Project description:Heterogeneity, shortage of material, and lack of progenitor-specific cell surface markers are major obstacles to elucidating the mechanisms underlying developmental processes. Here we report a proteomic platform that alleviates these difficulties and demonstrate its effectiveness in fractionating heterogeneous cultures of early endoderm derived from human embryonic stem cells. The approach, designated cell-capture antibody array, is based on highly parallel, comparative screening of live cell populations using hundreds of antibodies directed against cell-surface antigens. The results demonstrate the potential of the cell-capture antibody array as a powerful tool for detailed dissection of heterogeneous cellular systems. Genome-wide comparion of mRNA profile in hES-derived CD61+ cells versus CXCR4+ and CXCR4+/CD61- cells using Affymetrix arrays.
Project description:Endocrine therapy is the main therapeutic option for patients with estrogen receptor alpha positive (ER+) breast cancer. Nevertheless, most of them become estrogen-independent and relapse after the treatment. Ret is a tyrosine kinase receptor that shows elevated expression levels in ER+ human breast tumors. In this study, we demonstrate that activation of the Ret receptor promotes proliferation as well as cell migration irrespective of endocrine therapy. Microarray data show that Ret activation involves changes in the expression of inflammatory- and motility-related genes. In vivo treatment with a Ret pathway inhibitor in a ER+/Ret+ mouse mammary cancer model, reduces tumor growth and lung metastasis even after endocrine therapy. Additionally, we show a connection between Ret and inflammatory pathways. The pro-inflamatory cytokine IL6 lies at the core of this regulation, which involves a positive feedback loop with IL6 and the Ret pathway reciprocally stimulating each other to further leading metastasis risk. Our findings provide insight into endocrine resistance mechanism and point at the Ret pathway as a potential target for future therapies. In order to model letrozole-sensitive breast cancer we use aromatase expressing MCF7 cells (MCF7/Aro). Six-day treatment (6 days) of cultures with letrozole (L) or fulvestrant (F) reversed the proliferative effects of the exposure to the estrogen (E2) precursor androstenedione (D4A). The addition of only EtOH (E) to the cells was used as control condition of deprivation. Treatment with the Ret ligand GDNF (G) partially rescues the inhibition of estrogen-dependent proliferation in these cells. To go deeper insight into the pathways involved, we decided to perform a microarray following different treatments (1-8: E, E+G, D, D+G, L, L+G, F, F+G) used in proliferation assays. Three biological replicates (rep 1-3) were used to the array.
Project description:CD4+ dendritic cells are part of the innate immunity essential for priming and activating of CD4+ T cells To identify Runx3 responsive genes CD4+ dendritic cells were sorted from freshly isolated macs enriched splenic DCs taken from 6 weeks old mice. Six samples from six mice were sorted and analyzed where in each littermates pair consisted of a control and Runx3 KO.
Project description:Esam/CD4+ dendritic cells are part of the innate immunity essential for priming and activating of CD4+ T cells To identify Runx3 responsive genes Esam dendritic cells were freshly sorted from macs enriched splenic DCs taken from 6 weeks old mice. Four samples from four mice were sorted and analyzed where in each littermates pair consisted of a control and Runx3 conditional KO. Mice lacking Runx3 specifically in the DC compartment were produced by crossing Runx3fl/fl mice onto CD11c-Cre mice. This mating scheme generated Runx3fl/fl/CD11c:Cre (CD11c-DC-Runx3Δ) mice.
Project description:The supply of red blood cells (RBCs) is not sufficient in many developing countries or in developed countries for patients who need chronic transfusion from best-matched donors. Ex vivo expansion and maturation of human erythroid precursor cells (erythroblasts) could represent a potential solution. Proliferating erythroblasts can be expanded from human umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 10^6-10^7 fold (in ~50 days) before undergoing senescence. Here, we report that ectopic expression of three to four genetic factors that have been used for iPS cell derivation enables CB-derived erythroblasts to undergo extended ex vivo expansion (≥10^51 fold in ~9 months) in a defined suspension culture condition without change of cell identity or function. These vastly expanding erythroblasts maintain homogeneously immature erythroblast phenotypes, a normal diploid karyotype and dependence on specific combination of cytokines and hormone for survival and proliferation throughout the continuous expansion period. When switched to a culture condition for terminal maturation, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes. Our result may ultimately lead to the development of unlimited sources of cultured RBCs for optimally-matched or personalized transfusion medicine. We compared the global gene expression profiles of different human cell types: iE: immortalized erythroblasts generated by genetic reprogramming from pCBE; pCBE: primary cord blood-derived erythroblasts; CD34+: CD34+ purified hematopoietic stem/progenitor cells from adult blood or fetal liver; TF-1: a human erythroleukemia cell line; ESC: human embryonic stem cells; iPSCs: human induced pluripotent stem cells. We want to see the relationship among these cell types. We included multiple samples (biological replicates) for most cell types.
Project description:This investigation provides a robust multi-dimensional compendium of gene expression data relevant to mouse facial development. It profiles the transcriptome ofectoderm and mesenchyme from the three facial prominences in a time series encompassing their growth and fusion. Analysis of the dataset identified more than 8000 differentially expressed genes comprising dramatically different ectoderm and mesenchyme programs. The mesenchyme programs included many genes identified in earlier analyses as well hundreds of genes not previously implicated in craniofacial development. The ectoderm programs included over a thousand genes that highlight epithelial structure, cell-cell interactions and signaling. The dataset includes 45 .cel files, DABG probability and RMA log2 expression values for each probeset, and statistics for 9457 probesets representing 8575 genes. 45 total samples, with 15 conditions sampling three ages (E10.5, E11.5, E12.5), three facial prominences (mandibular, maxillary and fronto-nasal) and two tissue layers (ectoderm or mesenchyme), with 3 biological replicates per condition. Differential expression was determined after median filter for variance with three-way ANOVA, Benjamini-Hochberg multiple testing correction
Project description:A collection of 100 ovarian cancer sample gene expression data from Singapore. Frozen archival epithelial ovarian cancer tumors samples from Department of Obstetrics & Gynecology, National University of Singapore dated from 2006 to 2014 were collected and subjected to microarray analysis.
Project description:Variation in individuals' adaptive immune response is believed to influence susceptibility to complex diseases in humans. The genetic basis of such variation is poorly understood. We measured gene expression from resting and activated CD4+ T cells derived from the peripheral blood of healthy individuals. We activated the primary T cells with anti-CD3/CD28 beads. We collected peripheral blood from each human donor. We isolated peripheral blood mononuclear cells by Ficoll, and negatively selected for CD4+ T cells using RosettaSep. We then either left cells unstimulated or stimulated them with beads conjugated with anti-CD3 and anti-CD28. Cells from 15 individuals were harvested at up to 3 time points (0hr, 4hr or 48hr), lysed and RNA isolated to be profiled on microarray.
Project description:In lymphomas derived from mature B cells the expression of the transcription factor PAX5 is maintained whereas classical Hodgkin lymphoma displays significantly reduced PAX5 expression despite its derivation from mature B cells. To elucidate the functional role of PAX5 in classical Hodgkin lymphoma, we re-established the PAX5 expression in the Hodgkin cell line L428 with and without epigenetic modulation. To this end, we stably transfected the Hodgkin cell line L428 with an inducible PAX5 expression construct. Although the overexpressed PAX5 was transcriptionally active as demonstrated by synthetic reporter constructs, no induction of the B-cell phenotype was achieved. PAX5 chromatin immunoprecipitation with subsequent next generation sequencing in B-cell lines and the PAX5 overexpressing L428 cell line showed different binding patterns. Since epigenetic restrictions might affect PAX5 binding, combined DNA demethylation and histone acetylation was performed. However, no re-expression of B-cell genes was observed also under these conditions. Thus, PAX5 is not sufficient for the re-activation of the B-cell program in Hodgkin cells despite epigenetic opening of the chromatin. This clearly indicates that the repression of the B-cell identity of the Hodgkin cells is caused and secured by complex molecular mechanisms. 2 Burkitt lymphoma cell lines (Raji, Namalwa), the Hodgkin lymphoma cell line L428 and the PAX5-producing L428 (L428-PAX5) with or without 5-aza-2′-deoxycytidine/Trichostatin A treatment were analysed in triplicate.