Using ex vivo organotypic cultures to investigate donor cell integration in the gut
Lead Research Organisation:
University College London
Department Name: Institute of Child Health
Abstract
The correct functioning of the gut depends on the movement of the gut wall along the length of the bowel. This functional movement is coordinated by nerve cells within the gut called the enteric nervous system, or ENS for short. When there is an issue with any of these nerve cells this results in diseases called "enteric neuropathies". There are no cures for these diseases and successful treatment remains a challenge. Unfortunately, patients often have surgery to remove large pieces of gut. This can cause significant problems, as patients have to live with long-term, life changing symptoms. Therefore, new treatments for these diseases are vital.
Over the past 10 years scientists have suggested that stem cells may be a good option to treat these diseases. Work from our group, and others, have shown that it is possible to rescue nerve cells in mice by transplanting gut nervous system stem cells. However, we don't really understand how these stem cells do this.
One important factor is that most of these studies have performed surgery in mice and transplanted donor cells to the gut. While this has worked in showing donor stem cells can grow and function in the gut, there are a number of drawbacks. Normally, these studies use a large number of mice as it is only possible to transplant cells once. After the surgery it is also difficult to understand how the donor cells behave as it is difficult to see them inside the mouse.
Recently, our group have developed a method which allows us to take some mouse gut and keep this alive in a dish for up to 3 weeks. This system allows us to get 6 segments of bowel from any 1 mouse gut. By doing this we can reduce the number of mice used in any experiment by nearly 84%. Also using this approach we don't have to do surgery on the mice anymore. As we can then grow the gut in an incubator we can transplant any cells and learn how they behave in the gut more easily.
Therefore, in this study we hope to:
1) Investigate how different gut nervous system stem cells move in the gut
2) Determine what happens to the gut after transplantation
3) Assess how gut nervous system stem cells function after transplantation.
To do this, we want to use our new method to investigate how different gut nervous system stem cells behave in the gut after transplantation, while reducing the numbers of mice used.
To investigate how different gut nervous system stem cells move in the gut we will first tag mouse embryonic stem cells with a marker. This marker will allow us to follow the cells after transplantation. With help from colleagues in the University of Sheffield we will then make gut nervous system stem cells which contain the marker in a dish. We will then transplant these gut nervous system stem cells to mouse gut using our new system and look at what they do over 3 weeks. We will then study how transplantation affects the gut by looking at the protein code of the gut before and after we transplant donor cells. Finally, we will look at how the donor cells work in the gut by applying a dye which allows us to record activity in individual cells.
These studies will allow us to understand how different gut nervous system stem cells behave in the gut after transplantation, and what this does to the gut itself. This will be very important in helping to develop better treatments for gut diseases. However, these studies will also help to prove that our new system really works and that we can reduce the number of animals used in these types of experiment. By showing this we hope that we can encourage other scientists to use this system so that we can help to reduce the overall number of animals used in research.
Over the past 10 years scientists have suggested that stem cells may be a good option to treat these diseases. Work from our group, and others, have shown that it is possible to rescue nerve cells in mice by transplanting gut nervous system stem cells. However, we don't really understand how these stem cells do this.
One important factor is that most of these studies have performed surgery in mice and transplanted donor cells to the gut. While this has worked in showing donor stem cells can grow and function in the gut, there are a number of drawbacks. Normally, these studies use a large number of mice as it is only possible to transplant cells once. After the surgery it is also difficult to understand how the donor cells behave as it is difficult to see them inside the mouse.
Recently, our group have developed a method which allows us to take some mouse gut and keep this alive in a dish for up to 3 weeks. This system allows us to get 6 segments of bowel from any 1 mouse gut. By doing this we can reduce the number of mice used in any experiment by nearly 84%. Also using this approach we don't have to do surgery on the mice anymore. As we can then grow the gut in an incubator we can transplant any cells and learn how they behave in the gut more easily.
Therefore, in this study we hope to:
1) Investigate how different gut nervous system stem cells move in the gut
2) Determine what happens to the gut after transplantation
3) Assess how gut nervous system stem cells function after transplantation.
To do this, we want to use our new method to investigate how different gut nervous system stem cells behave in the gut after transplantation, while reducing the numbers of mice used.
To investigate how different gut nervous system stem cells move in the gut we will first tag mouse embryonic stem cells with a marker. This marker will allow us to follow the cells after transplantation. With help from colleagues in the University of Sheffield we will then make gut nervous system stem cells which contain the marker in a dish. We will then transplant these gut nervous system stem cells to mouse gut using our new system and look at what they do over 3 weeks. We will then study how transplantation affects the gut by looking at the protein code of the gut before and after we transplant donor cells. Finally, we will look at how the donor cells work in the gut by applying a dye which allows us to record activity in individual cells.
These studies will allow us to understand how different gut nervous system stem cells behave in the gut after transplantation, and what this does to the gut itself. This will be very important in helping to develop better treatments for gut diseases. However, these studies will also help to prove that our new system really works and that we can reduce the number of animals used in these types of experiment. By showing this we hope that we can encourage other scientists to use this system so that we can help to reduce the overall number of animals used in research.
Technical Summary
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.
Here, we propose to further develop this model to investigate the integration and interaction of donor ENS progenitor cells in murine gut tissue after ex vivo transplantation, whilst reducing the numbers of animals used.
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.
Here, we propose to further develop this model to investigate the integration and interaction of donor ENS progenitor cells in murine gut tissue after ex vivo transplantation, whilst reducing the numbers of animals used.
