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Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process [R. C. van der Wath, B. S. Gardiner, A. W. Burgess, D. W. Smith, 8, e73204 (2013); S. Kozar , 13, 626-633 (2013)], how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogen we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.
Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.
-induced gastritis is the strongest risk factor for gastric adenocarcinoma, a malignancy preceded by a series of well-defined histological stages, including metaplasia. One microbial constituent that augments cancer risk is the type 4 secretion system (T4SS), which translocates the oncoprotein CagA into host cells. Aberrant stem cell activation is linked to carcinogenesis, and Lrig1 (leucine-rich repeats and Ig-like domains 1) marks a distinct population of progenitor cells. We investigated whether microbial effectors with carcinogenic potential influence Lrig1 progenitor cells ex vivo and via lineage expansion within -infected gastric mucosa. Lineage tracing was induced in (Lrig1/YFP) mice that were uninfected or subsequently infected with or an isogenic mutant (nonfunctional T4SS). In contrast to infection with wild-type (WT) for 2 wk, infection for 8 wk resulted in significantly increased inflammation and proliferation in the corpus and antrum compared with uninfected or mice infected with the mutant. WT -infected mice harbored significantly higher numbers of Lrig1/YFP epithelial cells that coexpressed UEA1 (surface cell marker). The number of cells coexpressing intrinsic factor (chief cell marker), YFP (lineage marker), and GSII lectin (spasmolytic polypeptide-expressing metaplasia marker) were increased only by WT In human samples, Lrig1 expression was significantly increased in lesions with premalignant potential compared with normal mucosa or nonatrophic gastritis. In conclusion, chronic infection stimulates Lrig1-expressing progenitor cells in a -dependent manner, and these reprogrammed cells give rise to a full spectrum of differentiated cells.
In this chapter, heterogeneity is explored in the context of the ventricular-subventricular zone, the largest stem cell niche in the mammalian brain. This niche generates up to 10,000 new neurons daily in adult mice and extends over a large spatial area with dorso-ventral and medio-lateral subdivisions. The stem cells of the ventricular-subventricular zone can be subdivided by their anatomical position and transcriptional profile, and the stem cell lineage can also be further subdivided into stages of pre- and post-natal quiescence and activation. Beyond the stem cells proper, additional differences exist in their interactions with other cellular constituents of the niche, including neurons, vasculature, and cerebrospinal fluid. These variations in stem cell potential and local interactions are discussed, as well as unanswered questions within this system.
The ventricular-subventricular zone (V-SVZ) of the mammalian brain is a site of adult neurogenesis. Within the V-SVZ reside type B neural stem cells (NSCs) and type A neuroblasts. The V-SVZ is also a primary site for very aggressive glioblastoma (GBM). Standard-of-care therapy for GBM consists of safe maximum resection, concurrent temozolomide (TMZ), and X-irradiation (XRT), followed by adjuvant TMZ therapy. The question of how this therapy impacts neurogenesis is not well understood and is of fundamental importance as normal tissue tolerance is a limiting factor. Here, we studied the effects of concurrent TMZ/XRT followed by adjuvant TMZ on type B stem cells and type A neuroblasts of the V-SVZ in C57BL/6 mice. We found that chemoradiation induced an apoptotic response in type A neuroblasts, as marked by cleavage of caspase 3, but not in NSCs, and that A cells within the V-SVZ were repopulated given sufficient recovery time. 53BP1 foci formation and resolution was used to assess the repair of DNA double-strand breaks. Remarkably, the repair was the same in type B and type A cells. While Bax expression was the same for type A or B cells, antiapoptotic Bcl2 and Mcl1 expression was significantly greater in NSCs. Thus, the resistance of type B NSCs to TMZ/XRT appears to be due, in part, to high basal expression of antiapoptotic proteins compared with type A cells. This preclinical research, demonstrating that murine NSCs residing in the V-SVZ are tolerant of standard chemoradiation therapy, supports a dose escalation strategy for treatment of GBM. Stem Cells 2019;37:1629-1639.
© 2019 The Authors. Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press 2019.
OBJECTIVE - In the randomised scleroderma: Cyclophosphamide Or Transplantation (SCOT trial) (NCT00114530), myeloablation, followed by haematopoietic stem cell transplantation (HSCT), led to improved clinical outcomes compared with monthly cyclophosphamide (CYC) treatment in systemic sclerosis (SSc). Herein, the study aimed to determine global molecular changes at the whole blood transcript and serum protein levels ensuing from HSCT in comparison to intravenous monthly CYC in 62 participants enrolled in the SCOT study.
METHODS - Global transcript studies were performed at pretreatment baseline, 8 months and 26 months postrandomisation using Illumina HT-12 arrays. Levels of 102 proteins were measured in the concomitantly collected serum samples.
RESULTS - At the baseline visit, interferon (IFN) and neutrophil transcript modules were upregulated and the cytotoxic/NK module was downregulated in SSc compared with unaffected controls. A paired comparison of the 26 months to the baseline samples revealed a significant decrease of the IFN and neutrophil modules and an increase in the cytotoxic/NK module in the HSCT arm while there was no significant change in the CYC control arm. Also, a composite score of correlating serum proteins with IFN and neutrophil transcript modules, as well as a multilevel analysis showed significant changes in SSc molecular signatures after HSCT while similar changes were not observed in the CYC arm. Lastly, a decline in the IFN and neutrophil modules was associated with an improvement in pulmonary forced vital capacity and an increase in the cytotoxic/NK module correlated with improvement in skin score.
CONCLUSION - HSCT contrary to conventional treatment leads to a significant 'correction' in disease-related molecular signatures.
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
BACKGROUND - Male hypogonadism, arising from a range of etiologies including androgen-deprivation therapies (ADTs), has been reported as a risk factor for acquired long-QT syndrome (aLQTS) and torsades de pointes (TdP). A full description of the clinical features of aLQTS associated with ADT and of underlying mechanisms is lacking.
METHODS - We searched the international pharmacovigilance database VigiBase for men (n=6 560 565 individual case safety reports) presenting with aLQTS, TdP, or sudden death associated with ADT. In cardiomyocytes derived from induced pluripotent stem cells from men, we studied electrophysiological effects of ADT and dihydrotestosterone.
RESULTS - Among subjects receiving ADT in VigiBase, we identified 184 cases of aLQTS (n=168) and/or TdP (n=68; 11% fatal), and 99 with sudden death. Of the 10 ADT drugs examined, 7 had a disproportional association (reporting odds ratio=1.4-4.7; <0.05) with aLQTS, TdP, or sudden death. The minimum and median times to sudden death were 0.25 and 92 days, respectively. The androgen receptor antagonist enzalutamide was associated with more deaths (5430/31 896 [17%]; <0.0001) than other ADT used for prostate cancer (4208/52 089 [8.1%]). In induced pluripotent stem cells, acute and chronic enzalutamide (25 µM) significantly prolonged action potential durations (action potential duration at 90% when paced at 0.5 Hz; 429.7±27.1 (control) versus 982.4±33.2 (acute, <0.001) and 1062.3±28.9 ms (chronic; <0.001), and generated afterdepolarizations and/or triggered activity in drug-treated cells (11/20 acutely and 8/15 chronically). Enzalutamide acutely and chronically inhibited delayed rectifier potassium current, and chronically enhanced late sodium current. Dihydrotestosterone (30 nM) reversed enzalutamide electrophysiological effects on induced pluripotent stem cells.
CONCLUSIONS - QT prolongation and TdP are a risk in men receiving enzalutamide and other ADTs.
CLINICAL TRIAL REGISTRATION - URL: https://www.clinicaltrials.gov. Unique identifier: NCT03193138.
A novel, common, and potent cardiovascular risk factor has recently emerged: clonal hematopoiesis of indeterminate potential (CHIP). CHIP arises from somatic mutations in hematopoietic stem cells that yield clonal progeny of mutant leukocytes in blood. Individuals with CHIP have a doubled risk of coronary heart disease and ischemic stroke, and worsened heart failure outcomes independent of traditional cardiovascular risk factors. The recognition of CHIP as a nontraditional risk factor challenges specialists in hematology/oncology and cardiovascular medicine alike. Should we screen for CHIP? If so, in whom? How should we assess cardiovascular risk in people with CHIP? How should we manage the excess cardiovascular risk in the absence of an evidence base? This review explains CHIP, explores the clinical quandaries, strives to provide reasonable recommendations for the multidisciplinary management of cardiovascular risk in individuals with CHIP, and highlights current knowledge gaps.
Copyright © 2019 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.
p63 is a transcriptional regulator of ectodermal development that is required for basal cell proliferation and stem cell maintenance. p73 is a closely related p53 family member that is expressed in select p63-positive basal cells and can heterodimerize with p63. p73-/- mice lack multiciliated cells and have reduced numbers of basal epithelial cells in select tissues; however, the role of p73 in basal epithelial cells is unknown. Herein, we show that p73-deficient mice exhibit delayed wound healing despite morphologically normal-appearing skin. The delay in wound healing is accompanied by decreased proliferation and increased levels of biomarkers of the DNA damage response in basal keratinocytes at the epidermal wound edge. In wild-type mice, this same cell population exhibited increased p73 expression after wounding. Analyzing single-cell transcriptomic data, we found that p73 was expressed by epidermal and hair follicle stem cells, cell types required for wound healing. Moreover, we discovered that p73 isoforms expressed in the skin (ΔNp73) enhance p63-mediated expression of keratinocyte genes during cellular reprogramming from a mesenchymal to basal keratinocyte-like cell. We identified a set of 44 genes directly or indirectly regulated by ΔNp73 that are involved in skin development, cell junctions, cornification, proliferation, and wound healing. Our results establish a role for p73 in cutaneous wound healing through regulation of basal keratinocyte function.