Project description:Abstract Background: Bone marrow stromal cells (BMSCs) are being used for immune modulatory, anti-inflammatory and tissue engineering applications, but the properties responsible for these effects are not completely understood. Human BMSCs were characterized to identify factors that might be responsible for their clinical effects and biomarkers for assessing their quality. Methods: Early passage BMSCs prepared from marrow aspirates of 4 healthy subjects were compared to 3 human embryonic stem cell (hESC) samples, CD34+ cells from 3 healthy subjects and 3 fibroblast cell lines. The cells were analyzed with oligonucleotide expression microarrays with more than 35,000 probes. Results: BMSC gene expression signatures of BMSCs differed from those of hematopoietic stem cells (HSCs), hESCs and fibroblasts. Genes up-regulated in BMSCs were involved with cell movement, cell-to-cell signaling and interaction and proliferation. The BMSC up-regulated genes were most likely to belong to integrin signaling, integrin linked kinase (ILK) signaling, NFR2-mediated oxidative stress response, regulation of actin-based motility by Rho, actin cytoskeletal signaling, caveolar-mediated endocytosis, clathrin-mediated endocytosis and Wnt/beta catenin signaling pathways. Among the most highly up-regulated genes were structural extracellular (ECM) proteins: alpha1 and beta1 integrin chains, fibronectin, collagen type IIIalpha1, and collagen type Valpha1 and functional EMC proteins: connective tissue growth factor (CTGF) and transforming growth factor beta induced protein (TGFBI) and ADAM12. Conclusions: Global analysis of human BMSCs suggests that they are mobile, metabolically active, proliferative and interactive cells that make use of integrins and integrin signaling. They produce abundant ECM proteins; some of which may contribute to their clinical immune modulatory and anti-inflammatory effects. Seven samples from early passage BMSCs were prepared from marrow aspirates of healthy subjects and compared to 3 human embryonic stem cell (hESC) samples, CD34+ cells from 3 healthy subjects and 3 fibroblast cell lines. Total RNA from a pool of PBMCs from six healthy subjects was extracted and amplified into aRNA to serve as a reference.

Project description:miRNA expression profiling was performed on MM.1S MM cells cultured 8 hours in control media or 50nM RGB-286638, with or without BMSCs. The emerging role of miRNAs in the pathogenesis of multiple myeloma (MM) led us to hypothesize that the miRNA network might be among the inducible transcriptional alterations consequent to MM-bone marrow stromal cell (BMSC) interactions. Our data suggests that BMSC induced MM transcription led to aberrant miRNA expression. We therefore hypothesized that agents interfering with RNAPII transcription might inhibit aberrant miRNA expression in MM. To test this hypothesis we used RGB-286638, a novel protein kinase inhibitor, which works primarily via RNAPII inhibition followed by transcriptional arrest in MM cells. miRNA profiling of RGB-286638-exposed MM cells resulted in RNAPII arrest associated with reduced miRNA levels. RGB-286638 abrogated BMSCs-induced miRNAs, which correlated with growth arrest in MM cells. Analysis of RGB-286638-induced differentially-expressed miRNAs in MM cells, in the presence or absence of BMSCs, revealed RNAPII regulation of expression of BMSC-inducible miRNAs with established oncogenic functions in MM Our findings demonstrate the role of RNAPII in regulating miRNA network, suggesting a new rationale for using agents interfering with RNAPII transcription in the treatment of MM. TaqMan Low-Density Array (TLDA) using human miRNA version 2.0A and version 3.0B cards (Applied Biosystems) were applied to examine the global change of miRNA expression levels in MM.1S cells when co-cultured with BMSCs, with or without RGB-286638 treatment. A total of 756 mature miRNA updated in the Sanger miRBase v.15.0 were quantified according to the manufacturer's instructions as previously described. miRNAs with Ct values higher than 37 were excluded from the analysis. Normalization was carried out with the mean of RNU44 and RNU48. Relative quantification of miRNA expression was calculated with the 2M-bM-^HM-^RM-NM-^TM-NM-^TCt Ct method using the ddCt program (Shannon McCormack Advanced Molecular Diagnostics Laboratory Research Services). The data was presented as log10 of the relative quantity of each miRNA.

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MiRNA expression in BMSCs

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