Accumulation of Somatic Mutations in Normal Human Colonic Epithelium

I investigated stem cell dynamics in normal human colon by detecting somatic variants affecting X-linked and autosomal genes using immunohistochemistry. Applying neutral clonal marks to a large cohort of patients to interpret age-related trends in clone frequencies has established the baseline stem cell dynamics of the tissue. Analysis of a number of new clonal marks showing biased behaviours suggests that different gene-specific mutations can subvert constraints resulting from the tissue architecture in different ways. In respect of intra-gland competition of stem cell derived clones, a disadvantage is observed for the histone modifier HDAC6, while at the other end of the spectrum, loss of the cohesin member STAG2 strongly advantages affected stem cells. Subsequent clonal expansion beyond the boundary of a single crypt is recognised by clones occupying multi-crypt patches. Quantification of such events allows the rate of lateral expansion for different mutations to be measured. Moderate effects were found for PTEN, p53 and STAG2, while mutations in the histone demethylase KDM6A generate very large areas of mutant epithelium in aged humans. Further, targeted sequencing revealed dramatic expansion of KRAS-mutant clones in histologically normal colon. Patches may arise from crypt fission and fusion events. Using a clonal mark based on mild periodic acid-Schiff staining, the neutral crypt fission and fusion rates were quantified. Against this baseline, it was found that KDM6A-mutant clones expand mainly by fission, while fusion remains at homeostatic levels. The emerging picture is that of the aged human colon consisting of a mosaic of different mutations. The work presented in this thesis offers detailed insights into the rates at which different gene-specific mutations arising in colonic stem cells can become fixed within individual crypts and undergo subsequent lateral expansion through crypt fission and fusion events. This defines the timeframe taken for cancer drivers to achieve high mutant allele burden within the tissue, which can serve as a basis for cancer prevention strategies.Wellcome Trus

Fixation and Spread of Somatic Mutations in Adult Human Colonic Epithelium.

We investigated the means and timing by which mutations become fixed in the human colonic epithelium by visualizing somatic clones and mathematical inference. Fixation requires two sequential steps. First, one of approximately seven active stem cells residing within each colonic crypt has to be mutated. Second, the mutated stem cell has to replace neighbors to populate the entire crypt in a process that takes several years. Subsequent clonal expansion due to crypt fission is infrequent for neutral mutations (around 0.7% of all crypts undergo fission in a single year). Pro-oncogenic mutations subvert both stem cell replacement to accelerate fixation and clonal expansion by crypt fission to achieve high mutant allele frequencies with age. The benchmarking of these behaviors allows the advantage associated with different gene-specific mutations to be compared irrespective of the cellular mechanisms by which they are conferred

Cell population dynamics in the course of adult hippocampal neurogenesis: Remaining unknowns

Neural stem cells (NSCs) generate new neurons throughout life in the mammalian hippocampus. The distinct developmental steps in the course of adult neurogenesis, including NSC activation, expansion, and neuronal integration, are increasingly well characterized down to the molecular level. However, substantial gaps remain in our knowledge about regulators and mechanisms involved in this biological process. This review highlights three long-standing unknowns. First, we discuss potency and identity of NSCs and the quest for a unifying model of short- and long-term self-renewal dynamics. Next, we examine cell death, specifically focusing on the early demise of newborn cells. Then, we outline the current knowledge on cell integration dynamics, discussing which (if any) neurons are replaced by newly added neurons in the hippocampal circuits. For each of these unknowns, we summarize the trajectory of studies leading to the current state of knowledge. Finally, we offer suggestions on how to fill the remaining gaps by taking advantage of novel technology to reveal currently hidden secrets in the course of adult hippocampal neurogenesis

A Diffusion-like Process Accommodates New Crypts During Clonal Expansion in Human Colonic Epithelium.

BACKGROUND & AIMS: Colorectal cancer (CRC) is thought to arise when the cumulative mutational burden within colonic crypts exceeds a certain threshold that leads to clonal expansion and ultimately neoplastic transformation. Therefore, quantification of the fixation and subsequent expansion of somatic mutations in normal epithelium is key to understanding colorectal cancer initiation. The aim of the present study was to determine how advantaged expansions can be accommodated in the human colon. METHODS: Immunohistochemistry was used to visualize loss of the cancer driver KDM6A in formalin-fixed paraffin-embedded (FFPE) normal human colonic epithelium. Combining microscopy with neural network-based image analysis, we determined the frequencies of KDM6A-mutant crypts and fission/fusion intermediates as well as the spatial distribution of clones. Mathematical modeling then defined the dynamics of their fixation and expansion. RESULTS: Interpretation of the age-related behavior of KDM6A-negative clones revealed significant competitive advantage in intracrypt dynamics as well as a 5-fold increase in crypt fission rate. This was not accompanied by an increase in crypt fusion. Mathematical modeling of crypt spacing identifies evidence for a crypt diffusion process. We define the threshold fission rate at which diffusion fails to accommodate new crypts, which can be exceeded by KRAS activating mutations. CONCLUSIONS: Advantaged gene mutations in KDM6A expand dramatically by crypt fission but not fusion. The crypt diffusion process enables accommodation of the additional crypts up to a threshold value, beyond which polyp growth may occur. The fission rate associated with KRAS mutations offers a potential explanation for KRAS-initiated polyps