Fate plasticity in the intestine: the devil is in the detail

The intestinal epithelium possesses a remarkable ability for both proliferation and regeneration. The last two decades have generated major advances in our understanding of the stem cell populations responsible for its maintenance during homeostasis and more recently the events that occur during injury induced regeneration. These fundamental discoveries have capitalised on the use of transgenic mouse models and in vivo lineage tracing to make their conclusions. It is evident that maintenance is driven by rapidly proliferating crypt base stem cells, but complexities associated with the technicality of mouse modelling have led to several overlapping populations being held responsible for the same behaviour. Similarly, it has been shown that essentially any population in the intestinal crypt can revert to a stem cell state given the correct stimulus during epithelial regeneration. Whilst these observations are profound it is uncertain how relevant they are to human intestinal homeostasis and pathology. Here, these recent studies are presented, in context with technical considerations of the models used, to argue that their conclusions may indeed not be applicable in understanding ‘homeostatic regeneration’ and experimental suggestions presented for validating their results in human tissue.Supported by a Fellowship grant from Cancer Research UK C14094/A27178; and core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute

Mouse models of colorectal cancer as preclinical models.

In this review, we discuss the application of mouse models to the identification and pre-clinical validation of novel therapeutic targets in colorectal cancer, and to the search for early disease biomarkers. Large-scale genomic, transcriptomic and epigenomic profiling of colorectal carcinomas has led to the identification of many candidate genes whose direct contribution to tumourigenesis is yet to be defined; we discuss the utility of cross-species comparative 'omics-based approaches to this problem. We highlight recent progress in modelling late-stage disease using mice, and discuss ways in which mouse models could better recapitulate the complexity of human cancers to tackle the problem of therapeutic resistance and recurrence after surgical resection.REM, SJAB, MJA and DJA are funded by Cancer Research UK.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/bies.20150003

An analysis of SARS-CoV-2 cell entry genes identifies the intestine and colorectal cancer as susceptible tissues.

SARS-CoV-2 is the causative agent for the COVID-19 pandemic. COVID-19 has necessitated rapid changes in surgical practice and organisation through both the initial peak and ongoing recovery period 1. SARS-CoV-2 infects cells by interacting with the host cell surface protein ACE2 and utilises TMPRSS2 in viral spike protein priming to facilitate cell entry (Fig. 1a) 2. Whilst COVID-19 is predominantly a respiratory disease approximately 15% of patients have concurrent gastrointestinal symptoms 3. SARS-CoV-2 RNA and live virus have been identified in stool from COVID-19 patients and SARS-CoV-2 readily infects intestinal organoids 4-6. Despite these circumstantial data, gastrointestinal transmission has not yet been formally confirmed. Cancers commonly express different genes from the tissue of origin and it is largely unexplored whether tumours can be infected with SARS-CoV-2. We sought to explore the expression of ACE2 and TMPRSS2 in large publicly available normal tissue and pan-cancer expression data sets to understand whether levels of these genes identify susceptible tissues.SJAB is supported by an Advanced Clinician Scientist Fellowship grant from Cancer Research UK C14094/A27178; and core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute

The use of oral antibiotics and mechanical bowel preparation in elective colorectal resection for the reduction of surgical site infection.

Surgical site infection (SSI) is a major cause of morbidity following elective colorectal resection worldwide. Reduction in SSI rates can be achieved with the use of SSI reduction bundles. Debate about the role of mechanical bowel preparation and oral antibiotics (MOAB) in reducing SSI has persisted over decades with considerable variation in international practice. This article summarises the arguments for and against the routine use of MOAB in the elective setting, highlighting the areas of controversy and evidence gaps and provides pragmatic suggestions for colorectal practice.This topical debate paper was commissioned by the President and Executive of the Association of Coloproctology of Great Britain and Ireland. ACPGBI also funded the open access publication fee. The authors would like to thank Ms J Pipe for her comments from the patient perspective and Miss N Fearnhead for her support in developing this debate article

Isolation of Human Colon Stem Cells Using Surface Expression of PTK7

SummaryInsertion of reporter cassettes into the Lgr5 locus has enabled the characterization of mouse intestinal stem cells (ISCs). However, low cell surface abundance of LGR5 protein and lack of high-affinity anti-LGR5 antibodies represent a roadblock to efficiently isolate human colonic stem cells (hCoSCs). We set out to identify stem cell markers that would allow for purification of hCoSCs. In an unbiased approach, membrane-enriched protein fractions derived from in vitro human colonic organoids were analyzed by quantitative mass spectrometry. Protein tyrosine pseudokinase PTK7 specified a cell population within human colonic organoids characterized by highest self-renewal and re-seeding capacity. Antibodies recognizing the extracellular domain of PTK7 allowed us to isolate and expand hCoSCs directly from patient-derived mucosa samples. Human PTK7+ cells display features of canonical Lgr5+ ISCs and include a fraction of cells that undergo differentiation toward enteroendocrine lineage that resemble crypt label retaining cells (LRCs)

Targeting Acid Ceramidase to Improve the Radiosensitivity of Rectal Cancer.

Previous work utilizing proteomic and immunohistochemical analyses has identified that high levels of acid ceramidase (AC) expression confers a poorer response to neoadjuvant treatment in locally advanced rectal cancer. We aimed to assess the radiosensitising effect of biological and pharmacological manipulation of AC and elucidate the underlying mechanism. AC manipulation in three colorectal cancer cell lines (HT29, HCT116 and LIM1215) was achieved using siRNA and plasmid overexpression. Carmofur and a novel small molecular inhibitor (LCL521) were used as pharmacological AC inhibitors. Using clonogenic assays, we demonstrate that an siRNA knockdown of AC enhanced X-ray radiosensitivity across all colorectal cancer cell lines compared to a non-targeting control siRNA, and conversely, AC protein overexpression increased radioresistance. Using CRISPR gene editing, we also generated AC knockout HCT116 cells that were significantly more radiosensitive compared to AC-expressing cells. Similarly, two patient-derived organoid models containing relatively low AC expression were found to be comparatively more radiosensitive than three other models containing higher levels of AC. Additionally, AC inhibition using carmofur and LCL521 in three colorectal cancer cell lines increased cellular radiosensitivity. Decreased AC protein led to significant poly-ADP ribose polymerase-1 (PARP-1) cleavage and apoptosis post-irradiation, which was shown to be executed through a p53-dependent process. Our study demonstrates that expression of AC within colorectal cancer cell lines modulates the cellular response to radiation, and particularly that AC inhibition leads to significantly enhanced radiosensitivity through an elevation in apoptosis. This work further solidifies AC as a target for improving radiotherapy treatment of locally advanced rectal cancer

Isolation of Human Colon Stem Cells Using Surface Expression of PTK7.

Insertion of reporter cassettes into the Lgr5 locus has enabled the characterization of mouse intestinal stem cells (ISCs). However, low cell surface abundance of LGR5 protein and lack of high-affinity anti-LGR5 antibodies represent a roadblock to efficiently isolate human colonic stem cells (hCoSCs). We set out to identify stem cell markers that would allow for purification of hCoSCs. In an unbiased approach, membrane-enriched protein fractions derived from in vitro human colonic organoids were analyzed by quantitative mass spectrometry. Protein tyrosine pseudokinase PTK7 specified a cell population within human colonic organoids characterized by highest self-renewal and re-seeding capacity. Antibodies recognizing the extracellular domain of PTK7 allowed us to isolate and expand hCoSCs directly from patient-derived mucosa samples. Human PTK7+ cells display features of canonical Lgr5+ ISCs and include a fraction of cells that undergo differentiation toward enteroendocrine lineage that resemble crypt label retaining cells (LRCs)

An individual based computational model of intestinal crypt fission and its application to predicting unrestrictive growth of the intestinal epithelium.

Intestinal crypt fission is a homeostatic phenomenon, observable in healthy adult mucosa, but which also plays a pathological role as the main mode of growth of some intestinal polyps. Building on our previous individual based model for the small intestinal crypt and on in vitro cultured intestinal organoids, we here model crypt fission as a budding process based on fluid mechanics at the individual cell level and extrapolated predictions for growth of the intestinal epithelium. Budding was always observed in regions of organoids with abundant Paneth cells. Our data support a model in which buds are biomechanically initiated by single stem cells surrounded by Paneth cells which exhibit greater resistance to viscoelastic deformation, a hypothesis supported by atomic force measurements of single cells. Time intervals between consecutive budding events, as simulated by the model and observed in vitro, were 2.84 and 2.62 days, respectively. Predicted cell dynamics was unaffected within the original crypt which retained its full capability of providing cells to the epithelium throughout fission. Mitotic pressure in simulated primary crypts forced upward migration of buds, which simultaneously grew into new protruding crypts at a rate equal to 1.03 days-1 in simulations and 0.99 days-1 in cultured organoids. Simulated crypts reached their final size in 4.6 days, and required 40 6.2 days to migrate to the top of the primary crypt. The growth of the secondary crypt is independent of its migration along the original crypt. Assuming unrestricted crypt fission and multiple budding events, a maximal growth rate of the intestinal epithelium of 0.10 days-1 43 is predicted and thus approximately 22 days are required for a 10-fold increase of polyp size. These predictions are in agreement with the time reported to develop macroscopic adenomas in mice after loss of Apc in intestinal stem cells

Stem cell decisions:A twist of fate or a niche market?

AbstractEstablishing and maintaining cell fate in the right place at the right time is a key requirement for normal tissue maintenance. Stem cells are at the core of this process. Understanding how stem cells balance self-renewal and production of differentiating cells is key for understanding the defects that underpin many diseases. Both, external cues from the environment and cell intrinsic mechanisms can control the outcome of stem cell division. The role of the orientation of stem cell division has emerged as an important mechanism for specifying cell fate decisions. Although, the alignment of cell divisions can dependent on spatial cues from the environment, maintaining stemness is not always linked to positioning of stem cells in a particular microenvironment or `niche'. Alternate mechanisms that could contribute to cellular memory include differential segregation of centrosomes in asymmetrically dividing cells

Assessment of the response of Plasmodiophora brassicae in contaminated horticultural land, using lime‐based fertilizer concentrations

Infection of brassica crops with the clubroot pathogen, Plasmodiophora brassicae, can result in stunted plant growth and wilting, which can severely affect crop yield. Determining P. brassicae infection within a field prior to crop planting has long posed a problem for choosing appropriate control treatments. The options for control of this pathogen are limited and in the UK and are based on adjusting pH with soil amendments. In this study quantitative polymerase chain reaction (qPCR) was investigated for measurement of this pathogen in different control treatments. The qPCR was capable of reliably quantifying P. brassicae at levels greater than and including 103 resting spores/g soil. The assay was used to study the effect of lime‐based products (LimeX) on the incidence of the clubroot pathogen in field trials with broccoli crops grown on contaminated land. The results showed that variation occurred in clubroot resting spore levels in treated and untreated plots during the crop growing period. In year one there was a 96% decrease in spore load during the growth of the crop. Treatment with LimeX resulted in a greater marketable head weight of broccoli in 2 years of the field trials, and significantly reduced gall numbers on the roots in 1 year. The rate of lime (calcium carbonate) application was not found to have a significant effect in this study, however a greater reduction in clubroot was observed at higher LimeX concentrations