Using key models for developing stem cell-based therapies for aganglionic gut disorders

Life threatening developmental disorders of the gut enteric nervous system (ENS) such as Hirschsprung?s disease (HSCR) occur commonly and are characterised by absent (aganglionic) nerve cells in the gut wall. Current surgical treatments for HSCR are unsatisfactory due to persistent complications and are best classed as palliative therapies rather than definitive cures. The overall research goal is to develop curative therapies for these disorders by testing whether specialised stem cells isolated from the gut (ENS stem cells) are capable of restoring missing ENS following transplantation into, and restoring function of, affected gut. To date, the applicants? research has laid the groundwork for progression to the critical step of testing such therapeutic strategies in key animal models that closely resemble HSCR, prior to clinical trials in patients. This work includes the recent identification of a novel source of ENS stem cells from the gut lining (mucosa), which provides a safer, more accessible and reproducible source of stem cells. ENS stem cells isolated from the gut mucosa of post-natal animals and humans (including from the normal? segment of HSCR gut) have been shown to restore components of the ENS when transplanted into recipient aganglionc gut grown in the laboratory. In order to translate these findings into an effective cell replacement therapy in humans, the proposed research aims to study whether gut mucosal ENS stem cells can be safely and efficiently delivered into the gut of live animals suffering HSCR-like disorders. The research will study the best methods to deliver ENS stem cells to the affected gut and optimise their ability to colonise the gut, form ENS and integrate with other components of the gut musculature to restore function. In addition to access to the best available mouse models of HSCR, the applicants are well placed to carry out this research given their track record in the field of ENS development, stem cell biology and transplantation, and ultimately expertise in the clinical management of children suffering ENS disorders.

Current surgical treatments for common life threatening enteric nervous system (ENS) disorders such as Hirschsprung?s disease (HSCR) are unsatisfactory and palliative. The overall aim of the proposed research is to develop more effective and curative therapies utilising ENS stem cells. The applicants have an international track record in the field of ENS development and ENS stem cell biology and have already addressed key prerequisites for progressing the research most effectively towards definitive trials of ENS stem cells in humans. These include the isolation, harvesting, and characterisation of ENS stem cells from post-natal mouse and human gut (including from normal gut and the ganglionated portion of HSCR gut) and their effective transplantation into in vitro models of gut aganglionosis. The assessment of transplantation success in these in vitro studies, however, has been limited by a number of practical issues and the research needs to progress to definitive in vivo research employing robust animal models of HSCR. This research proposal aims to utilise both an established and well characterised mouse models of HSCR (monoisoformic Ret51) as well as a transgenic mouse line (Wnt1Cre;ROSA26YFPstop) which allows accurate and permanent lineage tracing of transplanted neural crest-derived ENS stem cells. The work will also use ENS stem cells isolated from gut mucosa as a novel, more accessible and reproducible source of cells. The applicants propose to utilise protocols routinely employed by the research teams to isolate and expand ENS stem cells obtained from wild-type or HSCR-like postnatal animals, and from gut mucosal biopsies from human patients. These will be transplanted in vivo into both embryonic and post-natal aganglionic gut segments of Ret51 mice. Transplant success will be assessed by characterising the ability of the transplanted ENS stem cells to colonise the aganglionic gut, generate an anatomically mature and integrated ENS, and ultimately to effect functional rescue of the dysmotile gut.