Defining and mining the intestinal stem cell signature

Abstract

The work in this thesis adds to our knowledge of intestinal stem cell physiology. After identification of Lgr5 as a specific marker for intestinal stem cells by Barker et al, we now set out to characterize them in depth. Taking advantage of the possibility to sort pure fractions of Lgr5+ stem cells based on EGFP expression from the Lgr5-ki mouse model, we performed both transcriptional profiling as well as quantitative proteomics to achieve a comprehensive overview over both the mRNA and the protein content of Lgr5 positive stem cells. A signature comprising 510 genes and proteins could be established: The intestinal stem cell signature was defined. Among the 510 genes and proteins defining the intestinal stem cell signature were a multitude of interesting candidates for further functional tests. A functional screening system needed to be established that recapitulated faithfully the in vivo situation while at the same time being easy to manipulate. The basis for this system was laid earlier by the development of the intestinal organoid culture system by Sato et al. Building on this, we established a retrovirus mediated approach that allowed us to conditionally manipulate the expression of any gene of interest in the organoid culture systems. Large scale mining of the intestinal stem cell signature for further candidates was now possible. Two genes were chosen to be interesting enough to generate mouse models. The first involved Lrig1, a negative feedback inhibitor of EGFR signaling. The EGFR signaling pathway is one of the most important pathways for the proliferative capacity of the intestine. Nevertheless, deregulation of this pathway is one of the most frequently found alterations in colorectal cancer. It therefore needs be tightly regulated. Using a Lrig1 knock-out mouse model, we could demonstrate that Lrig1 is essential in regulating EGFR signaling in intestinal stem cells, thereby balancing the need for tissue regeneration and suppression of malignant hyper-proliferation. The second mouse model concerned the gene Tnfrsf19, a Tnf receptor family member. Tnfrsf19 is expressed specifically in small intestinal Lgr5 stem cells. To explore if it is expressed in other adult stem cell populations, a Tnfrsf19-EGFR-ires-CreERT2 knock-in mouse was generated. This mouse allows for the detection and isolation of Tnfrsf19 expressing cell populations and at the same time for lineage tracing when crossed with a Cre reporter line. Tnfrsf19 expression was detected besides other organs in the corpus region of the stomach. Lineage tracing from the Tnfrsf19 locus resulted in the labeling of all differentiated cell types of the gastric corpus. We found that Tnfrsf19 positive cells are a sub-population of chief cells, one of the terminally differentiated cell types in the stomach. We could show that Tnfrsf19 positive cells are able to contribute to the normal homeostasis of the stomach by infrequently proliferating. Upon tissue damage, these cells can be activated. Tnfrsf19 positive chief cells therefore represent a quiescent pool of cells with multi-lineage stem cell potential both in homeostasis as well as in challenged situations.