CSI: Intestine: Investigating intestinal crypt and stem cell dynamics by in vivo imaging

Abstract

The intestinal epithelium is a highly dynamic tissue that continuously renews itself to maintain its barrier and absorptive functions. This process is driven by intestinal stem cells located at the base of crypts, where cells are generated, differentiate, and are eventually shed in a tightly regulated conveyor belt–like system. Although this rapid turnover is essential for tissue homeostasis and repair, it also increases the risk of DNA damage accumulation and tumorigenesis over time. In this thesis, the intestine was studied as a living and dynamic system, focusing on the behavior of individual stem cells within their native microenvironment. Using advanced intravital microscopy techniques, referred to as a “CSI: intestine” approach, stem cells and their progeny were tracked in real time in living mice. This allowed direct observation of cellular processes such as movement, competition, regeneration, and responses to inflammation and oncogenic mutations. Chapter 1 provided an overview of intestinal biology, including epithelial organization, stem cell hierarchies, and mechanisms that protect tissue integrity by limiting mutation accumulation. These concepts form the basis for understanding how disruption of homeostasis can predispose tissue to disease. In Chapter 2, we investigated the role of cell movement in stem cell function. We demonstrated that stem cells in the small intestine display considerable plasticity: cells displaced from the stem cell niche can return through Wnt-dependent retrograde movement and regain stem cell identity. This behavior increases the pool of cells capable of long-term tissue renewal. In contrast, retrograde movement is largely absent in the large intestine, resulting in a more restricted stem cell pool. These findings identify retrograde movement as an important regulator of stem cell dynamics, regeneration, and mutation fixation. Chapter 3 examined how inflammation alters stem cell behavior using a DSS-induced colitis model. Inflammatory injury accelerated stem cell competition and clonal fixation, particularly in the large intestine. Although this enhanced regeneration, it also favored the persistence and expansion of mutant cells. These effects were region-specific and associated with localized tissue damage, emphasizing the role of the microenvironment in early tumorigenesis. In Chapter 4, we developed an in vivo model combining multiphoton laser ablation with longitudinal intravital imaging to study intestinal regeneration. This approach enabled controlled induction of local damage and revealed dynamic remodeling processes such as crypt fission, fusion, and crypt loss, providing insight into how tissue architecture is restored after injury. Finally, Chapter 5 investigated the effects of oncogenic mutations on stem cell dynamics. KRAS mutations provided mutant cells with a competitive advantage, allowing clonal expansion and persistence within the epithelium. In contrast, HRAS-mutant cells were actively eliminated through mechanisms such as basal extrusion. These findings correspond with human colorectal cancer data, where KRAS mutations are common and HRAS mutations are rare. Together, this thesis demonstrates that intestinal homeostasis is governed by a balance between stem cell competition, positional plasticity, and tissue remodeling. Small changes in cellular behavior or genetic context can determine whether mutations are eliminated or retained, ultimately influencing the earliest stages of cancer development.