Why did we fund this project?
This award aims to use an ex vivo organotypic culture method to investigate therapeutic interventions used to treat enteric nervous system defects, reducing the number of mice in this research.
The enteric nervous system coordinates the function of the bowel including movement, hormone secretion and fluid exchange. Enteric neuropathies are degenerative conditions that can occur when the neurons making up this system degrade, and treatments are limited to managing symptoms. It has previously been shown that lost or damaged enteric nerve cells can be rescued in mice by transplanting gut nervous stem cells. However, the underlying mechanisms of how the stem cells integrate is not well understood. Researching stem cell treatment and integration typically requires the stem cells to be implanted using surgical procedures in vivo, usually using mice. Dr Conor McCann and colleagues have developed a culture method that allows portions of the murine gut to be grown ex vivo for up to three weeks. Importantly, six portions of bowel can be obtained from one mouse gut, reducing the number of mice needed overall for this type of research.
With NC3Rs funding, Conor will build confidence in the ex vivo culture method and demonstrate its utility in cell-based transplantation applications. Mouse stem cells will be tagged with a fluorescent marker and introduced into the ex vivo model to track the stem cells after implantation. Conor will then determine if these stem cells integrate into the ex vivo tissue. The transcriptomics of the gut before and after implantation will be analysed before the functionality of the implanted stem cells is determined.
Disruption of the enteric nervous system (ENS), the largest branch of the peripheral nervous system, impacts on critical intestinal functions such as motility, fluid exchange and gastric acid/hormone secretion. Unfortunately, therapeutic interventions to treat ENS defects, are mainly limited to surgical resection of the affected region. However, over the past decade there has been an increasing focus on stem cell-based therapies for treating disease. Recent studies from our group, and others, have highlighted the potential of ENS progenitor-based therapy, as a means of replacing neurons after in vivo transplantation to mouse colon. (Cooper et al, 2016. PLoS One. 2016; 11:e0147989; Cooper et al, 2017. Neurogastroenterol Motil. 2017 Jan;29(1):e12900; McCann et al, 2017. Nature Communications 3;8:15937; Frith TJ et al, 2019 bioRxiv doi: https://www.biorxiv.org/content/10.1101/819748v1).
However, the precise mechanisms by which donor cells integrate within recipient tissue remain unclear. Importantly, previous studies have relied heavily on in vivo surgical transplantation procedures to rodents. While this has provided proof-of-principal data that donor cells can integrate within organs after transplantation, technical limitations in tissue opacity and in vivo imaging have limited the mechanistic investigation of how donor cells integrate. Recently, we have developed an ex vivo organotypic culture method which allows for long-term culture of murine gut segments. Using this approach, it is possible to reduce animal usage by >84%, as each individual mouse can generate up to 6 cultured colonic segments, as well as providing refinement in experimental technique as in vivo surgical transplantation is not required.