Gutierrez-Gonzalez, L. et al. Analysis of the clonal architecture of the human small intestinal epithelium establishes a common stem cell for all lineages and reveals a mechanism for the fixation and spread of mutations. J. Pathol. 217, 489-496

Histopathology Unit, London Research Institute, Cancer Research UK, London, UK.
The Journal of Pathology (Impact Factor: 7.43). 03/2009; 217(4):489-96. DOI: 10.1002/path.2502
Source: PubMed


Little is known about the clonal structure or stem cell architecture of the human small intestinal crypt/villus unit, or how mutations spread and become fixed. Using mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion of stem cell progeny, we aimed to provide answers to these questions. Enzyme histochemistry (for cytochrome c oxidase and succinate dehydrogenase) was performed on frozen sections of normal human duodenum. Laser-capture microdissected cells were taken from crypts/villi. The entire mitochondrial genome was amplified using a nested PCR protocol; sequencing identified mutations and immunohistochemistry demonstrated specific cell lineages. Cytochrome c oxidase-deficient small bowel crypts were observed within all sections: negative crypts contained the same clonal mutation and all differentiated epithelial lineages were present, indicating a common stem cell origin. Mixed crypts were also detected, confirming the existence of multiple stem cells. We observed crypts where Paneth cells were positive but the rest of the crypt was deficient. We have demonstrated patches of deficient crypts that shared a common mutation, suggesting that they have divided by fission. We have shown that all cells within a small intestinal crypt are derived from one common stem cell. Partially-mutated crypts revealed some novel features of Paneth cell biology, suggesting that either they are long-lived or a committed Paneth cell-specific long-lived progenitor was present. We have demonstrated that mutations are fixed in the small bowel by fission and this has important implications for adenoma development.

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Available from: Stuart A.C. McDonald, Oct 07, 2015
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    • "Infrequent stochastic loss of CCO activity (CCOÀ) is observed in the human intestine and is attributed to an underlying somatic mitochondrial DNA (mtDNA) mutation (Taylor et al., 2003). mtDNA sequencing confirms that adjacent CCOÀ cells in the intestine are clonally derived (Fellous et al., 2009; Greaves et al., 2006; Gutierrez-Gonzalez et al., 2009; Taylor et al., 2003). CCO activity was assessed in en face serial sections of colonic mucosa (n = 9 patients; Table S1). "
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    ABSTRACT: Human intestinal stem cell and crypt dynamics remain poorly characterized because transgenic lineage-tracing methods are impractical in humans. Here, we have circumvented this problem by quantitatively using somatic mtDNA mutations to trace clonal lineages. By analyzing clonal imprints on the walls of colonic crypts, we show that human intestinal stem cells conform to one-dimensional neutral drift dynamics with a ‘‘functional’’ stem cell number of five to six in both normal patients and individuals with familial adenomatous polyposis (germline APC�/+). Furthermore, we show that, in adenomatous crypts (APC�/�), there is a proportionate increase in both functional stem cell number and the loss/replacement rate. Finally, by analyzing fields of mtDNA mutant crypts, we show that a normal colon crypt divides around once every 30–40 years, and the division rate is increased in adenomas by at least an order of magnitude. These data provide in vivo quantification of human intestinal stem cell and crypt dynamics.
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    • "In order to directly assess the in vivo identity and potency of individual human airway epithelial progenitor cells, we make use of the predisposition of mtDNA to develop spontaneous mutations that affect expression of the cytochrome c oxidase (CCO) gene. CCO gene mutations occur spontaneously in all cells in a stochastic manner, do not significantly affect cellular function, and are unrelated to cellular toxicant exposure (Elson et al., 2001; Taylor et al., 2001; Carew and Huang, 2002; Taylor et al., 2003; Taylor and Turnbull, 2005; Greaves et al., 2006; McDonald et al., 2008; Fellous et al., 2009; Gutierrez-Gonzalez et al., 2009; Lin et al., 2010; Gaisa et al., 2011a; Nicholson et al., 2011). Thus, the division and accumulation of CCO-deficient cells leads to the formation of clonal patches of CCO-deficient cells within tissues, including the normal airway, and their examination provides a unique, histologically traceable record of airway progenitor cell fate. "
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    ABSTRACT: Lineage tracing approaches have provided new insights into the cellular mechanisms that support tissue homeostasis in mice. However, the relevance of these discoveries to human epithelial homeostasis and its alterations in disease is unknown. By developing a novel quantitative approach for the analysis of somatic mitochondrial mutations that are accumulated over time, we demonstrate that the human upper airway epithelium is maintained by an equipotent basal progenitor cell population, in which the chance loss of cells due to lineage commitment is perfectly compensated by the duplication of neighbours, leading to “neutral drift” of the clone population. Further, we show that this process is accelerated in the airways of smokers, leading to intensified clonal consolidation and providing a background for tumorigenesis. This study provides a benchmark to show how somatic mutations provide quantitative information on homeostatic growth in human tissues, and a platform to explore factors leading to dysregulation and disease. DOI:
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    • "As new crypts arise from rapid expansion of resident stem cells and subsequent fission, these polyclonal crypts become monoclonal. This clonality is maintained throughout adulthood, and can serve to fix genetic and epigenetic changes geographically within the intestines (Fuller et al., 1990; Endo et al., 1995; Novelli et al., 1996) For example, mutations can arise in crypts and these mutant crypts can expand by fission to create patches of clonally-derived cells with identical mutations (Greaves et al., 2006; Gutierrez-Gonzalez et al., 2009). Epigenetic marks are similarly stable and can be used to study stem cell dynamics in the intestines (Yatabe et al., 2001). "
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