Final Report Summary - INFO (Organoids as infection model)
The mucosal surfaces of the human stomach and gut are in constant contact with bacteria and viruses. The study of host-microbe interactions is hampered by a lack of suitable model systems: many bacteria or viruses cannot be cultured or existing models do not sufficiently recount the human disease. Recently, a new culture system has been developed in which adult stem cells give rise to three-dimensional structures (‘organoids’) resembling mature, polarized epithelium. This project aims to test the organoids as model systems for infectious agents.
Helicobacter pylori (H. pylori) is a gastric pathogen, that currently lacks appropriate models to mimic human disease. To develop the human gastric epithelial culture, we used normal tissue from resection material from gastric resection from cancer patients, isolated gastric glands and embedded them in Matrigel. Culture conditions were established and the optimal conditions for indefinite (>1year tested) expansion require EGF, Noggin, Rspodin, Wnt, FGF10, Gastrin and TGFβ-inhibitor (ENRWFGTi). In these conditions, organoids grow as cystic 3-dimentional structures within the Matrigel and comprise of 4 different cell types of the human stomach: Mucin (MUC) 5AC-positive pit cells, MUC6-positive gland mucus cells, pepsinogen (PGC)-positive chief cells and somatostatin (SST) positive enteroendocrine cells. Using Wnt and Nicotinamide, we could direct the organoids into either the pit cell lineage (“pit-type”) or the gland cell lineages (“gland-type”).
In an infected individual, H. pylori resides in the stomach lumen and has there contact with the apical side of cells. In the organoids, the apical side faces the cysts lumen. To enable bacteria to reach their natural side of infection we microinjected bacteria into organoids. Successful injection was confirmed in confocal microscopy. Re-culturing bacteria from single infected organoids showed that the bacteria were viable inside the organoids. EM studies show that most bacteria reside in the lumen of gastric organoids but some bacteria can establish tight interaction with the epithelial cells.
We then globally assessed the cellular response to infection by genome wide RNA analysis using microarray. After 2h of infection, 25 genes were up-regulated. Many of them were known targets from the inflammatory signaling pathway NF-kappaB, such as the neutrophil attractor IL-8. Staining for the NF-kappaB subunit p65 demonstrated nuclear p65, indicating that indeed the pathway was activated. qPCR confirmed upregulation of IL-8. We then differentiated the organoids in either gland-type organoids or pit-type organoids, and found that while the pit-type organoids only react mildly to bacterial infection, the gland-type organoids mount a strong inflammatory response. This may have implications for the in vivo situation, where the stem cells are known to reside within the glands. The results are now published (Bartfeld et al. Gastroenterology, in press).
We could further show that gastric epithelial organoids can be established from single cells isolated from human gastric epithelium. These clonal organoids can differentiate into the four cell lines pit mucus cells, gland mucus cells, chief cells and enteroendocrine cells. The clonal organoids can be expanded in the same way as gland-derived organoids: they expand about 5-fold every 2 weeks and can be expanded indefinitely (1 year tested). Organoids can also be established from gastric tumors in parallel to healthy tissue from the same patient. The resulting organoids can be compared in drug testing (Gastroenterology in press).
We also tested the possibilities to use organoids as models for other microorganisms, that currently can not be cultured by any in vitro method, specifically Norovirus, or Segmented Filamentous Bacteria. Here, we have tested various methods for infection, including microinjection, but in both cases, the organoids do not support growth of the virus or bacteria respectively.
This work will have an impact on understanding gastric pathologies and the development of new therapies, including regenerative medicine. It is a basis for future patient-derived disease models, drug screens, gastric stem cell research and for the study of host pathogen interactions.
Correspondence should be addressed to bartfeld@gmx.de
Helicobacter pylori (H. pylori) is a gastric pathogen, that currently lacks appropriate models to mimic human disease. To develop the human gastric epithelial culture, we used normal tissue from resection material from gastric resection from cancer patients, isolated gastric glands and embedded them in Matrigel. Culture conditions were established and the optimal conditions for indefinite (>1year tested) expansion require EGF, Noggin, Rspodin, Wnt, FGF10, Gastrin and TGFβ-inhibitor (ENRWFGTi). In these conditions, organoids grow as cystic 3-dimentional structures within the Matrigel and comprise of 4 different cell types of the human stomach: Mucin (MUC) 5AC-positive pit cells, MUC6-positive gland mucus cells, pepsinogen (PGC)-positive chief cells and somatostatin (SST) positive enteroendocrine cells. Using Wnt and Nicotinamide, we could direct the organoids into either the pit cell lineage (“pit-type”) or the gland cell lineages (“gland-type”).
In an infected individual, H. pylori resides in the stomach lumen and has there contact with the apical side of cells. In the organoids, the apical side faces the cysts lumen. To enable bacteria to reach their natural side of infection we microinjected bacteria into organoids. Successful injection was confirmed in confocal microscopy. Re-culturing bacteria from single infected organoids showed that the bacteria were viable inside the organoids. EM studies show that most bacteria reside in the lumen of gastric organoids but some bacteria can establish tight interaction with the epithelial cells.
We then globally assessed the cellular response to infection by genome wide RNA analysis using microarray. After 2h of infection, 25 genes were up-regulated. Many of them were known targets from the inflammatory signaling pathway NF-kappaB, such as the neutrophil attractor IL-8. Staining for the NF-kappaB subunit p65 demonstrated nuclear p65, indicating that indeed the pathway was activated. qPCR confirmed upregulation of IL-8. We then differentiated the organoids in either gland-type organoids or pit-type organoids, and found that while the pit-type organoids only react mildly to bacterial infection, the gland-type organoids mount a strong inflammatory response. This may have implications for the in vivo situation, where the stem cells are known to reside within the glands. The results are now published (Bartfeld et al. Gastroenterology, in press).
We could further show that gastric epithelial organoids can be established from single cells isolated from human gastric epithelium. These clonal organoids can differentiate into the four cell lines pit mucus cells, gland mucus cells, chief cells and enteroendocrine cells. The clonal organoids can be expanded in the same way as gland-derived organoids: they expand about 5-fold every 2 weeks and can be expanded indefinitely (1 year tested). Organoids can also be established from gastric tumors in parallel to healthy tissue from the same patient. The resulting organoids can be compared in drug testing (Gastroenterology in press).
We also tested the possibilities to use organoids as models for other microorganisms, that currently can not be cultured by any in vitro method, specifically Norovirus, or Segmented Filamentous Bacteria. Here, we have tested various methods for infection, including microinjection, but in both cases, the organoids do not support growth of the virus or bacteria respectively.
This work will have an impact on understanding gastric pathologies and the development of new therapies, including regenerative medicine. It is a basis for future patient-derived disease models, drug screens, gastric stem cell research and for the study of host pathogen interactions.
Correspondence should be addressed to bartfeld@gmx.de