Crypt fusion as a homeostatic mechanism in the human colon.

OBJECTIVE: The crypt population in the human intestine is dynamic: crypts can divide to produce two new daughter crypts through a process termed crypt fission, but whether this is balanced by a second process to remove crypts, as recently shown in mouse models, is uncertain. We examined whether crypt fusion (the process of two neighbouring crypts fusing into a single daughter crypt) occurs in the human colon. DESIGN: We used somatic alterations in the gene cytochrome c oxidase (CCO) as lineage tracing markers to assess the clonality of bifurcating colon crypts (n=309 bifurcating crypts from 13 patients). Mathematical modelling was used to determine whether the existence of crypt fusion can explain the experimental data, and how the process of fusion influences the rate of crypt fission. RESULTS: In 55% (21/38) of bifurcating crypts in which clonality could be assessed, we observed perfect segregation of clonal lineages to the respective crypt arms. Mathematical modelling showed that this frequency of perfect segregation could not be explained by fission alone (p<10-20). With the rates of fission and fusion taken to be approximately equal, we then used the distribution of CCO-deficient patch size to estimate the rate of crypt fission, finding a value of around 0.011 divisions/crypt/year. CONCLUSIONS: We have provided the evidence that human colonic crypts undergo fusion, a potential homeostatic process to regulate total crypt number. The existence of crypt fusion in the human colon adds a new facet to our understanding of the highly dynamic and plastic phenotype of the colonic epithelium.

Inherited pathogenic mitochondrial DNA mutations and gastrointestinal stem cell populations.

Inherited mitochondrial DNA (mtDNA) mutations cause mitochondrial disease, but mtDNA mutations also occur somatically and accumulate during ageing. Studies have shown that the mutation load of some inherited mtDNA mutations decreases over time in blood, suggesting selection against the mutation. However, it is unknown whether such selection occurs in other mitotic tissues, and where it occurs within the tissue. Gastrointestinal epithelium is a canonical mitotic tissue rapidly renewed by stem cells. Intestinal crypts (epithelium) undergo monoclonal conversion with a single stem cell taking over the niche and producing progeny. We show: (1) that there is a significantly lower mtDNA mutation load in the mitotic epithelium of the gastrointestinal tract when compared to the smooth muscle in the same tissue in patients with the pathogenic m.3243A>G and m.8344A>G mutations; (2) that there is considerable variation seen in individual crypts, suggesting changes in the stem cell population; (3) that this lower mutation load is reflected in the absence of a defect in oxidative phosphorylation in the epithelium. This suggests that there is selection against inherited mtDNA mutations in the gastrointestinal stem cells that is in marked contrast to the somatic mtDNA mutations that accumulate with age in epithelial stem cells leading to a biochemical defect. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Analysis of clonal expansions through the normal and premalignant human breast epithelium reveals the presence of luminal stem cells.

It is widely accepted that the cell of origin of breast cancer is the adult mammary epithelial stem cell; however, demonstrating the presence and location of tissue stem cells in the human breast has proved difficult. Furthermore, we do not know the clonal architecture of the normal and premalignant mammary epithelium or its cellular hierarchy. Here, we use deficiency in the mitochondrial enzyme cytochrome c oxidase (CCO), typically caused by somatic mutations in the mitochondrial genome, as a means to perform lineage tracing in the human mammary epithelium. PCR sequencing of laser-capture microdissected cells in combination with immunohistochemistry for markers of lineage differentiation was performed to determine the clonal nature of the mammary epithelium. We have shown that in the normal human breast, clonal expansions (defined here by areas of CCO deficiency) are typically uncommon and of limited size, but can occur at any site within the adult mammary epithelium. The presence of a stem cell population was shown by demonstrating multi-lineage differentiation within CCO-deficient areas. Interestingly, we observed infrequent CCO deficiency that was restricted to luminal cells, suggesting that niche succession, and by inference stem cell location, is located within the luminal layer. CCO-deficient areas appeared large within areas of ductal carcinoma in situ, suggesting that the rate of clonal expansion was altered in the premalignant lesion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Clonal architecture of human prostatic epithelium in benign and malignant conditions.

The location of stem cells in the epithelium of the prostatic acinus remains uncertain, as does the cellular origin of prostatic neoplasia. Here, we apply lineage tracing to visualize the clonal progeny of stem cells in benign and malignant human prostates and understand the clonal architecture of this epithelium. Cells deficient for the mitochondrially-encoded enzyme cytochrome c oxidase (CCO) were identified in 27 frozen prostatectomy specimens using dual colour enzyme histochemistry and individual CCO-normal and -deficient cell areas were laser-capture microdissected. PCR-sequencing of the entire mitochondrial genome (mtDNA) of cells from CCO-deficient areas found to share mtDNA mutations not present in adjacent CCO-normal cells, thus proving a clonal origin. Immunohistochemistry was performed to visualize the three cell lineages normally present in the prostatic epithelium. Entire CCO-deficient acini, and part-deficient acini were found. Deficient patches spanned either basal or luminal cells, but sometimes also both epithelial cell types in normal, hyperplastic or atrophic epithelium, and prostatic intraepithelial neoplasia (PIN). Patches comprising both PIN and invasive cancer were observed. Each cell area within a CCO-deficient patch contained an identical mtDNA mutation, defining the patch as a clonal unit. CCO-deficient patches in benign epithelium contained basal, luminal and endocrine cells, demonstrating multilineage differentiation and therefore the presence of a stem cell. Our results demonstrate that the normal, atrophic, hypertrophic and atypical (PIN) epithelium of human prostate contains stem cell-derived clonal units that actively replenish the epithelium during ageing. These deficient areas usually included the basal compartment indicating the basal layer as the location of the stem cell. Importantly, single clonal units comprised both PIN and invasive cancer, supporting PIN as the pre-invasive lesion for prostate cancer.

Field cancerization in the GI tract.

The widely accepted paradigm for tumorigenesis begins with rate-limiting mutations in a key growth control gene resulting in immediate lesion growth. Tumor progression occurs as cells within the tumor acquire additional carcinogenic mutations. However, there is clear evidence that the road to cancer can begin long before the growth of a clinically detectable lesion - indeed, long before any of the usual morphological correlates of preneoplasia are recognizable. Field cancerization, the replacement of the normal cell population by a histologically nondysplastic but protumorigenic mutant cell clone, underlies the development of many cancer types, and in this article we review field cancerization in the GI tract. We present the evidence that field cancerization can underpin tumorigenesis in all gastrointestinal compartments, discuss the homeostatic mechanisms that could permit clone spread and highlight how an understanding of the mechanisms driving field cancerization is a means to study human stem cell biology. Finally, we discuss how appropriate recognition of the role of field cancerization in tumorigenesis could impact patient care.

Age-associated mitochondrial DNA mutations cause metabolic remodelling that contributes to accelerated intestinal tumorigenesis.

Oxidative phosphorylation (OXPHOS) defects caused by somatic mitochondrial DNA (mtDNA) mutations increase with age in human colorectal epithelium and are prevalent in colorectal tumours, but whether they actively contribute to tumorigenesis remains unknown. Here we demonstrate that mtDNA mutations causing OXPHOS defects are enriched during the human adenoma/carcinoma sequence, suggesting they may confer a metabolic advantage. To test this we deleted the tumour suppressor Apc in OXPHOS deficient intestinal stem cells in mice. The resulting tumours were larger than in control mice due to accelerated cell proliferation and reduced apoptosis. We show that both normal crypts and tumours undergo metabolic remodelling in response to OXPHOS deficiency by upregulating the de novo serine synthesis pathway (SSP). Moreover, normal human colonic crypts upregulate the SSP in response to OXPHOS deficiency prior to tumorigenesis. Our data show that age-associated OXPHOS deficiency causes metabolic remodelling that can functionally contribute to accelerated intestinal cancer development.