Pluripotent Stem Cell-Derived Hematopoietic Progenitors Are Unable to Downregulate Key Epithelial-Mesenchymal Transition-Associated miRNAs.

Hematopoietic stem cells derived from pluripotent stem cells could be used as an alternative to bone marrow transplants. Deriving these has been a long-term goal for researchers. However, the success of these efforts has been limited with the cells produced able to engraft in the bone marrow of recipient animals only in very low numbers. There is evidence that defects in the migratory and homing capacity of the cells are due to mis-regulation of miRNA expression and are responsible for their failure to engraft. We compared the miRNA expression profile of hematopoietic progenitors derived from pluripotent stem cells to those derived from bone marrow and found that numerous miRNAs are too highly expressed in hematopoietic progenitors derived from pluripotent stem cells, and that most of these are inhibitors of epithelial-mesenchymal transition or metastasis (including miR-200b, miR-200c, miR-205, miR-148a, and miR-424). We hypothesize that the high expression of these factors, which promote an adherent phenotype, may be causing the defect in hematopoietic differentiation. However, inhibiting these miRNAs, individually or in multiplex, was insufficient to improve hematopoietic differentiation in vitro, suggesting that other miRNAs and/or genes may be involved in this process. Stem Cells 2018;36:55-64.

Integrated gene set analysis for microRNA studies.

MOTIVATION: Functional interpretation of miRNA expression data is currently done in a three step procedure: select differentially expressed miRNAs, find their target genes, and carry out gene set overrepresentation analysis Nevertheless, major limitations of this approach have already been described at the gene level, while some newer arise in the miRNA scenario.Here, we propose an enhanced methodology that builds on the well-established gene set analysis paradigm. Evidence for differential expression at the miRNA level is transferred to a gene differential inhibition score which is easily interpretable in terms of gene sets or pathways. Such transferred indexes account for the additive effect of several miRNAs targeting the same gene, and also incorporate cancellation effects between cases and controls. Together, these two desirable characteristics allow for more accurate modeling of regulatory processes. RESULTS: We analyze high-throughput sequencing data from 20 different cancer types and provide exhaustive reports of gene and Gene Ontology-term deregulation by miRNA action. AVAILABILITY AND IMPLEMENTATION: The proposed methodology was implemented in the Bioconductor library mdgsa http://bioconductor.org/packages/mdgsa For the purpose of reproducibility all of the scripts are available at https://github.com/dmontaner-papers/gsa4mirna CONTACT: : david.montaner@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A Novel Role for miR-1305 in Regulation of Pluripotency-Differentiation Balance, Cell Cycle, and Apoptosis in Human Pluripotent Stem Cells.

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are defined as pluripotent in view of their self-renewal ability and potential to differentiate to cells of all three germ layers. Recent studies have indicated that microRNAs (miRNAs) play an important role in the maintenance of pluripotency and cell cycle regulation. We used a microarray based approach to identify miRNAs that were enriched in hESCs when compared to differentiated cells and at the same time showed significant expression changes between different phases of cell cycle. We identified 34 candidate miRNAs and performed functional studies on one of these, miR-1305, which showed the highest expression change during cell cycle transition. Overexpression of miR-1305 induced differentiation of pluripotent stem cells, increased cell apoptosis and sped up G1/S transition, while its downregulation facilitated the maintenance of pluripotency and increased cell survival. Using target prediction software and luciferase based reporter assays we identified POLR3G as a downstream target by which miR-1305 regulates the fine balance between maintenance of pluripotency and onset of differentiation. Overexpression of POLR3G rescued pluripotent stem cell differentiation induced by miR-1305 overexpression. In contrast, knock-down of POLR3G expression abolished the miR-1305-knockdown mediated enhancement of pluripotency, thus validating its role as miR-1305 target in human pluripotent stem cells. Together our data point to an important role for miR-1305 as a novel regulator of pluripotency, cell survival and cell cycle and uncovers new mechanisms and networks by which these processes are intertwined in human pluripotent stem cells. Stem Cells 2016;34:2306-2317.

Brief Report: Inhibition of miR-145 Enhances Reprogramming of Human Dermal Fibroblasts to Induced Pluripotent Stem Cells.

MicroRNA (miRNAs) are short noncoding RNA molecules involved in many cellular processes and shown to play a key role in somatic cell induced reprogramming. We performed an array based screening to identify candidates that are differentially expressed between dermal skin fibroblasts (DFs) and induced pluripotent stem cells (iPSCs). We focused our investigations on miR-145 and showed that this candidate is highly expressed in DFs relative to iPSCs and significantly downregulated during reprogramming process. Inhibition of miR-145 in DFs led to the induction of "cellular plasticity" demonstrated by: (a) alteration of cell morphology associated with downregulation of mesenchymal and upregulation of epithelial markers; (b) upregulation of pluripotency-associated genes including SOX2, KLF4, C-MYC; (c) downregulation of miRNA let-7b known to inhibit reprogramming; and (iv) increased efficiency of reprogramming to iPSCs in the presence of reprogramming factors. Together, our results indicate a direct functional link between miR-145 and molecular pathways underlying reprogramming of somatic cells to iPSCs.

Babelomics 5.0: functional interpretation for new generations of genomic data.

Babelomics has been running for more than one decade offering a user-friendly interface for the functional analysis of gene expression and genomic data. Here we present its fifth release, which includes support for Next Generation Sequencing data including gene expression (RNA-seq), exome or genome resequencing. Babelomics has simplified its interface, being now more intuitive. Improved visualization options, such as a genome viewer as well as an interactive network viewer, have been implemented. New technical enhancements at both, client and server sides, makes the user experience faster and more dynamic. Babelomics offers user-friendly access to a full range of methods that cover: (i) primary data analysis, (ii) a variety of tests for different experimental designs and (iii) different enrichment and network analysis algorithms for the interpretation of the results of such tests in the proper functional context. In addition to the public server, local copies of Babelomics can be downloaded and installed. Babelomics is freely available at: http://www.babelomics.org.

Family-based genome-wide association study in Patagonia confirms the association of the DMD locus and cleft lip and palate.

The etiology of cleft lip with or without cleft palate (CL±P) is complex and heterogeneous, and multiple genetic and environmental factors are involved. Some candidate genes reported to be associated with oral clefts are located on the X chromosome. At least three genes causing X-linked syndromes [midline 1 (MID1), oral-facial-digital syndrome 1 (OFD1), and dystrophin (DMD)] were previously found to be associated with isolated CL±P. We attempted to confirm the role of X-linked genes in the etiology of isolated CL±P in a South American population through a family-based genome-wide scan. We studied 27 affected children and their mothers, from 26 families, in a Patagonian population with a high prevalence of CL±P. We conducted an exploratory analysis of the X chromosome to identify candidate regions associated with CL±P. Four genomic segments were identified, two of which showed a statistically significant association with CL±P. One is an 11-kb region of Xp21.1 containing the DMD gene, and the other is an intergenic region (8.7 kb; Xp11.4). Our results are consistent with recent data on the involvement of the DMD gene in the etiology of CL±P. The MID1 and OFD1 genes were not included in the four potential CL±P-associated X-chromosome genomic segments.

A new overgrowth syndrome is due to mutations in RNF125.

Overgrowth syndromes (OGS) are a group of disorders in which all parameters of growth and physical development are above the mean for age and sex. We evaluated a series of 270 families from the Spanish Overgrowth Syndrome Registry with no known OGS. We identified one de novo deletion and three missense mutations in RNF125 in six patients from four families with overgrowth, macrocephaly, intellectual disability, mild hydrocephaly, hypoglycemia, and inflammatory diseases resembling Sjögren syndrome. RNF125 encodes an E3 ubiquitin ligase and is a novel gene of OGS. Our studies of the RNF125 pathway point to upregulation of RIG-I-IPS1-MDA5 and/or disruption of the PI3K-AKT and interferon signaling pathways as the putative final effectors.

Quantitative modeling of clinical, cellular, and extracellular matrix variables suggest prognostic indicators in cancer: a model in neuroblastoma.

BACKGROUND: Risk classification and treatment stratification for cancer patients is restricted by our incomplete picture of the complex and unknown interactions between the patient's organism and tumor tissues (transformed cells supported by tumor stroma). Moreover, all clinical factors and laboratory studies used to indicate treatment effectiveness and outcomes are by their nature a simplification of the biological system of cancer, and cannot yet incorporate all possible prognostic indicators. METHODS: A multiparametric analysis on 184 tumor cylinders was performed. To highlight the benefit of integrating digitized medical imaging into this field, we present the results of computational studies carried out on quantitative measurements, taken from stromal and cancer cells and various extracellular matrix fibers interpenetrated by glycosaminoglycans, and eight current approaches to risk stratification systems in patients with primary and nonprimary neuroblastoma. RESULTS: New tumor tissue indicators from both fields, the cellular and the extracellular elements, emerge as reliable prognostic markers for risk stratification and could be used as molecular targets of specific therapies. CONCLUSION: The key to dealing with personalized therapy lies in the mathematical modeling. The use of bioinformatics in patient-tumor-microenvironment data management allows a predictive model in neuroblastoma.

Large-scale transcriptional profiling and functional assays reveal important roles for Rho-GTPase signalling and SCL during haematopoietic differentiation of human embryonic stem cells.

Understanding the transcriptional cues that direct differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells to defined and functional cell types is essential for future clinical applications. In this study, we have compared transcriptional profiles of haematopoietic progenitors derived from hESCs at various developmental stages of a feeder- and serum-free differentiation method and show that the largest transcriptional changes occur during the first 4 days of differentiation. Data mining on the basis of molecular function revealed Rho-GTPase signalling as a key regulator of differentiation. Inhibition of this pathway resulted in a significant reduction in the numbers of emerging haematopoietic progenitors throughout the differentiation window, thereby uncovering a previously unappreciated role for Rho-GTPase signalling during human haematopoietic development. Our analysis indicated that SCL was the 11th most upregulated transcript during the first 4 days of the hESC differentiation process. Overexpression of SCL in hESCs promoted differentiation to meso-endodermal lineages, the emergence of haematopoietic and erythro-megakaryocytic progenitors and accelerated erythroid differentiation. Importantly, intrasplenic transplantation of SCL-overexpressing hESC-derived haematopoietic cells enhanced recovery from induced acute anaemia without significant cell engraftment, suggesting a paracrine-mediated effect.

The protease MT1-MMP drives a combinatorial proteolytic program in activated endothelial cells.

The mechanism by which proteolytic events translate into biological responses is not well understood. To explore the link of pericellular proteolysis to events relevant to capillary sprouting within the inflammatory context, we aimed at the identification of the collection of substrates of the protease MT1-MMP in endothelial tip cells induced by inflammatory stimuli. We applied quantitative proteomics to endothelial cells (ECs) derived from wild-type and MT1-MMP-null mice to identify the substrate repertoire of this protease in TNF-α-activated ECs. Bioinformatics analysis revealed a combinatorial MT1-MMP proteolytic program, in which combined rather than single substrate processing would determine biological decisions by activated ECs, including chemotaxis, cell motility and adhesion, and vasculature development. MT1-MMP-deficient ECs inefficiently processed several of these substrates (TSP1, CYR61, NID1, and SEM3C), validating the model. This novel concept of MT1-MMP-driven combinatorial proteolysis in angiogenesis might be extendable to proteolytic actions in other cellular contexts.

Initial genomics of the human nucleolus.

We report for the first time the genomics of a nuclear compartment of the eukaryotic cell. 454 sequencing and microarray analysis revealed the pattern of nucleolus-associated chromatin domains (NADs) in the linear human genome and identified different gene families and certain satellite repeats as the major building blocks of NADs, which constitute about 4% of the genome. Bioinformatic evaluation showed that NAD-localized genes take part in specific biological processes, like the response to other organisms, odor perception, and tissue development. 3D FISH and immunofluorescence experiments illustrated the spatial distribution of NAD-specific chromatin within interphase nuclei and its alteration upon transcriptional changes. Altogether, our findings describe the nature of DNA sequences associated with the human nucleolus and provide insights into the function of the nucleolus in genome organization and establishment of nuclear architecture.

Automatization of metabolite extraction for high-throughput metabolomics: case study on transgenic isoprene-emitting birch.

Metabolomics studies are becoming increasingly common for understanding how plant metabolism responds to changes in environmental conditions, genetic manipulations and treatments. Despite the recent advances in metabolomics workflow, the sample preparation process still limits the high-throughput analysis in large-scale studies. Here, we present a highly flexible robotic system that integrates liquid handling, sonication, centrifugation, solvent evaporation and sample transfer processed in 96-well plates to automatize the metabolite extraction from leaf samples. We transferred an established manual extraction protocol performed to a robotic system, and with this, we show the optimization steps required to improve reproducibility and obtain comparable results in terms of extraction efficiency and accuracy. We then tested the robotic system to analyze the metabolomes of wild-type and four transgenic silver birch (Betula pendula Roth) lines under unstressed conditions. Birch trees were engineered to overexpress the poplar (Populus × canescens) isoprene synthase and to emit various amounts of isoprene. By fitting the different isoprene emission capacities of the transgenic trees with their leaf metabolomes, we observed an isoprene-dependent upregulation of some flavonoids and other secondary metabolites as well as carbohydrates, amino acid and lipid metabolites. By contrast, the disaccharide sucrose was found to be strongly negatively correlated to isoprene emission. The presented study illustrates the power of integrating robotics to increase the sample throughput, reduce human errors and labor time, and to ensure a fully controlled, monitored and standardized sample preparation procedure. Due to its modular and flexible structure, the robotic system can be easily adapted to other extraction protocols for the analysis of various tissues or plant species to achieve high-throughput metabolomics in plant research.

Babelomics: an integrative platform for the analysis of transcriptomics, proteomics and genomic data with advanced functional profiling.

Babelomics is a response to the growing necessity of integrating and analyzing different types of genomic data in an environment that allows an easy functional interpretation of the results. Babelomics includes a complete suite of methods for the analysis of gene expression data that include normalization (covering most commercial platforms), pre-processing, differential gene expression (case-controls, multiclass, survival or continuous values), predictors, clustering; large-scale genotyping assays (case controls and TDTs, and allows population stratification analysis and correction). All these genomic data analysis facilities are integrated and connected to multiple options for the functional interpretation of the experiments. Different methods of functional enrichment or gene set enrichment can be used to understand the functional basis of the experiment analyzed. Many sources of biological information, which include functional (GO, KEGG, Biocarta, Reactome, etc.), regulatory (Transfac, Jaspar, ORegAnno, miRNAs, etc.), text-mining or protein-protein interaction modules can be used for this purpose. Finally a tool for the de novo functional annotation of sequences has been included in the system. This provides support for the functional analysis of non-model species. Mirrors of Babelomics or command line execution of their individual components are now possible. Babelomics is available at http://www.babelomics.org.

FM19G11, a new hypoxia-inducible factor (HIF) modulator, affects stem cell differentiation status.

The biology of the alpha subunits of hypoxia-inducible factors (HIFalpha) has expanded from their role in angiogenesis to their current position in the self-renewal and differentiation of stem cells. The results reported in this article show the discovery of FM19G11, a novel chemical entity that inhibits HIFalpha proteins that repress target genes of the two alpha subunits, in various tumor cell lines as well as in adult and embryonic stem cell models from rodents and humans, respectively. FM19G11 inhibits at nanomolar range the transcriptional and protein expression of Oct4, Sox2, Nanog, and Tgf-alpha undifferentiating factors, in adult rat and human embryonic stem cells, FM19G11 activity occurs in ependymal progenitor stem cells from rats (epSPC), a cell model reported for spinal cord regeneration, which allows the progression of oligodendrocyte cell differentiation in a hypoxic environment, has created interest in its characterization for pharmacological research. Experiments using small interfering RNA showed a significant depletion in Sox2 protein only in the case of HIF2alpha silencing, but not in HIF1alpha-mediated ablation. Moreover, chromatin immunoprecipitation data, together with the significant presence of functional hypoxia response element consensus sequences in the promoter region of Sox2, strongly validated that this factor behaves as a target gene of HIF2alpha in epSPCs. FM19G11 causes a reduction of overall histone acetylation with significant repression of p300, a histone acetyltransferase required as a co-factor for HIF-transcription activation. Arrays carried out in the presence and absence of the inhibitor showed the predominant involvement of epigenetic-associated events mediated by the drug.

Hypoxia promotes efficient differentiation of human embryonic stem cells to functional endothelium.

Early development of mammalian embryos occurs in an environment of relative hypoxia. Nevertheless, human embryonic stem cells (hESC), which are derived from the inner cell mass of blastocyst, are routinely cultured under the same atmospheric conditions (21% O(2)) as somatic cells. We hypothesized that O(2) levels modulate gene expression and differentiation potential of hESC, and thus, we performed gene profiling of hESC maintained under normoxic or hypoxic (1% or 5% O(2)) conditions. Our analysis revealed that hypoxia downregulates expression of pluripotency markers in hESC but increases significantly the expression of genes associated with angio- and vasculogenesis including vascular endothelial growth factor and angiopoitein-like proteins. Consequently, we were able to efficiently differentiate hESC to functional endothelial cells (EC) by varying O(2) levels; after 24 hours at 5% O(2), more than 50% of cells were CD34+. Transplantation of resulting endothelial-like cells improved both systolic function and fractional shortening in a rodent model of myocardial infarction. Moreover, analysis of the infarcted zone revealed that transplanted EC reduced the area of fibrous scar tissue by 50%. Thus, use of hypoxic conditions to specify the endothelial lineage suggests a novel strategy for cellular therapies aimed at repair of damaged vasculature in pathologies such as cerebral ischemia and myocardial infarction.

SNOW, a web-based tool for the statistical analysis of protein-protein interaction networks.

Understanding the structure and the dynamics of the complex intercellular network of interactions that contributes to the structure and function of a living cell is one of the main challenges of today's biology. SNOW inputs a collection of protein (or gene) identifiers and, by using the interactome as scaffold, draws the connections among them, calculates several relevant network parameters and, as a novelty among the rest of tools of its class, it estimates their statistical significance. The parameters calculated for each node are: connectivity, betweenness and clustering coefficient. It also calculates the number of components, number of bicomponents and articulation points. An interactive network viewer is also available to explore the resulting network. SNOW is available at http://snow.bioinfo.cipf.es.

Gene set-based analysis of polymorphisms: finding pathways or biological processes associated to traits in genome-wide association studies.

Genome-wide association studies have become a popular strategy to find associations of genes to traits of interest. Despite the high-resolution available today to carry out genotyping studies, the success of its application in real studies has been limited by the testing strategy used. As an alternative to brute force solutions involving the use of very large cohorts, we propose the use of the Gene Set Analysis (GSA), a different analysis strategy based on testing the association of modules of functionally related genes. We show here how the Gene Set-based Analysis of Polymorphisms (GeSBAP), which is a simple implementation of the GSA strategy for the analysis of genome-wide association studies, provides a significant increase in the power testing for this type of studies. GeSBAP is freely available at http://bioinfo.cipf.es/gesbap/.

DNA methylation epigenotypes in breast cancer molecular subtypes.

INTRODUCTION: Identification of gene expression based breast cancer subtypes is considered as a critical means of prognostication. Genetic mutations along with epigenetic alterations contribute to gene expression changes occurring in breast cancer. So far, these epigenetic contributions to sporadic breast cancer subtypes have not been well characterized, and there is only a limited understanding of the epigenetic mechanisms affected in those particular breast cancer subtypes. The present study was undertaken to dissect the breast cancer methylome and deliver specific epigenotypes associated with particular breast cancer subtypes. METHODS: Using a microarray approach we analyzed DNA methylation in regulatory regions of 806 cancer related genes in 28 breast cancer paired samples. We subsequently performed substantial technical and biological validation by Pyrosequencing, investigating the top qualifying 19 CpG regions in independent cohorts encompassing 47 basal-like, 44 ERBB2+ overexpressing, 48 luminal A and 48 luminal B paired breast cancer/adjacent tissues. Using all-subset selection method, we identified the most subtype predictive methylation profiles in multivariable logistic regression analysis. RESULTS: The approach efficiently recognized 15 individual CpG loci differentially methylated in breast cancer tumor subtypes. We further identify novel subtype specific epigenotypes which clearly demonstrate the differences in the methylation profiles of basal-like and human epidermal growth factor 2 (HER2)-overexpressing tumors. CONCLUSIONS: Our results provide evidence that well defined DNA methylation profiles enables breast cancer subtype prediction and support the utilization of this biomarker for prognostication and therapeutic stratification of patients with breast cancer.

Babelomics: advanced functional profiling of transcriptomics, proteomics and genomics experiments.

We present a new version of Babelomics, a complete suite of web tools for the functional profiling of genome scale experiments, with new and improved methods as well as more types of functional definitions. Babelomics includes different flavours of conventional functional enrichment methods as well as more advanced gene set analysis methods that makes it a unique tool among the similar resources available. In addition to the well-known functional definitions (GO, KEGG), Babelomics includes new ones such as Biocarta pathways or text mining-derived functional terms. Regulatory modules implemented include transcriptional control (Transfac, CisRed) and other levels of regulation such as miRNA-mediated interference. Moreover, Babelomics allows for sub-selection of terms in order to test more focused hypothesis. Also gene annotation correspondence tables can be imported, which allows testing with user-defined functional modules. Finally, a tool for the 'de novo' functional annotation of sequences has been included in the system. This allows using yet unannotated organisms in the program. Babelomics has been extensively re-engineered and now it includes the use of web services and Web 2.0 technology features, a new user interface with persistent sessions and a new extended database of gene identifiers. Babelomics is available at http://www.babelomics.org.

GEPAS, a web-based tool for microarray data analysis and interpretation.

Gene Expression Profile Analysis Suite (GEPAS) is one of the most complete and extensively used web-based packages for microarray data analysis. During its more than 5 years of activity it has continuously been updated to keep pace with the state-of-the-art in the changing microarray data analysis arena. GEPAS offers diverse analysis options that include well established as well as novel algorithms for normalization, gene selection, class prediction, clustering and functional profiling of the experiment. New options for time-course (or dose-response) experiments, microarray-based class prediction, new clustering methods and new tests for differential expression have been included. The new pipeliner module allows automating the execution of sequential analysis steps by means of a simple but powerful graphic interface. An extensive re-engineering of GEPAS has been carried out which includes the use of web services and Web 2.0 technology features, a new user interface with persistent sessions and a new extended database of gene identifiers. GEPAS is nowadays the most quoted web tool in its field and it is extensively used by researchers of many countries and its records indicate an average usage rate of 500 experiments per day. GEPAS, is available at http://www.gepas.org.