Canonical Notch signalling has been demonstrated to regulate proliferation and neuronal differentiation of human enteric nervous system progenitor cells (ENSPCs) cultured as neurospheres in vitro. Unexpectedly, we have recently shown that ENSPCs can also be isolated from the thickened nerve trunks characteristic of aganglionic gut of Hirschsprung patients. Here we aim to determine if Notch signalling is also present in aganglionic bowel and cultured ENSPCs to assess whether modulation of Notch signalling may offer a novel therapy for treatment of Hirschsprung's disease.
Ethical approval was obtained from the UK North West 3 Research Ethics Committee (Ref: 10/H1002/77). Expression of key Notch receptors was determined using immunofluorescence in aganglionic and ganglionic Hirschsprung's gut samples obtained from four patients with different lengths of aganglionosis during pull-through surgery or bowel resection. Tertiary aganglionic neurospheres were formed following primary culture of ENSPCs derived from the myenteric plexus and subsequent dissociation of primary and secondary neurospheres. The Notch inhibitor DAPT was added to cultures for 4 days.
Notch 1,2,3 and 4 receptors were generally distributed within thickened nerve trunks in aganglionic colon regardless of Hirschsprung's phenotype, similarly to the cells in normal enteric nervous system ganglia. Notch 2,3 and 4 expression was retained by the cells in cultured tertiary aganglionic neurospheres. Preliminary data using the Notch inhibitor DAPT suggest a reduction in proliferation and an increase in differentiation in both ganglionic and aganglionic neurospheres.
Given that Notch receptors were expressed in aganglionic gut we hypothesize that Notch signalling can regulate the maintenance and differentiation of ENSPCs from aganglionic Hirschsprung bowel in vivo and thereby offer a potential tool to help develop novel therapies for children with Hirschsprung's disease. Our current work will now further assess the functional effects of Notch signalling in ENSPCs dissociated from aganglionic and ganglionic bowel-derived neurospheres.