Gastric organoids as an in vitro model to study H. pylori and its exotoxin VacA

Abstract

The inherent limitations of studying gastric pathogens in vivo, prompted me to develop a 3-dimensional gastric organoid model originating from primary murine gastric tissue. In vitro cultivation of glands from primary gastric tissue robustly yields spheroidal organoids that share structural and assembly characteristics with authentic gastric tissue. Fluorescent confocal microscopy of organoids stained with cell-type specific antibodies revealed several distinct cell types that normally occupy the glandular structure of gastric tissue, including gastric stem cells, surface mucous pit cells, epithelial cells, parietal cells, D cells and chief cells. In our laboratory, I use this 3-dimensional gastric organoid model to evaluate alterations in gastric physiology and function caused by the vacuolating cytotoxin (VacA), secreted by the human gastric pathogen, Helicobacter pylori. Exposure of organoids to VacA resulted in a visible increase in cellular vacuolation, which is a stress response of most cell types, and one of the canonical markers of VacA intoxication. Additionally cell death was clearly visible upon VacA intoxication. The use of gastric organoids is an exceptionally suitable model to study the interaction of VacA inside the stomach, by providing both the gland structure and physiological functions normally associated with the gastric environment.

Popular Abstract

Helicobacter pylori is a bacteria which increases the risk of developing stomach ulcer and stomach adenocarcinoma and without knowing it 50% of the human population, all around the world, are carrying this bacteria. However 80-90% of the infected people can live their entire life without knowing that they harbor this bacterium as it is not causing any damage and no symptoms are being developed. It is thought that this bacterium evolved with the humans thousands of years ago.

Helicobacter pylori can through different mechanisms damage the tissue and cells in the human stomach. One way the bacterium does the damage is by releasing a compound, a toxin, which damages different types of cell layers in the stomach. In the laboratory it is quite simple to grow one type of cells on a dish and then add the bacterium to the cells to see what kind of damage they are doing. One of the end results could be that the cells undergo apoptosis, which is when the cells die, but other effects can be seen as well. However there is one problem with this system. Only one type of cells is used at a time, but the human stomach contains different types of cells that work together to create the environment of the stomach. We lose the complexity of the stomach when we are working in the laboratory. We do not know if the toxin prefers a certain part of the stomach, how is it causing different types of damages or what is the reason behind the increased chance of developing stomach ulcer and stomach adenocarcinoma. A better model is needed to grasp the consequences of this toxin.

In the last 5 years a new model has been developed that has had tremendous success. This model utilizes organs, from living mice and humans, and by chopping the organ into small pieces and adding different nutrients and growth factors we can regrow the tissue of the organ into small spheres. We can think of these spheres as “mini-organs” grown on a dish. These “mini-organs” can be originated from any organ of choice, and since the toxin is specific to the stomach, these “mini-organs” can e grown from mice stomachs, which is more convenient than using tissue from humans.

I constructed these 3-dimensional “mini-organs” by isolating the stomach from a mouse and growing it on a dish. I also used a special microscope to demonstrate that the “mini-organ” consist of many cells as well as different layers of cells that are quite different from each other. I also demonstrated that the “mini-organ” contains many different cell types just like the real stomach and all these cells work together to create the environment seen in the stomach. This shows that these “mini-organs” are quite complex just like the stomach. Finally I showed that the toxin released by Helicobacter pylori causes damage to these “mini-organs” and ultimately causes the cells to undergo apoptosis, meaning the cells die.

With this model we can get a closer look on what is happening in the stomach and my positive results have demonstrated that this model is quite similar to the complexity of the stomach environment.

• author: Koliana, Marianne
• supervisor: Johannes Hedman
• organization: Applied Microbiology; Biotechnology
• course: KMB820 20161
• year: 2016
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: vacuolation confocal microscopy, VacA, 3D structures, Gastric organoids, applied microbiology, teknisk mikrobiologi
• language: English
• id: 8892137
• date added to LUP: 2016-09-30 09:35:05
• date last changed: 2016-09-30 09:35:05