data generated by a genome-wide association study on HSCR disease, which indicates that specific genotype constellations of Patched (PTCH1) (which encodes a receptor for Hedgehog) and delta-like 3 (DLL3) (which encodes a receptor for Notch) SNPs confer higher risk to HSCR. Importantly, deletion of Ptch1 in mouse ENCCs induced robust Dll1 expression and activation of the Notch pathway, leading to premature gliogenesis and reduction of ENCC progenitors in mutant bowels. Dll1 integrated Hedgehog and Notch pathways to coordinate neuronal and glial cell differentiation during enteric nervous system development. In addition, Hedgehog-mediated gliogenesis was found to be highly conserved, such that Hedgehog was consistently able to promote gliogenesis of human neural crest-related precursors. Collectively, we defined PTCH1 and DLL3 as HSCR susceptibility genes and suggest that Hedgehog/Notch-induced premature gliogenesis may represent a new disease mechanism for HSCR.