Characterizing intestinal stem cells. An important part of the puzzle

Abstract

The aim of this thesis is to gain better understanding of intestinal stem cells in normal and malignant conditions. In chapter 2 we take our first steps in the characterization of intestinal stem cells. We determine that they have telomerase activity but still suffer from telomere loss. In addition we pinpoint their cell cycle length and demonstrate that they randomly segregate their chromosomes. In order to accurately visualize the chromosome segregations we developed a sectioning technique that maintains optimal tissue and cellular morphology and allows detection of DNA-intercalating analogs and genetically expressed fluorescence. This technique is described in chapter 3 and is also used for imaging in the latter chapters. In chapter 4 we re-examine the expression of Bmi1, a proposed marker for a distinct stem cell population in the crypt. We demonstrate that, in contradiction to what was originally published, Bmi1 is not predominantly expressed in cells at the +4 position. We show that Bmi1 marks cells at all positions in the crypt and villus, including CBCs, transit-amplifying cells and even differentiated goblet cells and Paneth cells. In chapter 5 we investigate the presence of stem cell activity within primary intestinal adenomas in mice. We introduce a novel strategy of lineage re-tracing to visualize Lgr5+ stem cells and their progeny within adenomas. We demonstrate that Lgr5+ adenoma cells self-renew and give rise to all other adenoma cell types. In chapter 6 we examine the potential spread of stem cells with oncogenic mutations. We demonstrate that activating oncogenic K-ras in stem cells gives them a competition advantage over their wild-type counterparts in the crypt. We further show that K-ras mutant cells spread through the epithelium by enhanced crypt fission. Chapter 7 gives a summarizing discussion of the work presented in this thesis.