Date of Award
Doctor of Philosophy (PhD)
Sherif M. Karam
Stem cells are powerful tools in different aspects of biomedical and translational research, including disease modelling, drug testing, and tissue engineering for regenerative medicine. However, isolation and culture of organ-specific stem cells are challenging tasks. Therefore, the biological features of many adult stem cells are not well studied and their involvement in the development of cancer is still controversial. Some immortal stem cell lines have been established and used as an alternative to studying features of organ-specific stem cells. The ability to grow cells in a scaffold-free, three-dimensional (3D) model system that mimics in vivo conditions would help in revealing more and more properties of stem cells.
In this study, two types of 3D culture models were established to define specific properties of gastric stem cells. In the first model, the hanging drop method was used to grow immortalized mouse gastric epithelial progenitor/stem (mGS) cells with molecular and morphological features similar to those of stomach stem cells. Within a day, mGS cells hanging in RPMI media containing 10% serum without adding any growth factors formed a small cluster. By day 2, when transferred onto the surface of agarose using the same media, each cell cluster developed into a small spherical organoid with a central lumen, characterized by electron microscopy. Due to cell proliferation, these organoids progressively grew in size and were maintained for six months. The second type of organoids was developed from incipient gastric glands freshly isolated from neonatal mouse stomach using the Matrigel method. Organoids were developed within a day and were maintained for up to 10 days. The stem cell contribution and cellular dynamics during the formation of these organoids were investigated using bromodeoxyuridine labelling. Organoids were further characterized at different time points by using calcein-propidium iodide labelling, electron microscopy, lectin histochemistry, immunohistochemistry and quantitative reverse-transcription polymerase chain reactions. Evidence of differentiation into gastric mucus-producing epithelial cells was detected.
To use the gastric organoids as a model for investigating the role of aryl hydrocarbon receptors (AhR), one of the important factors involved in stem cell control as well as in the pathogenesis of cancer. Their expression levels were first tested using immunohistochemistry. Data revealed the localization of AhR in mGS cells and cells forming the gastric primary organoids. These findings correlated with the cellular expression of AhR in gastric mucosa of mice, rats and humans. Immunolabeled cells were located in the middle of gastric glands where dividing stem cells are located. To test the consequences of AhR activation, two-day-old organoids were incubated with 0.1 and 1.0 nM of dioxin for two days. Upregulation of cytochrome P450 indicated activation of AhR. This was associated with upregulation of Oct-4 expression which suggested enhancement of self-renewal of gastric stem cells. Similar findings were observed in human gastric precancerous and cancer tissues.
In conclusion, gastric organoids are useful models to study the regulation of gastric stem cells. Activation of AhR plays an important role in gastric stem cell self-renewal via Oct-4 upregulation. This could also explain the role of AhR in gastric cancer development. This study provides new insights into gastric stem cells which help in better understanding of their roles in tissue engineering and gastric cancer.
Afroz Taleb, Shakila, "ESTABLISHMENT OF ORGANOIDS FROM GASTRIC STEM CELLS AND THEIR USE TO STUDY THE ROLE OF ARYL-HYDROCARBON RECEPTORS" (2020). Dissertations. 149.