Hematopoietic stem and progenitor cells are a rare, self-renewing bone marrow resident population capable of giving rise to all circulating hematopoietic cells. They can be used therapuetically for reconstituting defective or ablated hematopoietic systems following chemotherapy, and for inducing tolerance toward allografts of the same haplotype as the HSC donor. There are several sources for HSCs, such as the adult bone marrow, or umblical cord blood, which is more replete with such HSCs. However, HSCs obtained from such sources may be immunogenic, especially if isolated from adult bone marrow. To overcome this issue, our lab has establsihed human induced pluripotent stem cell-derived HPCs with the hope of creating a nonimmunogenic, readily available and unlimited source of HSCs to use for therapy.
Human iPS cell-derived hematopoietic progenitor cells induce T-cell anergy in in vitro-generated alloreactive CD8(+) T cells.
Disease
View SamplesWe 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.
Osteogenic Stimulation of Human Adipose-Derived Mesenchymal Stem Cells Using a Fungal Metabolite That Suppresses the Polycomb Group Protein EZH2.
Specimen part, Subject
View SamplesPmr1 is a cis-Golgi Mn/Ca transporter with a key role in protein glycosylation and manganese detoxification.
Manganese redistribution by calcium-stimulated vesicle trafficking bypasses the need for P-type ATPase function.
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View SamplesTo exert regulatory function, miRNAs guide Argonaute (AGO) proteins to partially complementary sites on target RNAs. Crosslinking and immunoprecipitation (“re state-of-the-art to map AGO binding sites, but assigning the targeting miRNA to these sites relies on bioinformatics predictions and is therefore indirect. To directly and unambiguously identify miRNA:target site interactions, we modified our CLIP methodology in C. elegans to experimentally ligate miRNAs to their target sites. Unexpectedly, ligation reactions also occurred in absence of the exogenous ligase. Our in vivo dataset and re-analysis of published mammalian AGO-CLIP data for miRNA-chimeras yielded >17,000 miRNA:target site interactions. Analysis of interactions and extensive experimental validation of chimera-discovered targets of viral miRNAs suggest that our strategy identifies canonical, non-canonical, and non-conserved miRNA interactions. Our data suggest that ~80% of miRNA:targets have perfect or partial seed complementarity. In summary, analysis of miRNA:target chimeras enables the systematic, context-specific, in vivo discovery of miRNA interactions. Overall design: In vivo PAR-CLIP basically as described previously (Jungkamp et al. 2011) using GFP-tagged ALG-1 expressing worms in L3 stage. Worm lysate was treated with RNase T1. Following immunoprecipitation and a second RNase T1 digest, it was proceeded as described in Hafner et al. 2010. For the modified iPAR-CLIP ligation samples and its control samples immuno-purified complexes were treated with PNK phospathase minus, subjected to ligation with T4 RNA ligase/no ligase added and subsequently phosphorylated with PNK. Protein purification and RNA library preparation essentially as described in Hafner et al., but with the selection of longer RNA products.
Unambiguous identification of miRNA:target site interactions by different types of ligation reactions.
Specimen part, Subject
View SamplesTo more closely reproduce key cellular and stromal features of the desmoplastic reaction of cholangiocarcinoma in vitro, we developed a novel 3-dimensional culture modeling of cancer and stromal cells as a strategy for targeted therapies
Transforming Growth Factors α and β Are Essential for Modeling Cholangiocarcinoma Desmoplasia and Progression in a Three-Dimensional Organotypic Culture Model.
No sample metadata fields
View SamplesTh17 cells are believed to be a critical cell population for driving autoimmune diseases. However, environmental factors that are directly related to the development of Th17 cells are largely unknown.
Sodium chloride drives autoimmune disease by the induction of pathogenic TH17 cells.
Specimen part
View Samplesp63 mutations have been associated with several human hereditary disorders characterized by ectodermal dysplasia such as EEC syndrome, ADULT syndrome and AEC syndrome . The location and functional effects of the mutations that underlie these syndromes reveal a striking genotype-phenotype correlation. Unlike EEC and ADULT that result from missense mutations in the DNA-binding domain of p63, AEC is solely caused by missense mutations in the SAM domain of p63. We report a study on the TAp63a isoform, the first to be expressed during development of the embryonic epithelia, and on its naturally occurring Q540L mutant derived from an AEC patient. To assess the effects of the Q540L mutation, we generated stable cell lines expressing TAp63a wt, DeltaNp63 alpha or the TAp63 alpha-Q540L mutant protein and used them to systematically compare the cell growth regulatory activity of the mutant and wt p63 proteins and to generate, by microarray analysis, a comprehensive profile of differential gene expression. We found that the Q540L substitution impairs the transcriptional activity of TAp63a and causes misregulation of genes involved in the control of cell growth and epidermal differentiation.
The Hay Wells syndrome-derived TAp63alphaQ540L mutant has impaired transcriptional and cell growth regulatory activity.
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View SamplesUridylation of diverse RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms and mammals. Here, we report the first uridylyltransferase that acts on small RNAs in Drosophila, which we refer to as Tailor. Tailor is the source for the majority of 3´ end-modifications in microRNAs and predominantly targets precursor-hairpins. Uridylation modulates the characteristic two-nucleotide 3´ overhangs of microRNA hairpins, which regulates processing by Dicer-1 and destabilizes RNA hairpins. Furthermore, Tailor preferentially uridylates mirtron-hairpins, thereby impeding the production of non-canonical microRNAs. Mirtron-selectivity is explained by unique primary sequence specificity of Tailor, selecting RNA substrates ending with a 3´ guanosine, a feature not previously observed for TUTases. In contrast to mirtrons, conserved Drosophila pre-miRNAs are significantly depleted in 3´ guanosine, thereby escaping regulatory uridylation. Our data support the hypothesis that evolutionary adaptation to pre-miRNA uridylation shapes the nucleotide composition of pre-miRNA 3´ ends. Hence, hairpin-uridylation may serve as a barrier for the de novo creation of miRNAs in Drosophila. Overall design: mRNA sequencing of Drosophila S2 cells (3-times; control libraries) and three biological replicates of S2 cells stably depleted of CG1091/Tailor by CRISPR/Cas9
Uridylation of RNA Hairpins by Tailor Confines the Emergence of MicroRNAs in Drosophila.
Cell line, Subject
View SamplesOchratoxin A gene expression profiling in liver and kidney, with time points of exposure from 7 days to 12 motnhs
A toxicogenomics approach to identify new plausible epigenetic mechanisms of ochratoxin a carcinogenicity in rat.
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View SamplesThe Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types. However, we still have limited knowledge about the role of Notch in early preimplantation stages of mammalian development, or how it interacts with other signalling pathways active at these stages such as Hippo. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos and pluripotent cell culture, we have found that Notch is active from the 4-cell stage. Transcriptomic analysis in single morula identified novel Notch targets, such as early naïve pluripotency markers or transcriptional repressors such as TLE4. Our results reveal a previously undescribed role for Notch in driving transitions during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions. Overall design: Transcriptomic analysis comparing single Rbpj mutant and control mouse morulae. RNA was isolated from individual E2.5 embryos from two litters. 3 mutant and 3 control embryos were used for analysis.
Transitions in cell potency during early mouse development are driven by Notch.
Specimen part, Subject
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