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 Sequence library of miRNAs from a single sample of human foetal mesenchymal stem cells. Results tested and confirmed by northern blotting. Please note that only raw data files are available for the embryonic and neual samples and thus, directly submitted to SRA (SRX547311, SRX548700, respectively under SRP042115/PRJNA247767)
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:In pluripotential reprogramming, a pluripotent state is established within somatic cells. In this study, we have generated induced pluripotent stem (iPS) cells from bi-maternal (uniparental) parthenogenetic neural stem cells (pNSCs) by transduction with four (Oct4, Klf4, Sox2, and c-Myc) or two (Oct4 and Klf4) transcription factors. The parthenogenetic iPS (piPS) cells directly reprogrammed from pNSCs were able to generate germline-competent himeras, and hierarchical clustering analysis showed that piPS cells were clustered more closer to parthenogenetic ES cells than normal female ES cells. Interestingly, piPS cells showed loss of parthenogenetic-specific imprinting patterns of donor cells. Microarray data also showed that the maternally imprinted genes, which were not expressed in pNSCs, were upregulated in piPS cells, indicating that pluripotential reprogramming lead to induce loss of imprinting as well as re-establishment of various features of pluripotent cells in parthenogenetic somatic cells. 5 samples were analyzed by microarray, each one them in duplicate. fNSC: Mouse female NSC (Neural Stem Cell) pNSC: Mouse parthenogenetic NSC (Neural Stem Cell) piPS-2F: Mouse parthenogenetic induced pluripotent cells derived from NSC overexpressing Oct4 and Klf4 pESC-B: Mouse parthenogenetic ESC (Embryonic Stem Cell) SSEA-1 sorted fESC: Mouse female ESC (Embryonic Stem Cell) OG2
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: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:RNA-seq samples from 3 species across a differentiation from induced pluripotent stem cells to neural progenitor cells were generated to study gene expression evolution. Briefly, previously generated urinary stem cell derived iPSCs of 3 human (Homo sapiens) individuals (3 clones), 1 gorilla (Gorilla gorilla) individual and fibroblast derived cynomolgus macaque (Macaca fascicularis) iPSCs of 2 individuals (4 clones) (Geuder et al. 2021) were differentiated to neural progenitor cells via dual-SMAD inhibition as three-dimensional aggregation culture (Chambers et al. 2009; Ohnuki et al. 2014). Bulk RNA-seq libraries of iPSCs and NPCs were generated using prime-seq protocol (Janjic et al. 2022).
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.