Project description:Gene expression profile of p53 knockdown MSCs or p53 knockdown+TERT MSCs was compared with that of control MSCs. Our data show p53 knockdown prolongs the lifespan of MSCs, and a combination of p53 knockdown and TERT overexpression is sufficient to immortalize MSCs. The results provide important information about the molecular basis underlying p53 knockdown in MSCs and immortalization-related genes of MSCs. Total RNA obtained from p53 knockdown MSCs or p53 knockdown+TERT MSCs from three patients were compared with control MSCs.
Project description:Gene expression profile of p53 knockdown MSCs or p53 knockdown+TERT MSCs was compared with that of control MSCs. Our data show p53 knockdown prolongs the lifespan of MSCs, and a combination of p53 knockdown and TERT overexpression is sufficient to immortalize MSCs. The results provide important information about the molecular basis underlying p53 knockdown in MSCs and immortalization-related genes of MSCs.
Project description:siRNA-mediated knockdown of MED31 and Negative siRNA control in human mesenchymal stem cells Total RNA was collected 72 hours after transfection with siRNA.
Project description:Analysis of gene changes in different genes modulation in MSCs and compared to primary human osteosarcoma cells Total RNA isolated from human parental MSCs and from MSCs siRB, OeMyc, SiRb-OeMyc and primary human osteosarcoma cells
Project description:The instrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterised. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts with only minor fluctuations over time in culture (from day 15 to day 48). We used microarray to compare the gene-expression profile of cultured human fetal cardiac MSCs over time (from day 15 to day 48). MSCs from human fetal hearts were cultured on GelTrex in a defined medium stimulating the canonical Wnt/beta-catenin pathway. Samples from three different time points (day 15, 27 and 48) were compared on microarray.
Project description:The goal of this study was to determine expression profiles of microRNAs (miRNAs) in whole cell extracts of human bone marrow-derived mesenchymal stem/stromal cells (MSCs) as well as in MSCs during osteogenic differentiation. MicroRNAs are epigenetic regulators that commonly function by targeting specific mRNAs resulting in suppression of protein expression and modulation of a number of cellular pathways. This experiment is part of a larger study analyzing the expression of mitochondria-associated miRNAs in MSCs during osteogenesis that we recently submitted to GEO (Series GSE134946). Here, the same three human MSC lines were used in this study, under the same osteogenic induction conditions, to generate expression profiles of miRNAs present in whole cell extracts. A standard in vitro osteogenesis assay system was used to differentiate MSCs toward the osteoblast lineage.Purified whole cell extracts were obtained from MSCs or from MSCs at specific time points of osteogenic induction. RNA was isolated from whole cell extracts and then biotin-labeled in preparation for microRNA array (Affymetrix miRNA array 4.0). Array data was analyzed to generate information on most abundantly-expressed miRNAs in non-induced MSCs as well as in MSCs at set time points (day 3, 7, or 14) of osteogenic induction. Information on significantly differentially-expressed miRNAs during osteogenesis (comparing day 0 with either day 3, 7 or 14) was also obtained.
Project description:Human mesenchymal stem cells or multipotent stromal cells (MSCs) are of interest for clinical therapy, in part because of their capacity for proliferation and differentiation. However, results from clinical trials and in vitro models have been variable, possibly due to MSC heterogeneity and a lack of standardization between MSC in vitro expansion protocols. Here we defined changes in MSCs during expansion in vitro. In low density cultures, MSCs expand through distinct lag, exponential growth and stationary phases. We assayed cultures of passage 2 human MSCs from three donors at low density (50 cells/cm2) at about 5% confluence on Day 2 and after the cultures had expanded to about 70% confluence on Day 7. On Day 2 genes involved in cell division were up-regulated. On Day 7 genes for cell development were up-regulated. The variations between three donors were less than the variation within the expansion of MSCs from a single donor. The microarray data for selected genes were confirmed by real-time PCR, ELISA and FACScan. About 50% of cells at Day 2 were in S-phase compared to 10% at Day 7. The results demonstrated major differences in early and late stage cultures of MSCs that should be considered in using the cells in experiments and clinical applications. Experiment Overall Design: We assayed cultures of passage 2 human MSCs from three donors at low density (50 cells/cm2) at about 5% confluence on Day 2 and after the cultures had expanded to about 70% confluence on Day 7.
Project description:mRNA from bone marrow-derived MSCs stably expressing CTGF-specific shRNA (or empty vector control) was analyzed for differential gene expression. Significant differences were found in cell proliferation-related genes, especially genes related to the M phase of the cell cycle, which were down-regulated in CTGF-knockdown-MSCs compared to control MSCs. Bone marrow-derived MSCs were stably transduced with lentivirus expressing CTGF-specific shRNA or an empty vector control. After antibiotic selection, total RNA was amplified and hybridized to Illumina HT12 version 3 human whole-genome arrays (Illumina, San Diego, CA).