Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis.

The formation of new blood vessel networks occurs via angiogenesis during development, tissue repair, and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation, and migration. Extracellular signal-regulated kinase (Erk) is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response, however, involves a Vegfr-dependent mechanism that initiates concomitantly with the wound inflammatory response. This work reveals a highly dynamic sequence of signalling events in regenerative angiogenesis and validates a new resource for the study of vascular Erk-signalling in real-time.

Unique properties of a subset of human pluripotent stem cells with high capacity for self-renewal.

Archetypal human pluripotent stem cells (hPSC) are widely considered to be equivalent in developmental status to mouse epiblast stem cells, which correspond to pluripotent cells at a late post-implantation stage of embryogenesis. Heterogeneity within hPSC cultures complicates this interspecies comparison. Here we show that a subpopulation of archetypal hPSC enriched for high self-renewal capacity (ESR) has distinct properties relative to the bulk of the population, including a cell cycle with a very low G1 fraction and a metabolomic profile that reflects a combination of oxidative phosphorylation and glycolysis. ESR cells are pluripotent and capable of differentiation into primordial germ cell-like cells. Global DNA methylation levels in the ESR subpopulation are lower than those in mouse epiblast stem cells. Chromatin accessibility analysis revealed a unique set of open chromatin sites in ESR cells. RNA-seq at the subpopulation and single cell levels shows that, unlike mouse epiblast stem cells, the ESR subset of hPSC displays no lineage priming, and that it can be clearly distinguished from gastrulating and extraembryonic cell populations in the primate embryo. ESR hPSC correspond to an earlier stage of post-implantation development than mouse epiblast stem cells.

Frizzled-7 Is Required for Wnt Signaling in Gastric Tumors with and Without Apc Mutations.

A subset of patients with gastric cancer have mutations in genes that participate in or regulate Wnt signaling at the level of ligand (Wnt) receptor (Fzd) binding. Moreover, increased Fzd expression is associated with poor clinical outcome. Despite these findings, there are no in vivo studies investigating the potential of targeting Wnt receptors for treating gastric cancer, and the specific Wnt receptor transmitting oncogenic Wnt signaling in gastric cancer is unknown. Here, we use inhibitors of Wnt/Fzd (OMP-18R5/vantictumab) and conditional gene deletion to test the therapeutic potential of targeting Wnt signaling in preclinical models of intestinal-type gastric cancer and ex vivo organoid cultures. Pharmacologic targeting of Fzd inhibited the growth of gastric adenomas in vivo. We identified Fzd7 to be the predominant Wnt receptor responsible for transmitting Wnt signaling in human gastric cancer cells and mouse models of gastric cancer, whereby Fzd7-deficient cells were retained in gastric adenomas but were unable to respond to Wnt signals and consequently failed to proliferate. Genetic deletion of Fzd7 or treatment with vantictumab was sufficient to inhibit the growth of gastric adenomas with or without mutations to Apc. Vantictumab is currently in phase Ib clinical trials for advanced pancreatic, lung, and breast cancer. Our data extend the scope of patients that may benefit from this therapeutic approach as we demonstrate that this drug will be effective in treating patients with gastric cancer regardless of APC mutation status. SIGNIFICANCE: The Wnt receptor Fzd7 plays an essential role in gastric tumorigenesis irrespective of Apc mutation status, therefore targeting Wnt/Fzd7 may be of therapeutic benefit to patients with gastric cancer.

A molecular classification of human mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) are widely used for the study of mesenchymal tissue repair, and increasingly adopted for cell therapy, despite the lack of consensus on the identity of these cells. In part this is due to the lack of specificity of MSC markers. Distinguishing MSC from other stromal cells such as fibroblasts is particularly difficult using standard analysis of surface proteins, and there is an urgent need for improved classification approaches. Transcriptome profiling is commonly used to describe and compare different cell types; however, efforts to identify specific markers of rare cellular subsets may be confounded by the small sample sizes of most studies. Consequently, it is difficult to derive reproducible, and therefore useful markers. We addressed the question of MSC classification with a large integrative analysis of many public MSC datasets. We derived a sparse classifier (The Rohart MSC test) that accurately distinguished MSC from non-MSC samples with >97% accuracy on an internal training set of 635 samples from 41 studies derived on 10 different microarray platforms. The classifier was validated on an external test set of 1,291 samples from 65 studies derived on 15 different platforms, with >95% accuracy. The genes that contribute to the MSC classifier formed a protein-interaction network that included known MSC markers. Further evidence of the relevance of this new MSC panel came from the high number of Mendelian disorders associated with mutations in more than 65% of the network. These result in mesenchymal defects, particularly impacting on skeletal growth and function. The Rohart MSC test is a simple in silico test that accurately discriminates MSC from fibroblasts, other adult stem/progenitor cell types or differentiated stromal cells. It has been implemented in the www.stemformatics.org resource, to assist researchers wishing to benchmark their own MSC datasets or data from the public domain. The code is available from the CRAN repository and all data used to generate the MSC test is available to download via the Gene Expression Omnibus or the Stemformatics resource.

The pluripotent state in mouse and human.

In the mouse, naïve pluripotent stem cells (PSCs) are thought to represent the cell culture equivalent of the late epiblast in the pre-implantation embryo, with which they share a unique defining set of features. Recent studies have focused on the identification and propagation of a similar cell state in human. Although the capture of an exact human equivalent of the mouse naïve PSC remains an elusive goal, comparative studies spurred on by this quest are lighting the path to a deeper understanding of pluripotent state regulation in early mammalian development.

Gene expression variability as a unifying element of the pluripotency network.

Heterogeneity is a hallmark of stem cell populations, in part due to the molecular differences between cells undergoing self-renewal and those poised to differentiate. We examined phenotypic and molecular heterogeneity in pluripotent stem cell populations, using public gene expression data sets. A high degree of concordance was observed between global gene expression variability and the reported heterogeneity of different human pluripotent lines. Network analysis demonstrated that low-variability genes were the most highly connected, suggesting that these are the most stable elements of the gene regulatory network and are under the highest regulatory constraints. Known drivers of pluripotency were among these, with lowest expression variability of POU5F1 in cells with the highest capacity for self-renewal. Variability of gene expression provides a reliable measure of phenotypic and molecular heterogeneity and predicts those genes with the highest degree of regulatory constraint within the pluripotency network.

Ontogeny of small RNA in the regulation of mammalian brain development.

BACKGROUND: MicroRNAs (miRNAs) play a pivotal role in coordinating messenger RNA (mRNA) transcription and stability in almost all known biological processes, including the development of the central nervous system. Despite our broad understanding of their involvement, we still have a very sparse understanding of specifically how miRNA contribute to the strict regional and temporal regulation of brain development. Accordingly, in the current study we have examined the contribution of miRNA in the developing rat telencephalon and mesencephalon from just after neural tube closure till birth using a genome-wide microarray strategy. RESULTS: We identified temporally distinct expression patterns in both the telencephalon and mesencephalon for both miRNAs and their target genes. We demonstrate direct miRNA targeting of several genes involved with the migration, differentiation and maturation of neurons. CONCLUSIONS: Our findings suggest that miRNA have significant implications for the development of neural structure and support important mechanisms that if disrupted, may contribute to or drive neurodevelopmental disorders.

Concise review: new paradigms for Down syndrome research using induced pluripotent stem cells: tackling complex human genetic disease.

Down syndrome (DS) is a complex developmental disorder with diverse pathologies that affect multiple tissues and organ systems. Clear mechanistic description of how trisomy of chromosome 21 gives rise to most DS pathologies is currently lacking and is limited to a few examples of dosage-sensitive trisomic genes with large phenotypic effects. The recent advent of cellular reprogramming technology offers a promising way forward, by allowing derivation of patient-derived human cell types in vitro. We present general strategies that integrate genomics technologies and induced pluripotent stem cells to identify molecular networks driving different aspects of DS pathogenesis and describe experimental approaches to validate the causal requirement of candidate network defects for particular cellular phenotypes. This overall approach should be applicable to many poorly understood complex human genetic diseases, whose pathogenic mechanisms might involve the combined effects of many genes.

Construct validity of three depersonalization measures in trauma-exposed college students.

Depersonalization is a type of dissociation characterized by feelings of unreality and detachment from one's sense of self. Despite a history rich in clinical description, the construct of depersonalization has proven difficult to define and measure. Available measures vary substantially in content, and all have relatively limited psychometric support. In this study the content validity, internal consistency, and convergent and discriminant validity of 3 depersonalization measures were compared in a sample of 209 trauma-exposed college students. Measures were the Dissociative Experiences Scale ( E. M. Bernstein & F. W. Putnam, 1986 ), Cambridge Depersonalization Scale (CDS; M. Sierra & G. E. Berrios, 2000 ), and Multiscale Dissociation Inventory (MDI; J. Briere, 2002 ). All 3 measures exhibited adequate to high internal consistency for the depersonalization-derealization items. Based on D. Westen and R. Rosenthal's (2003) procedure for quantifying construct validity, the CDS and MDI demonstrated the best fit with the predicted pattern of correlations with measures of other constructs. The CDS and MDI also demonstrated the strongest evidence of content validity. Overall, the results most strongly support the use of the CDS and MDI for assessing depersonalization in this population.

Association of life threat and betrayal with posttraumatic stress disorder symptom severity.

The Diagnostic and Statistical Manual of Mental Disorders (4th ed., text rev.; DSM-IV-TR; American Psychiatric Association [APA], 2000) emphasizes life threat as the defining feature of psychological trauma. Recent theoretical and empirical work, however, indicates the need to identify and evaluate other key aspects of trauma. Betrayal has been proposed as a pertinent, distinct, and complementary factor that can explain effects of trauma not accounted for by life threat alone. This study examined the relationship between injury, perceived life threat (PLT), and betrayal with posttraumatic stress disorder (PTSD) symptom severity. Trauma-exposed college students (N = 185) completed self-report measures of trauma exposure and PTSD, as well as items regarding life threat, betrayal, and level of medical care received. In hierarchical regressions incorporating injury, PLT, and betrayal, betrayal was associated with all PTSD symptom clusters and PTSD total severity (f(2) = .08), whereas PLT was associated with hyperarousal (f(2) = .05) and PTSD total (f(2) = .03), and injury had no association with PTSD symptoms. In a revised model with trauma type as an additional variable, betrayal was associated with avoidance (f(2) = .03), numbing (f(2) = .04), and PTSD total (f(2) = .03), whereas PLT was associated with reexperiencing (f(2) = .04), hyperarousal (f(2) = .04), and PTSD total (f(2) = .03), and injury was associated with avoidance (f(2) = .03). These findings support the idea that betrayal is a core dimension of psychological trauma that may play an important role in the etiology of PTSD.

The validity of predicting maximal oxygen uptake from perceptually regulated graded exercise tests of different durations.

The purpose of this study was to assess the validity of predicting maximal oxygen uptake [V(.-)((O)(2)(max))] from sub-maximal V(.-)((O)(2)) values elicited during perceptually regulated exercise tests of 2- and 4-min duration. Nineteen physically active men and women (age range 19-23 years) volunteered to participate in two graded exercise tests to volitional exhaustion to measure V(.-)((O)(2)(max)) [V(.-)((O)(2)(max))(GXT)], at the beginning and end of a 2-week period, and four incremental, perceptually regulated tests to predict [V(.-)((O)(2)(max))] in the intervening period. Effort production tests comprised 2 x 2-min and 2 x 4-min bouts on a cycle ergometer, perceptually regulated at intensities of 9, 11, 13, 15 and 17 on the Borg 6-20 rating of perceived (RPE) scale, in that order. Individual linear relationships between RPE and V(.-)((O)(2) for RPE ranges of 9-17, 11-17 and 9-15 were extrapolated to RPE 20 to predict [V(.-)((O)(2)(max))]. The prediction of [V(.-)((O)(2)(max))] was not moderated by gender. Although, [V(.-)((O)(2)(max))] estimated from RPE 9-17 of trial 1 of the 2-min protocol was significantly lower (P < 0.05) than [V(.-)((O)(2)(max))(GXT)], and V(.-)((O)(2)(max)) predicted from the 4-min trials, the V(.-)((O)(2)(max)) predicted from trial 2 of the 2-min protocol was a more accurate prediction of [V(.-)((O)(2)(max))(GXT)], across all trials. The intraclass correlation coefficient (R) was also higher between [V(.-)((O)(2)(max))(GXT)], and [V(.-)((O)(2)(max))] predicted from trial 2 of the 2-min protocol compared to both trials in the 4-min protocol (R = 0.95, 0.88 and 0.79, respectively). Similar results were observed for RPE ranges 9-15 and 11-17. Results suggest that a sub-maximal, perceptually guided, graded exercise protocol, particularly of a 2-min duration, provides acceptable estimates of maximal aerobic power, which are not moderated by gender.