Using ex vivo organotypic cultures to investigate donor cell integration in the gut

肠道的正常功能取决于肠壁沿着肠道长度的运动。这种功能性运动是由肠道内被称为肠神经系统（简称ENS）的神经细胞协调的。当这些神经细胞中的任何一个出现问题时，就会导致一种叫做“肠性神经病”的疾病。这些疾病无法治愈，成功的治疗仍然是一个挑战。不幸的是，患者通常需要手术切除大块的肠道。这可能会导致严重的问题，因为患者必须长期忍受改变生活的症状。因此，这些疾病的新疗法至关重要。在过去的10年里，科学家们认为干细胞可能是治疗这些疾病的一个很好的选择。我们小组和其他人的研究表明，通过移植肠道神经系统干细胞来拯救老鼠的神经细胞是可能的。然而，我们并不真正了解这些干细胞是如何做到这一点的。一个重要的因素是，这些研究大多是在小鼠身上进行手术，并将供体细胞移植到肠道中。虽然这表明供体干细胞可以在肠道中生长和发挥作用，但也有一些缺点。通常，这些研究使用大量的老鼠，因为只有一次移植细胞的可能性。手术后，很难了解供体细胞的行为，因为很难在小鼠体内看到它们。最近，我们的团队开发了一种方法，可以让我们取一些老鼠的肠道，并使其在培养皿中存活长达3周。这个系统可以让我们从任何一只老鼠的肠道中获得6段肠道。通过这样做，我们可以将任何实验中使用的小鼠数量减少近84%。用这种方法我们就不用再给老鼠做手术了。因为我们可以在培养箱中培养肠道，我们可以移植任何细胞，更容易地了解它们在肠道中的行为。因此，在本研究中，我们希望：1)研究不同的肠道神经系统干细胞如何在肠道中移动；2)确定移植后肠道发生了什么；3)评估肠道神经系统干细胞在移植后的功能。为了做到这一点，我们想用我们的新方法来研究不同的肠道神经系统干细胞在移植后在肠道中的表现，同时减少使用的小鼠数量。为了研究不同的肠道神经系统干细胞如何在肠道内移动，我们将首先用标记物标记小鼠胚胎干细胞。这个标记可以让我们追踪移植后的细胞。在谢菲尔德大学同事的帮助下，我们将在培养皿中制造含有这种标记物的肠道神经系统干细胞。然后，我们将用我们的新系统将这些肠道神经系统干细胞移植到小鼠肠道中，观察它们在三周内的表现。然后我们将通过观察供体细胞移植前后肠道的蛋白质编码来研究移植是如何影响肠道的。最后，我们将观察供体细胞是如何在肠道中工作的，通过使用一种染料，我们可以记录单个细胞的活动。这些研究将使我们了解移植后不同的肠道神经系统干细胞在肠道中的表现，以及这对肠道本身的影响。这对于帮助开发更好的肠道疾病治疗方法非常重要。然而，这些研究也将有助于证明我们的新系统确实有效，我们可以减少在这类实验中使用的动物数量。通过展示这一点，我们希望我们可以鼓励其他科学家使用这个系统，这样我们就可以帮助减少用于研究的动物总数。

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 gut2) Determine what happens to the gut after transplantation3) 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.