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SRP158590
Homo sapiens
138 Downloadable Samples
Description
Multiple myeloma (MM) is a malignant plasma cell disorder with well-defined clonal genetic/cytogenetic abnormalities. However, cellular heterogeneity is a key factor in MM's progression, therapeutic decision, and response to treatment. Single cell whole transcriptome profiling (scRNA-Seq) offers an opportunity to dissect this molecular heterogeneity during MM progression to better understand the disease and guide rational therapy. Here, we examined 597 CD138 positive cells from 15 patients at different stages of MM progression using scRNA-Seq. We selected 790 genes based on a Coefficient of Variation (CV) approach which organized cells into four clusters (L1-L4) based on unsupervised clustering. Plasma cells from each patient contained a mixed population of plasma cells at different state of aggressiveness based on gene expression signature reflecting the inter-cellular heterogeneous nature of MM. Cells in the L1 group is characterized by low level expression of genes involved in the oxidative phosphorylation, Myc targets, and mTORC1 signaling pathway having most cells from MGUS patients (p < 1.2x10-14). In contrast, low level of these genes in L1 group increased progressively and were the highest in the L4 group containing only cells from high-risk MM patients with t(4;14) translocations. Furthermore, 44 genes consistently overexpressed by pair-wised comparisons of the four groups strongly associated with a reduced overall survival in MM patients (APEX trial, p < 0.0001; Hazard Ratio (HR), 1.83; 95% CI, 1.33 to 2.52), particularly those in the bortezomib treated group (p < 0.0001; HR, 2.00; 95% CI, 1.39 to 2.89). No survival significance was observed for the dexamethasone treated group. Our study at the resolution of single cells showed that there is a mixed population of cells in each patient at different stages of MM progression and these cells can be organized into four different subgroups (L1 to L4). Consistent overexpression of the 44 genes from L1 to L4 groups is associated with patient outcome and treatment response. Our results show that oxidative phosphorylation, Myc target, and mTORC1 signaling genes are significant pathways for MM progression and affect MM prognosis and treatment stratification. Overall design: 597 single cell libraries passed QC and were included in the downstream analysis
Publication Title
Molecular signatures of multiple myeloma progression through single cell RNA-Seq.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 9 Downloadable Samples
Affymetrix Mouse Gene 1.0 ST Array (mogene10st)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Genomic occupancy of Runx2 with global expression profiling identifies a novel dimension to control of osteoblastogenesis.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 9 Downloadable Samples
- Affymetrix Mouse Gene 1.0 ST Array (mogene10st)
Description
Osteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcription program essential for bone formation through both genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed. By performing genome-wide characterization of Runx2 binding at the three major stages of osteoblast differentiation: proliferation, matrix deposition and mineralization, we identified Runx2-dependent regulatory networks driving bone formation. Using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) over the course of these stages, we discovered close to 80,000 significantly enriched regions of Runx2 binding throughout the mouse genome. These binding events exhibited distinct patterns during osteogenesis, and were associated with proximal promoters as well as a large percentage of Runx2 occupancy in non-promoter regions: upstream, introns, exons, transcription termination site (TTS) regions, and intergenic regions. These peaks were partitioned into clusters that are associated with genes in complex biological processes that support bone formation. Using Affymetrix expression profiling of differentiating osteoblasts depleted of Runx2, we identified novel Runx2 targets including Ezh2, a critical epigenetic regulator; Crabp2, a retinoic acid signaling component; Adamts4 and Tnfrsf19, two remodelers of extracellular matrix. We demonstrated by luciferase assays that these novel biological targets are regulated by Runx2 occupancy at non-promoter regions. Our data establish that Runx2 interactions with chromatin across the genome reveal novel genes, pathways and transcriptional mechanisms that contribute to the regulation of osteoblastogenesis.
Publication Title
Genomic occupancy of Runx2 with global expression profiling identifies a novel dimension to control of osteoblastogenesis.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 6 Downloadable Samples
- Illumina HiSeq 2000
Description
We report genome-wide expression changes that occur in adipose-derived mesenchymal stem cells upon treatment with CytoD cytoskeletal drug. mRNA-Seq analysis shows that CytoD-treated samples cluster together. In addition, we also see that cells treated with CytoD show upregulation of osteogenic markers, epiregulators, and a number of key molecular function pathways including extracellular matrix, cell membrane gene expression. Overall design: Adipose MSCs were cultured in Advanced-MEM base (Life Technologies), 5% platelet lysate, and 1% non-essential amino acids (Life Technologies), and 2U/ml heparin. Cells used for experiments were of passage 6. Adipose MSCs were seeded at 3,000 cells per cm2 in maintenance medium in 6-well plates and incubated under standard culture conditions for 24 hours before being changed to osteogenic medium containing vehicle (DMSO) or 0.1 µg/ml cytochalasin D (Sigma). Osteogenic medium maintenance media supplemented with 10 nM dexamethasone, 25 µg/ml ascorbic acid, and 10 mM ß-glycerophosphate. Cells in culture were prepared for RNA isolation by lysing with Qiazol. Purified RNA was then submitted for RNA-sequencing.
Publication Title
Osteogenic Stimulation of Human Adipose-Derived Mesenchymal Stem Cells Using a Fungal Metabolite That Suppresses the Polycomb Group Protein EZH2.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 2 Downloadable Samples
- IlluminaHiSeq2000
Description
Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage-essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt-domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal-derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI-responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma. Overall design: RNA-seq to compare transcriptiome of control A673 ewing sarcoma cells stably expression a non-target or RUNX3 shRNA
Publication Title
RUNX3 facilitates growth of Ewing sarcoma cells.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 1 Downloadable Sample
- Illumina MouseRef-8 v2.0 expression beadchip
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Runx2 is required for early stages of endochondral bone formation but delays final stages of bone repair in Axin2-deficient mice.
Sample Metadata Fields
Sex
View Samples- Homo sapiens
- 5 Downloadable Samples
- Affymetrix Human Gene 1.0 ST Array (hugene10st)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Identifying Nuclear Matrix-Attached DNA Across the Genome.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Mus musculus
- 1 Downloadable Sample
- Illumina MouseRef-8 v2.0 expression beadchip
Description
Runx2 and Axin2 regulate skeletal development. We recently determined that Axin2 and Runx2 molecularly interact in differentiating osteoblasts to regulate intramembranous bone formation, but the relationship between these factors in endochondral bone formation was unresolved. To address this, we examined the effects of Axin2 deficiency on the cleidocranial dysplasia (CCD) phenotype of Runx2+/-mice, focusing on skeletal defects attributed to improper endochondral bone formation. Axin2 deficiency unexpectedly exacerbated calvarial components of the CCD phenotype in the Runx2+/-mice; the endocranial layer of the frontal suture, which develops by endochondral bone formation, failed to mineralize in the Axin2-/-:Runx2+/-mice, resulting in a cartilaginous, fibrotic and larger fontanel than observed in Runx2+/-mice. Transcripts associated with cartilage development (e.g., Acan, miR140) were expressed at higher levels, whereas blood vessel morphogenesis transcripts (e.g., Slit2) were suppressed in Axin2-/-:Runx2+/-calvaria. Cartilage maturation was impaired, as primary chondrocytes from double mutant mice demonstrated delayed differentiation and produced less calcified matrix in vitro. The genetic dominance of Runx2 was also reflected during endochondral fracture repair, as both Runx2+/-and double mutant Axin2-/-:Runx2+/-mice had enlarged fracture calluses at early stages of healing. However, by the end stages of fracture healing, double mutant animals diverged from the Runx2+/-mice, showing smaller calluses and increased torsional strength indicative of more rapid end stage bone formation as seen in the Axin2-/-mice. Taken together, our data demonstrate a dominant role for Runx2 in chondrocyte maturation, but implicate Axin2 as an important modulator of the terminal stages of endochondral bone formation.
Publication Title
Runx2 is required for early stages of endochondral bone formation but delays final stages of bone repair in Axin2-deficient mice.
Sample Metadata Fields
Sex
View Samples- Mus musculus
- 12 Downloadable Samples
- Illumina HiSeq 2000
Description
We report genome-wide expression changes that occur in mouse bone marrow-derived mesenchymal stem cells treated in triplicate for 24 hours with or without Cytochalasin D and/or CK666. mRNA-Seq analysis shows that both cell surface and the nucleus undergo phenotypic changes. Cytochalasin D enhanced expression of genes involved in pathways known to regulate osteoblast differentiation, including genes involved in development and cell signaling, including calcium ion binding, WNT and PI3K/AKT pathway. In summary, RNA-seq data reveal that the CytoD activates genes linked to osteogenesis, while CK666stimulates adipogenic genes. Overall design: Bone marrow-derived MSCs were maintained in MEM containing 10% fetal bovine serum, 100 µg/ml penicillin/streptomycin. For experiments, the cells were plated at a density of 10,000 cells/cm2 in 6-well culture plates and cultured for 1 day prior to application of treatments. Cells were treated with CytochalasinD and/or CK666 for 24h followed by preparation for RNA isolation. Purified RNA was then submitted for RNA-sequencing.
Publication Title
Intranuclear Actin Structure Modulates Mesenchymal Stem Cell Differentiation.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 8 Downloadable Samples
- Illumina HiSeq 2000
Description
We report genome-wide expression changes that occur in H9-iMSCs frozen with different freezing methods that include DMSO and non-DMSO experimental solutions such as SGC (sucrose-glycerol-creatinine, SMC (sucrose-mannitol-creatinine), and SGI (sucrose-mannitol-isoleucine). mRNA-Seq analysis shows that DMSO samples cluster with fresh samples in the same clade, while all samples using the experimental solutions cluster together. In addition, we also see that cells frozen using experimental solutions have upregulation of a number of key molecular function pathways including extracellular matrix structural genes, receptor binding, and growth factor expression. Overall design: H9 MSCs were cultured in alpha-MEM base (Life Technologies), 10% FBS (qualified), and 1% non-essential amino acids (Life Technologies). Culture flasks were coated with 0.01% porcine gelatin (Fisher) for a minimum of 2 hours before H9 MSC seeding. H9 MSCs were seeded in gelatin-coated flasks at a density of approximately 2500 cells/cm2. Cells were split when they reached 70% confluence and were used for experiments only from passages 8 to 12. Control cells in media were similarly combined stepwise with DMSO at a 1:1 final volume ratio. Each of these vials was incubated at room temperature for 0, 1, or 2 hours. Experimental solutions were frozen using a 3°C/min cooling rate while DMSO solutions were frozen using a 1°C/min cooling rate. Samples were submerged in a 37ºC bath to just under cap level, and agitated until only a small ice crystal was present. The cells were combined with acridine orange/propidium iodide (AO/PI) and enumerated using a hemocytometer. Samples were diluted, centrifuged and supernatant was aspirated, followed by preparation for RNA isolation. Purified RNA was then submitted for RNA-sequencing.
Publication Title
Improved Post-Thaw Function and Epigenetic Changes in Mesenchymal Stromal Cells Cryopreserved Using Multicomponent Osmolyte Solutions.
Sample Metadata Fields
Cell line, Subject
View Samples