Lineage selection and plasticity in the intestinal crypt.

We know more about the repertoire of cellular behaviours that define the stem and progenitor cells maintaining the intestinal epithelium than any other renewing tissue. Highly dynamic and stochastic processes define cell renewal. Historically the commitment step in differentiation is viewed as a ratchet, irreversibly promoting a given fate and corresponding to a programme imposed at the point of cell division. However, the emerging view of intestinal self-renewal is one of plasticity in which a stem cell state is easily reacquired. The pathway mediators of lineage selection are largely known but how they interface within highly dynamic populations to promote different lineages and yet permit plasticity is not. Advances in understanding gene regulation in the nervous system suggest possible mechanisms.AP was supported by Medical Research Council Research grant MR/K018329/1. DJW is funded by Cancer Research UK.This is the final published manuscript. It has been published by Elsevier in Current Opinion in Cell Biology here: http://www.sciencedirect.com/science/article/pii/S0955067414000866

Formula SAE Final Project Report

The Formula SAE team at Trinity University has been working on a race car project since 2015 and has made significant progress in constructing a nearly complete car. This year, the team focused on continuing that progress by working towards implementing a new design, an airfoil, and redesigning suspension components, while also ensuring compliance with various regulations and standards. This year’s team has faced several constraints along the way, including time and budget limitations, complying with safety, technical, and environmental regulations, and following specific design constraints for the airfoil. To achieve set goals and eventually participate in FSAE competitions, the team must also follow applicable codes and standards, including the General Regulations and Rules of Conduct in the 2023 Formula SAE rules and specific standards related to the subsystems of the car, such as bodywork and aerodynamic devices. The team identified incomplete subsystems that needed to be addressed, one of which was the engine\u27s ability to idle. The team tested the spark and injector timing relative to the crank position using a 120 frame per second high-speed camera. Then using TunerStudio, a software for tuning an aftermarket MegaSquirt ECU, the team came up with four separate tunes that had varying spark and injector timings to get the car to start and idle. Despite getting combustion to occur and for the car to run for a few power strokes, the team was unsuccessful in achieving a consistent and steady idle. The team had ambitious goals for the project, but unforeseen difficulties prevented many of the design requirements from being met. Requirements such as maximum speed, user control, safety belts and seat, steering system, and airfoil mounting system were not fully tested or implemented. The team identified components that need to be fabricated by future teams, including a brake failure emergency shut off switch and a brake light. The team developed a CFD wind tunnel model to test the proposed airfoil design and conducted a validation test for the CFD model using literature results as the subsonic wind tunnel facility on campus was not available. The FSAE team planned to compare the downforce generated by a 3D printed model of an airfoil to the Ansys CFD model by testing the 3D printed model in a subsonic wind tunnel, but access to the wind tunnel was not available. Instead, the team compared the Ansys coefficients to those obtained from an experiment, and the results show promising accuracy of the Ansys model. However, the team suggests focusing on the performance and accuracy at higher angles of attack to improve the model. Furthermore, the team created a hypothetical racetrack to analyze the performance benefit of the airfoil and made several assumptions to simplify the process. The team calculated the lap times by dividing the distance traveled by the velocity of the car at different points of the racetrack, accounting for the aerodynamic effects of the airfoil, and the effect of downforce on the car. Overall, the 2022-23 Formula SAE team at Trinity University has faced numerous challenges in their race car project, including adhering to regulations, addressing incomplete subsystems, and conducting validation tests without proper facilities. However, the team made significant progress and will continue to work towards implementing a new design and analyzing the performance benefits of an airfoil

Quantifying microsatellite mutation rates from intestinal stem cell dynamics in Msh2-deficient murine epithelium

Microsatellite sequences have an enhanced susceptibility to mutation and can act as sentinels indicating elevated mutation rates and increased risk of cancer. The probability of mutant fixation within the intestinal epithelium is dictated by a combination of stem cell dynamics and mutation rate. Here we exploit this relationship to infer microsatellite mutation rates. First a sensitive, multiplexed and quantitative method for detecting somatic changes in microsatellite length was developed that allowed the parallel detection of mutant [CA]n sequences from hundreds of low-input tissue samples at up to 14 loci. The method was applied to colonic crypts in Mus musculus and enabled detection of mutant subclones down to 20% of the cellularity of the crypt (around 50 of 250 cells). By quantifying age-related increases in clone frequencies for multiple loci, microsatellite mutation rates in wild-type and Msh2- deficient epithelium were established. An average 388-fold increase in mutation per mitosis rate was observed in Msh2-deficient epithelium (2.4 x10-2) compared to wild- type epithelium (6.2 x10-5)

Side population sorting separates subfractions of cycling and non-cycling intestinal stem cells

Isolation of quiescent intestinal stem cells (ISCs) has remained a challenge.By side population (SP) sorting we show cycling ISCs to be in the upper SP.The lower SP is non-cycling and has markers of the quiescent ISCs.In culture both upper and lower SP cells yield all 4 intestinal lineages.Evidence for a role of lower SP cells in repair of intestinal damage is discussed.We report here that side population (SP) sorting allows for the simultaneous isolation of two intestinal stem cell (ISC) subsets from wild-type (WT) mice which are phenotypically different and represent cycling and non-cycling pools of cells. Following 5-ethynyl-2′-deoxyuridine (EdU) injection, in the upper side population (USP) the percentage of EdU + was 36% showing this fraction to be highly proliferative. In the lower side population (LSP), only 0.4% of cells were EdU +, indicating this fraction to be predominantly non-cycling. Using Lgr5-EGFP mice, we show that Lgr5-EGFPhi cells, representing actively cycling ISCs, are essentially exclusive to the USP. In contrast, using histone 2B-YFP mice, SP analysis revealed YFP label retaining cells (LRCs) in both the USP and the LSP. Correspondingly, evaluation of the SP fractions for mRNA markers by qRT-PCR showed that the USP was enriched in transcripts associated with both quiescent and active ISCs. In contrast, the LSP expressed mRNA markers of quiescent ISCs while being de-enriched for those of the active ISC. Both the USP and LSP are capable of generating enteroids in culture which include the four intestinal lineages. We conclude that sorting of USP and LSP fractions represents a novel isolation of cycling and non-cycling ISCs from WT mice.Figure optionsDownload full-size imageDownload high-quality image (243 K)Download as PowerPoint slid

Characterization of a heat resistant beta-glucosidase as a new reporter in cells and mice.

BACKGROUND: Reporter genes are widely used in biology and only a limited number are available. We present a new reporter gene for the localization of mammalian cells and transgenic tissues based on detection of the bglA (SYNbglA) gene of Caldocellum saccharolyticum that encodes a thermophilic beta-glucosidase. RESULTS: SYNbglA was generated by introducing codon substitutions to remove CpG motifs as these are associated with gene silencing in mammalian cells. SYNbglA expression can be localized in situ or detected quantitatively in colorimetric assays and can be co-localized with E. coli beta-galactosidase. Further, we have generated a Cre-reporter mouse in which SYNbglA is expressed following recombination to demonstrate the general utility of SYNbglA for in vivo analyses. SYNbglA can be detected in tissue wholemounts and in frozen and wax embedded sections. CONCLUSIONS: SYNbglA will have general applicability to developmental and molecular studies in vitro and in vivo.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

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)

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)

Metaplastic Cdx2-depleted cells can be very disruptive neighbors.

In this issue of JEM, Balbinot et al. (https://doi.org/10.1084/jem.20170934) describe an original mechanism where Cdx2 inactivation regulates intestinal metaplastic to neoplastic transition in a paracrine fashion. Surprisingly, the target cells are neighboring "normal" Cdx2-positive cells

5-hydroxymethylcytosine and gene activity in mouse intestinal differentiation

Abstract: Cytosine hydroxymethylation (5hmC) in mammalian DNA is the product of oxidation of methylated cytosines (5mC) by Ten-Eleven-Translocation (TET) enzymes. While it has been shown that the TETs influence 5mC metabolism, pluripotency and differentiation during early embryonic development, the functional relationship between gene expression and 5hmC in adult (somatic) stem cell differentiation is still unknown. Here we report that 5hmC levels undergo highly dynamic changes during adult stem cell differentiation from intestinal progenitors to differentiated intestinal epithelium. We profiled 5hmC and gene activity in purified mouse intestinal progenitors and differentiated progeny to identify 43425 differentially hydroxymethylated regions and 5325 differentially expressed genes. These differentially marked regions showed both losses and gains of 5hmC after differentiation, despite lower global levels of 5hmC in progenitor cells. In progenitors, 5hmC did not correlate with gene transcript levels, however, upon differentiation the global increase in 5hmC content showed an overall positive correlation with gene expression level as well as prominent associations with histone modifications that typify active genes and enhancer elements. Our data support a gene regulatory role for 5hmC that is predominant over its role in controlling DNA methylation states