Mechanisms mediating adult enteric neurogenesis are largely unknown. Using inflammation-associated neurogenesis models and a transgenic approach, we aimed to understand the cell-source for new neurons in infectious and inflammatory colitis. Dextran sodium sulfate (DSS) and Citrobacter rodentium colitis (CC) was induced in adult mice and colonic neurons were quantified. Sox2GFP and PLP1GFP mice confirmed the cell-type specificity of these markers. Sox2CreER:YFP and PLP1creER:tdT mice were used to determine the fate of these cells after colitis. Sox2 expression was investigated in colonic neurons of human patients with Clostridium difficile or ulcerative colitis. Both DSS and CC led to increased colonic neurons. Following colitis in adult Sox2CreER:YFP mice, YFP initially expressed predominantly by glia becomes expressed by neurons following colitis, without observable DNA replication. Similarly in PLP1CreER:tdT mice, PLP1 cells that co-express S100b but not RET also give rise to neurons following colitis. In human colitis, Sox2-expressing neurons increase from 1–2% to an average 14% in colitis. The new neurons predominantly express calretinin, thus appear to be excitatory. These results suggest that colitis promotes rapid enteric neurogenesis in adult mice and humans through differentiation of Sox2- and PLP1-expressing cells, which represent enteric glia and/or neural progenitors. Further defining neurogenesis will improve understanding and treatment of injury-associated intestinal motility/sensory disorders.

These results show that colitis promotes enteric neurogenesis in the adult colon through a serotonin-dependent mechanism that drives glial cells to transdifferentiate into neurons.

Background Transplanting autologous patient-derived enteric neuronal stem/progenitor cells (ENSCs) is an innovative approach to replacing missing enteric neurons in patients with Hirschsprung disease (HSCR). Using autologous cells eliminates immunologic and ethical concerns raised by other cell sources. However, whether postnatal aganglionic bowel is permissive for transplanted ENSCs and whether ENSCs from HSCR patients can be successfully isolated, cultured, and transplanted in vivo remains unknown. Methods ENSCs isolated from the ganglionic intestine of Ednrb−/− mice (HSCR-ENSCs) were characterized immunohistochemically and evaluated for their capacity to proliferate and differentiate in vitro. Fluorescently-labeled ENSCs were co-cultured ex vivo with aganglionic Ednrb−/− colon. For in vivo transplantation, HSCR-ENSCs were labeled with lentivirus expressing GFP and implanted into aganglionic embryonic chick gut in ovo and postnatal aganglionic Ednrb−/− rectum in vivo. Key Results HSCR-ENSCs maintain normal capacity self-renewal and neuronal differentiation. Moreover, the Ednrb−/− aganglionic environment is permissive to engraftment by wild-type ENSCs ex vivo and supports migratrion and neuroglial differentiation of these cells following transplantation in vivo. Lentiviral-GFP-labeled HSCR-ENSCs populated embryonic chick hindgut and postnatal colon of Ednrb−/− HSCR, with cells populating the intermuscular layer and forming enteric neurons and glia. Conclusions & Inferences ENSCs can be isolated and cultured from mice with HSCR, and transplanted into the aganglionic bowel of HSCR littermates to generate enteric neuronal networks. These results in an isogenic model establish the potential of using autologous-derived stem cells to treat HSCR and other intestinal neuropathies.