Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:Since the role of cord blood (CB) regulatory T cells (Tregs) for the suppression of the allogeneic T-cell response is under investigation, we analyzed and compared the functional properties and gene expression profile of Tregs expanded from CB units or from the peripheral blood (PB) of helathy donors. Tregs were purified from 23 CB units and from the PB of 13 donors and expanded for 6 days with anti-CD3, anti-CD28 and IL-2. Immunophenotypic analyses were performed, and suppressor activity of expanded Tregs was measured in mixed lymphocyte reaction (MLR) cultures. The IL-10 production capacity was tested and gene expression profile experiments were performed on 6 Tregs from PB and 4 from CB. CB and PB Tregs had similar immunophenotypic features. Tregs from CB presented a higher expansion capacity and genomic characterization showed in CB-derived Tregs a significant enrichments of genes involved in cell proliferation, chromatin modification and regulation of gene expression in CB-derived Tregs. All samples were positive for the Foxp3 gene and protein after expansion. CB and PB expanded Tregs exerted a comparable and potent suppressive function of MLR and presented a high in vitro IL-10 production capacity. Gene profile analysis also revealed for PB Tregs a significant enrichments of genes involved in the adaptive immune response. Comparison of the transcriptional profile of T-REGS derived either from cord blood or peripheral blood. 6 highly purified T-regs samples derived from peripheral blood of healthy donors were compared to highly purified T-regs from cord blood.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Our study shows that after extended stimulation of human CD4+CD25+CD127- Tregs in vitro, a stable cell subset that downregulates CD27 and upregulates CD70 develops. Expanded CD27+CD70- Tregs display a gene profile which closely relates to the previously described “Treg gene signature”, whereas CD27-CD70+ Tregs express some genes associated with Treg gene signature and some genes overexpressed in conventional T cells.
Project description:Since the role of cord blood (CB) regulatory T cells (Tregs) for the suppression of the allogeneic T-cell response is under investigation, we analyzed and compared the functional properties and gene expression profile of Tregs expanded from CB units or from the peripheral blood (PB) of helathy donors. Tregs were purified from 23 CB units and from the PB of 13 donors and expanded for 6 days with anti-CD3, anti-CD28 and IL-2. Immunophenotypic analyses were performed, and suppressor activity of expanded Tregs was measured in mixed lymphocyte reaction (MLR) cultures. The IL-10 production capacity was tested and gene expression profile experiments were performed on 6 Tregs from PB and 4 from CB. CB and PB Tregs had similar immunophenotypic features. Tregs from CB presented a higher expansion capacity and genomic characterization showed in CB-derived Tregs a significant enrichments of genes involved in cell proliferation, chromatin modification and regulation of gene expression in CB-derived Tregs. All samples were positive for the Foxp3 gene and protein after expansion. CB and PB expanded Tregs exerted a comparable and potent suppressive function of MLR and presented a high in vitro IL-10 production capacity. Gene profile analysis also revealed for PB Tregs a significant enrichments of genes involved in the adaptive immune response.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs. One-condition experment, gene expression of 3A6
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.