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Troy+ stomach stem cells in homeostasis, repair and pathogenesis

Periodic Reporting for period 3 - Troy Stem Cells (Troy+ stomach stem cells in homeostasis, repair and pathogenesis)

Reporting period: 2017-10-01 to 2019-03-31

In the adult mammalian stomach, due to constant exposure to mechanical stress and to hostile contents of the lumen, highly specialized cell types are constantly reproduced in order to maintain the function of the gastrointestinal tract. Recently, Troy+ chief cells are identified as a novel stem cell population in the corpus epithelium. Troy+ chief cells displayed a very low proliferation rate indicating their quiescent nature compared to other known gastro-intestinal tract stem cells. Interestingly, these stem cells can actively divide upon tissue damage, suggesting distinctive statuses under conditions of homeostasis and injury.

As Troy+ stomach stem cells exhibit interconvertible characteristics i.e. quiescent and proliferative, they represent a unique model of adult stem cells with which we can study the dynamics of stem cell propagation in homeostasis and regeneration and the underlying mechanism of this switch by analysing molecular and epigenetic profiles. We also study the stomach stem cell programme in homeostasis and regeneration using in vitro and in vivo functional genetics. Based on our current knowledge, we can understand how human stomach stem cells maintain the stomach in normal and pathological conditions, which will eventually help people with stomach diseases.

Here we pursue three main aims:
- Investigating Troy+ stem cell dynamics during homeostasis and injury repair
- Unmasking the stomach stem cell programme using in vitro and in vivo functional genetics
- Characterising human stomach stem cells
Task I: Troy+ stem cell dynamics during homeostasis and injury repair
I-1) Tracking Troy+ stem cells under homeostasis and conditions of specific cell lineage loss: Using Troy-eGFP-ires-CreER;Rosa-YFP (reporter) mice, we have collected clonal growth dynamics at population level. This result is currently under mathematical model analysis with my collaborator – Prof. Ben Simons (University of Cambridge). In order to understand which part of the propagation dynamics is changed in response to the loss of a certain cell type, we have developed a cell type specific diphtheria toxin Receptor (DTR) knockin mouse lines.
I-2) Investigating expression changes of Troy+ cells in quiescent and activated states: The Troy+ StSC showed a rapid response upon tissue damage, implying a molecular switch from quiescence to active cycling. We have obtained Troy+StSC expression profiles during tissue damage and repair using RNAseq analysis. A comprehensive bioinformatics analysis has been carried out and we found several candidate molecules that may serve as the molecular switch between stem cell quiescence and active proliferation.
I-3) Analysing epigenetic changes of Troy+ cells in quiescent and activated states: This part of research will be followed soon after the completion of Task I-2.
I-4) Determining the stem cell hierarchy in stomach corpus epithelium: We have performed unbiased random genetic labelling and lineage tracing in the stomach corpus epithelium and found the fundamental structure of gastric corpus glands. We are about to test several new lineage tracers in order to confirm our new hypothesis.

Task II: Unmasking the stomach stem cell programme using in vitro and in vivo functional genetics
II-1) Investigating the tumour suppressor role of RNF43 and ZNRF3 in stomach epithelium: We have developed stomach epithelium-specific CreERT2 knockin mouse. By crossing this new Cre driver with our RNF43;ZNRF3 (RZ) conditional knockout mice, we have tested the effect of RZ knockout in the stomach epithelium.
II-2) Rapid gain- and loss-of-function analyses of Troy+ stem cell-specific genes in gastric organoid culture: Two sets of CRISPR screening are currently being done with our gastric organoid model. In one scheme, we are testing a number of gastric cancer-related genes and in the other scheme, we are applying a genome-wide library.
II-3) Conditional knockout modelling of selected candidates: As mentioned in Task II-1, we have developed stomach epithelium-specific CreERT2 knockin mouse with Dr Daniel Stange (TUD, Dresden). Several candidate genes have been subjected to conditional mouse generation and we will be able to analyse their phenotype starting from late 2017.

Task III: Characterising human stomach stem cells
III-1) Isolation and characterisation of human stomach stem cells from human gastric organoids: In order to identify human stem cell markers we have taken advantage of the organoid cell culture. Normal human stomach from five patients has been successfully grown out into independent organoid lines. In normal gastric organoids growth factor deprivation in the medium induces profound differentiation including downregulation of the Wnt signalling pathway. We have performed MassSpec of membrane enriched proteins in organoids (grown in normal medium) vs. organoids (grown in differentiation medium). 400 differentially regulated proteins have thus been identified.
III-2) Isolation and characterisation of human stomach stem cells from various pathological conditions: We are currently waiting for the identification of suitable stem cell marker from Task III-1.
III-3) Investigating the function of candidate genes using human organoids: We are testing the role of RNF43 and ZNRF3 in the human stomach organoid line. We expect to apply more interesting gene candidates to human stomach organoids (e.g. genes identified from Task III-2 as well as Task I-2 and II-2).
A fundamentally important question in adult stem cell biology relates to how adult stem cells can make decisions in various contexts. Under homeostatic conditions, it could be considered preferable to protect genetic information by restricting the frequency of cell division. However, upon tissue damage, these quiescent stem cells need to sense and integrate extrinsic signals to facilitate the required cellular response in order to repair the tissue. Depending on the type of injury, stem cells may also activate appropriate differentiation programmes to specifically replace lost cell types. In the current project, my team has been trying to address all these questions. We will be able to identify the functional switch that regulates quiescence and active cell cycle in the adult stem cells. This work can provide novel insights on to the hidden nature of dormant cancer stem cells that often escape chemotherapy in clinic.

Stomach cancer and related diseases have a great impact on the society and economy. Worldwide stomach cancer ranges at position three in the number of new cancer cases and position two for the number of cancer related death. Of note, the numbers of gastric cancer at the esophageal junction are continuously on the rise. Technically, it was not feasible to genetically study the stomach in mice due to the lack of appropriate stomach-specific Cre lines. Our team has overcome this problem by establishing a stomach-specific CreERT2 for modelling gastric cancer in mice. Our progress in basic science will clearly improve our understanding of human gastric cancer.
Stomach epithelium labelled with four different fluorescent proteins