Generating human serotonergic neurons from fibroblasts
Generation of functional human serotonergic neurons from fibroblasts.
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Analysis of mRNA profiles after MEK1/2 inhibition in human pancreatic cancer cell lines reveals pathways involved in drug sensitivity.
Specimen part, Cell line
View SamplesAnalysis of mRNA profiles after MEK1/2 inhibition in human pancreatic cancer cell lines reveals pathways involved in drug sensitivity.
Analysis of mRNA profiles after MEK1/2 inhibition in human pancreatic cancer cell lines reveals pathways involved in drug sensitivity.
Specimen part, Cell line
View SamplesAnalysis of mRNA profiles after MEK1/2 inhibition in human pancreatic cancer cell lines reveals pathways involved in drug sensitivity.
Analysis of mRNA profiles after MEK1/2 inhibition in human pancreatic cancer cell lines reveals pathways involved in drug sensitivity.
Specimen part, Cell line
View SamplesThe ATP-dependent DExH/D-box helicase DHX9 is a key participant in a number of gene regulatory steps, including transcriptional, translational, microRNA-mediated control, DNA replication, and maintenance of genomic stability. DHX9 has also been implicated in maintenance of the tumorigenic process and in drug response. Here, we report that inhibition of DHX9 expression is lethal to multiple human and mouse cancer cell lines. In contrast, using a novel conditional shDHX9 mouse model, we demonstrate that sustained and prolonged suppression of DHX9 is well tolerated at the organismal level. Our results demonstrate a robust tolerance for DHX9 knockdown in non-transformed cells and supports the targeting of DHX9 as an effective and specific chemotherapeutic approach.
Tumor cell survival dependence on the DHX9 DExH-box helicase.
Specimen part
View SamplesThese cultures were grown to examine the differences in Agr-regulated virulence factor gene expression between wild-type S. aureus FRI1169 and a non-hemolytic variant isolated from a biofilm inoculated with FRI1169. The study is described more thoroughly in the paper "Generation of virulence factor variants in Staphylococcus aureus biofilms", Yarwood et al., J. Bacteriol. 2007.
Generation of virulence factor variants in Staphylococcus aureus biofilms.
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View SamplesDespite widespread interest in using human stem cells in neurological disease modeling, a suitable model system to study human neuronal connectivity is lacking. Here, we report a protocol for efficient differentiation of hippocampal pyramidal neurons and an in vitro model for hippocampal neuronal connectivity. We developed an embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-based protocol to differentiate human CA3 pyramidal neurons from patterned hippocampal neural progenitor cells (NPCs). This differentiation induces a comprehensive patterning and generates multiple CA3 neuronal subtypes. The differentiated CA3 neurons are functionally active and readily form neuronal connection with dentate granule (DG) neurons in vitro, recapitulating the synaptic connectivity within the hippocampus. When we applied this neuronal co-culture approach to study connectivity in schizophrenia, we found deficits in spontaneous activity in patient iPSC derived DG–CA3 co-culture by multi-electrode array recording. In addition, both multi-electrode array recording and whole cell patch clamp electrophysiology revealed a reduction in spontaneous and evoked neuronal activity in CA3 neurons derived from schizophrenia patients. Altogether these results underscore the relevance of this new model in studying diseases with hippocampal vulnerability. Overall design: 4 technical replicates were used and later pooled together for the bioinformatic analysis.
Efficient Generation of CA3 Neurons from Human Pluripotent Stem Cells Enables Modeling of Hippocampal Connectivity In Vitro.
Specimen part, Subject
View SamplesSingle-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully re-capitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc, and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes . In addition, we observe a continuum of activation states, revealing a pseudo-temporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs,allowing for novel insights into neuronal activation patterns in vivo. Overall design: Examination of 1) 82 whole-cell (WC) dentate granule cells from a PTZ- or saline-treated mouse, and 2) 23 single-nuclei (SN) from dentate granule cells of a homecage (HC) mouse or 96 nuclei from a mouse exposed to a novel environment (NE)
Nuclear RNA-seq of single neurons reveals molecular signatures of activation.
Specimen part, Cell line, Treatment, Subject
View SamplesPreviously, using a forward genetic approach we identified B. burgdorferi arthritis-associated locus 1 (Bbaa1), a quantitative trait locus on Chr4, which physically encompasses the type I IFN gene cluster and regulates Lyme arthritis through heightened type I IFN production. Reciprocal radiation chimeras between B6.C3-Bbaa1 and B6 mice revealed that arthritis is initiated by radiation-sensitive cells, but orchestrated by radiation-resistant components of joint tissue. Advanced congenic lines were developed to reduce the physical size of the Bbaa1 interval, and RNA-seq of resident CD45- joint cells from advanced interval specific recombinant congenic lines (ISRCL4 and ISRCL3) identified myostatin as uniquely upregulated in association with Bbaa1 arthritis development. Our manuscript further demonstrates that myostatin expression is linked to IFN-ß production, and in vivo inhibition of myostatin suppresses Lyme arthritis in the reduced interval Bbaa1 congenic mice, formally implicating myostatin as a novel downstream mediator of joint-specific inflammatory response to B. burgdorferi. Overall design: 22 days following infection with B. burgdorferi, mouse rear ankle joints were gently digested into single-cell suspensions and CD45 negative cells were isolated by magnetic bead separation. CD45 negative cells from both rear ankle joints of two mice were pooled for each n sample in order to increase RNA concentration for gene expression analysis (n=5 per genotype). Gene expression comparisons were made between B6 (control group) and ISRCL4/ISRCL3 congenic lines.
Genetic Control of Lyme Arthritis by <i>Borrelia burgdorferi</i> Arthritis-Associated Locus 1 Is Dependent on Localized Differential Production of IFN-β and Requires Upregulation of Myostatin.
Specimen part, Cell line, Subject
View SamplesThe small G-protein KRAS is crucial for mediating gonadotropin-induced events associated with ovulation. However, constitutive expression of KrasG12D in granulosa cells disrupted normal follicle development leading to the persistence of abnormal follicle-like structures containing non-mitotic cells. To determine what factors mediate this potent effect of KrasG12D, gene profiling analyses were done. We also analyzed KrasG12D;Cyp19-Cre and KrasG12D;Pgr-Cre mutant mouse models that express Cre prior to or after the initiation of granulosa cell differentiation, respectively. KrasG12D induced cell cycle arrest in granulosa cells of the KrasG12D;Cyp19-Cre mice but not in the KrasG12D;Pgr-Cre mice, documenting the cell context specific effect of KrasG12D. Expression of KrasG12D silenced the Kras gene, reduced cell cycle activator genes and impaired expression of granulosa cell and oocyte specific genes. Conversely, levels of PTEN and phosphorylated p38MAPK increased markedly in the mutant granulosa cells. Because disrupting Pten in granulosa cells leads to increased proliferation and survival, Pten was disrupted in the KrasG12D mutant mice. The Pten/Kras mutant mice were infertile but lacked GCTs. By contrast, the Ptenfl/fl;KrasG12D;Amhr2-Cre mice developed aggressive ovarian surface epithelial (OSE) cell tumors that did not occur in the Ptenfl/fl;KrasG12D;Cyp19-Cre or Ptenfl/fl;KrasG12D;Pgr-Cre mouse strains. These data document unequivocally that Amhr2-Cre is expressed in and mediates allelic recombination of oncogenic genes in OSE cells. That KrasG12D/Pten mutant granulosa cells do not transform but rather undergo cell cycle arrest indicates that they resist the oncogenic insults of Kras/Pten by robust self-protecting mechanisms that silence the Kras gene and elevate PTEN and phospho-p38MAPK.
Cell type-specific targeted mutations of Kras and Pten document proliferation arrest in granulosa cells versus oncogenic insult to ovarian surface epithelial cells.
Age, Specimen part
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