Genome-wide mapping of DNA hydroxymethylation in osteoarthritic chondrocytes [expression]
Ontology highlight
ABSTRACT: Examination of the genome-wide distribution of 5hmC in osteoarthritic chondrocytes compared to normal chondrocytes in order to elucidate the effect on OA-specific gene expression. 5hmC-sequencing was performed and data was compared with microarray gene expression data to identify genes with differential expression between normal and OA chondrocytes that are potentially under epigenetic regulation. Gene expression patterns were examined by comparing the 5 normal samples to the 2 OA samples to assess the changing expression profiles between normal and OA chondrocytes. We analyzed the changes in gene expression in OA; genes with a fold-change ≥ or ≤1.5 or 1.2, with a difference in intensity of >100 and within the lower 90% confidence bound, were selected.
Project description:Examination of the genome-wide distribution of 5hmC in osteoarthritic chondrocytes compared to normal chondrocytes in order to elucidate the effect on OA-specific gene expression. 5hmC-sequencing was performed and data was compared with microarray gene expression data to identify genes with differential expression between normal and OA chondrocytes that are potentially under epigenetic regulation. High-throughput sequencing of 5hmC in 4 normal and 4 OA chondrocyte samples.
Project description:Regeneration of human cartilage is inherently inefficient; an abundant autologous source like human induced pluripotent stem cells (hiPSC) is therefore attractive for engineering cartilage. Here, we report a defined growth factor based protocol for differentiating hiPSC into articular-like chondrocytes within two weeks with a high efficiency. The hiPSC-derived chondrocytes (hiChondrocytes) are stable and comparable to adult articular chondrocytes in global gene expression, extracellular matrix production and in their ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early Sox9lowCD44lowCD140low pre-chondrogenic mesodermal population during hiPSC differentiation that eventually generates a homogenous population of Sox9highCD44highCD140high hiChondrocytes. Additionally, global gene expression analyses revealed two distinct Sox9-regulated gene networks in the Sox9low and Sox9high populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generation of hiPSC-derived articular chondrocytes in terms of safety and efficiency. 10 samples were analysed (duplicate sets of 5 time points) to assess changing gene expression over the course of differentiation from iPSC to hiChondrocyte. All samples were compared relative to the undifferentiated iPSC. Adult chondrocytes (2 samples) were also included for comparison. We analyzed the changes in gene expression with differentiation; genes with a fold-change â?¥ or â?¤1.5, with a difference in intensity of >100 and within the lower 90% confidence bound were selected.
Project description:Although information on the molecular pathogenesis of Waldenström’s Macroglobulinemia (WM) has greatly improved in recent years, the exact cellular origin and the mechanisms behind WM transformation from IgM MGUS remain undetermined. Here, we undertook an integrative phenotypic, molecular and genomic approach to study clonal B-cells from newly-diagnosed patients with IgM MGUS (n=22), smoldering (n=17), and symptomatic WM (n=10). Through principal-component-analysis of multidimensional flow cytometry data, we demonstrated overlapping phenotypic profiles between clonal B-cells from IgM MGUS, smoldering and symptomatic WM patients. Similarly, virtually no genes were significantly deregulated between FACS-sorted clonal B-cells from the three disease stages. Interestingly, while the transcriptome of the Waldenström’s clone was highly deregulated as compared to CD25-CD22+ normal B-cells, significantly less genes were differentially expressed and specific WM pathways down-regulated while comparing the transcriptome of the Waldenström’s clone vs. its normal phenotypic counterpart: CD25+CD22+dim B-cells. The frequency of specific copy number abnormalities [+4, del(6q23.3-6q25.3), +12, and +18q11-18q23] progressively increased from IgM MGUS and smoldering WM vs. symptomatic WM (18% vs. 20% and 73%, respectively; P =.008), suggesting a multistep transformation of clonal B-cells that albeit benign (i.e.: IgM MGUS and smoldering WM), already harbor the phenotypic and molecular signatures of the malignant Waldenström’s clone. Normal bone marrow CD25+ B-cells, Clonal B-Cells from IgM Monoclonal Gammopathy of Undetermined Significance, and Clonal B-Cells from Waldenström's Macroglobulinemia
Project description:Immunoglobulin light-chain amyloidosis (AL) is a rare clonal plasma cell (PC) disorder that remains largely incurable. AL and multiple myeloma (MM) share the same cellular origin, but while knowledge about MM PC biology has improved significantly, the same does not apply for AL. Here, we undertook an integrative phenotypic, molecular, and genomic approach to study clonal PCs from 22 newly-diagnosed AL patients. Through principal-component-analysis, we demonstrated highly overlapping phenotypic profiles between AL and MGUS or MM patients. However, in contrast to MM, highly-purified FACSs-sorted clonal PCs in AL (n=9/22) show virtually normal transcriptomes with only 68 deregulated genes as compared to normal PCs, including a few tumor suppressor (CDH1, RCAN) and pro-apoptotic (GLIPR1, FAS) genes. Notwithstanding, clonal PCs in AL (n=11/22) were genomically unstable with a median of 9 copy-number-abnormities (CNAs) per case; many of which similar to those found in MM. Whole-exome sequencing (WES) was performed in three AL patients and revealed a median of 10 non-recurrent mutations per case. Altogether, we showed that although clonal PCs in AL display phenotypic and CNA profiles similar to MM, their transcriptome is remarkably similar to that of normal PCs. First-ever WES revealed the lack of a unifying mutation in AL A total of 22 patients with confirmed diagnosis of AL based on the presence of amyloid-related systemic syndrome, positive amyloid tissue staining with Congo red, and evidence of PC clonality were studied. Samples were collected after informed consent was given, in accordance with local ethical committee guidelines and the Helsinki Declaration. GEP was performed in 9/22 AL cases with adequate RNA extracted from FACS-purified clonal PCs according to patient-specific aberrant phenotypes, and compared to that of normal PCs from 5 healthy individuals (FACSAriaIIb, BDB; â¥95% purity). RNA was hybridized to the Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA) and normalization was carried using the expression console (Affymetrix) with the RMA algorithm which includes background correction, normalization and calculation of expression values (log2). Differentially expressed genes between classes were identified using the Significant Analysis of Microarrays (SAM) algorithm (http://www-stat.standford.edu/-tibs/SAM), and significant genes were selected based on the lowest q-value (<10-5).
Project description:Endothelial cells from nine steady state tissues and two regenerating tissues (bone marrow and liver) were intravitally labeld, isolated via flow sorting, and immediately processed for RNA extraction. When of sufficient quality, the RNA was amplified and hybridized. For comparison, Human Emybryonic Stem Cell-derived Endothelial cells (hESC-ECs) were differentiated and isolated based on similarities to the adult mouse counterparts. Endothelial cells were labeled via intravitally labeling of the vascular bed 8 minutes prior to sacrifice with minimally three markers to identify endothelial cells followed by flow sorting.
Project description:DNA hypomethylation could lead to activation of alternate promoters in GBM. We profiled DNA methylation and H3K4me3 genome-wide, and also performed expression and copy number analysis on the same samples In this dataset, we include all expression data obtained for five GBMs and one normal brain. We identified loci with concurrent DNA hypomethylation and H3K4me3 in GBM, and analyzed expression of the nearest gene in the same sample using these data Six total samples were analyzed. Each GBM sample was compared to normal brain to obtain expression changes
Project description:Expression Profiling on Human Cell Lines with Different Acetylation Levels Expression profiling of human cell lines with different acetylation levels.
Project description:Patients undergoing either partial or radical nephrectomy at William Beaumont Hospital (Royal Oak, MI) were consented prior to surgery with local IRB oversight. Samples were collected at time of surgery and stored at -80°C according to CAP (College of American Pathologist)-accredited standard operating procedures. Disease pathology of frozen samples was validated with hematoxylin and eosin stained tissue sections from adjacently collected formalin fixed paraffin embedded tissue. The objective of this study was to examine the gene expression patterns of clear cell carcinoma in relation to adjacent normal kidney as well as other benign kidney conditions. In particular, the goal was to identify gene expression that could differentiate high grade ccRCC from low grade ccRCC. Samples from patients with high grade ccRCC (Fuhrman grades 3 & 4, n=16) were compared to normal kidney samples (n=14) and also to samples from patients with benign kidney conditions (n=6). Samples from patients with low grade ccRCC (Fuhrman grades 1 & 2, n=13) were then also compared to normal kidney samples (n=14) and also to samples from patients with benign kidney conditions (n=6).
Project description:Whole transcript expression was profiled using the Affymetrix 1.0 array in human bronchial epithelial cells exposed to PM collected from Saudi Arabia for 1 or 4 days. The differentially expressed genes were identified and analyzed for enriched networks and pathways using Ingenuity Pathway Analysis (IPA). We have identified 140 and 230 genes that significantly changed more than 1.5 fold after PM exposure for 1 or 4 days, respectively. IPA analysis revealed that different exposure durations triggered distinct pathways. Genes involved in NRF2-mediated response to oxidative stress were up-regulated after 1 day exposure. In contrast, cells exposed for 4 days exhibited significantly changes in genes related to cholesterol and lipid synthesis pathways. We analyzed gene expression profiles from 12 samples collected at two different time points, including 2 untreated controls, 2 normal PM treated samples and 2 storm PM treated samples for each time point.