To 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.
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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
View SamplesWe have generated “reprogrammable” transgenic mice that ubiquitously express the four Yamanaka factors in an inducible manner. Transitory induction of the transgene results in multiple teratomas emerging from a variety of organs, thus indicating that full reprogramming into iPSCs can occur in vivo. By performing bone marrow transplant experiments, we demonstrate that both hematopoietic cells, as well as non-hematopoietic cells can be reprogrammed in vivo. Remarkably, reprogrammable mice also present circulating iPSCs in the bloodstream (in vivo-iPSCs) with all the expected properties of bona fide iPSCs. Moreover, in contrast to in vitro-iPSCs or embryonic stem cells (ESCs), in vivo-iPSCs have an increased capacity to undergo trophectoderm lineage differentiation, which suggests that in vivo-iPSCs are more plastic or primitive than in vitro-generated iPSCs or ESCs. Overall design: 6 clones of in vivo-generated iPSCs, 5 indendent clones of in vitro-generated iPSCs, and 3 clones of established ESCs
Reprogramming in vivo produces teratomas and iPS cells with totipotency features.
Specimen part, Cell line, Subject
View SamplesProgenitors of the first hematopoietic cells in the mouse arise in the early embryo from Brachyury-positive multipotent cells in the posterior-proximal region of the epiblast, but the mechanisms that specify primitive blood cells are still largely unknown. Pluripotency factors maintain uncommitted cells of the blastocyst and embryonic stem cells in the pluripotent state. However, little is known about the role played by these factors during later development, despite their being expressed in the postimplantation epiblast. Using a dual transgene system for controlled expression at postimplantation stages, we found that Nanog blocks primitive hematopoiesis in the gastrulating embryo, resulting in a loss of red blood cells and downregulation of erythropoietic genes. Accordingly, Nanog deficient embryonic stem cells are prone to erythropoietic differentiation. Moreover, Nanog expression in adults prevents the maturation of erythroid cells. By analysis of previous data for NANOG binding during stem cell differentiation and CRISPR/Cas9 genome editing, we found that Tal1 is a direct NANOG target. Our results show that Nanog regulates primitive hematopoiesis by directly repressing critical erythroid lineage specifiers. Overall design: MEPs mRNA profiles of adult mice Nanog-tg treated and untreated with doxycycline were generated by deep sequencing, in triplicate, using Illumina GAIIx.
The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates <i>Tal1</i>.
Cell line, Subject
View SamplesAnalysis of chromatin architecture suggests that the 3D structure of the genome plays a major role in regulating gene expression, orchestrating the compartmentalization of chromatin and facilitating specific enhancer-promoter interactions. However, the mechanisms that control this structuring of the genome are not fully understood. We have addressed this issue by analyzing the role of CTCF, a major architectural factor in chromatin structure, in the embryonic heart. Loss of CTCF triggered an overall downregulation of the cardiac developmental program, suggesting that CTCF facilitates enhancer-promoter interactions in the developing heart. Detailed analysis of the IrxA gene cluster showed that CTCF loss leads to disruption of the heart-specific regulatory domain that surrounds Irx4, resulting in changes in expression of IrxA cluster genes and neighboring genes. In contrast to the critical role proposed for CTCF in organizing large-scale chromatin domains, our results show that CTCF preferentially mediates local regulatory interactions. Overall design: RNAseq of mouse embryonic E10.5 hearts in three conditions: 1) control (labeled as WT), 2) heterozygous (labeled as HET) and 3) homozygous (labeled as KO). Three replicates were performed for each condition, each consisting of a pool of 6 hearts. Tissue was mechanically disaggregated and RNA extracted with trizol and purified through columns.
CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart.
Specimen part, Cell line, 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 SamplesHematopoietic 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 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 SamplesIL-22 acts on epithelial cells and has been shown to induce tissue protective and wound healing responses in these cells. But it has recently been decribed that IL-22 exacerbates ileatis after infection with T. gondii.
Interleukin-22 induces interleukin-18 expression from epithelial cells during intestinal infection.
Specimen part, Time
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