Specific Genetic Manipulation Research Articles

Abstract 2003: Epigenetic And Phenotypic Modulation Of Adventitial Fibroblasts In Atherosclerosis By Coronary Artery Risk Gene Tcf21

The vascular adventitia has long been postulated to play a key role in atherosclerosis, but the relevant adventitial cell populations and the mechanisms by which they influence atherosclerosis have remained elusive. To characterize relevant adventitial cells and their regulators, we have combined single cell multi-omic murine and human data with human genetics. Single cell transcriptomic and epigenetic analysis of both murine and human arterial tissue revealed a unique population of adventitial fibroblasts (AdvFib). Transcriptome and epigenome analysis revealed disproportionate enrichment for coronary artery disease (CAD) GWAS risk loci and genes in AdvFib, suggesting that AdvFib actively contributes to disease. Leveraging this rich information, we created a novel AdvFib specific CreERT2 murine model to enable AdvFib specific genetic manipulation. Murine lineage tracing using this novel AdvFib-specific model of atherosclerosis revealed early expansion and transcriptomic alteration of AdvFib during plaque development, akin to phenotypic modulation of SMC. Cell-cell communication analysis demonstrated an increase in AdvFib-driven crosstalk with other cell types during early phases of atherosclerosis, including activation of pro-inflammatory (Il6, Ccl2/7, Cxcl12) genes. Computational enrichment analysis of regulatory region of genes associated with AdvFib modulation points to a prominent role for TCF21 , a CAD GWAS risk gene. Single cell time course data of atherosclerosis demonstrates that Tcf21 is expressed primarily in AdvFib with dynamic alteration of its expression during atherosclerosis. AdvFib-specific Tcf21 loss in murine model of atherosclerosis resulted in altered expression of ECM and inflammatory genes, with corresponding changes in recruitment of inflammatory cells. Mechanistically, through histone modification cut-and-tag in human coronary AdvFib, we showed that TCF21 regulates AdvFib activation through epigenetic reprograming of key cytokine and cell fate regulators by binding and altering H3K27Acetylation. Collectively, our results begin to establish the role of AdvFib in atherosclerosis and highlight the importance of TCF21 in this process through a precise epigenetic mechanism.

Cystic Fibrosis and the Cells of the Airway Epithelium: What Are Ionocytes and What Do They Do?

Cystic fibrosis (CF) is caused by defects in an anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Recently, a new airway epithelial cell type has been discovered and dubbed the pulmonary ionocyte. Unexpectedly, these ionocytes express higher levels of CFTR than any other airway epithelial cell type. However, ionocytes are not the sole CFTR-expressing airway epithelial cells, and CF-associated disease genes are in fact expressed in multiple airway epithelial cell types. The experimental depletion of ionocytes perturbs epithelial physiology in the mouse trachea, but the role of these rare cells in the pathogenesis of human CF remains mysterious. Ionocytes have been described in diverse tissues(kidney and inner ear) and species (frog and fish). We draw on these prior studies to suggest potential roles of airway ionocytes in health and disease. A complete understanding of ionocytes in the mammalian airway will ultimately depend on cell type-specific genetic manipulation.

Using CRISPR-Cas9-based genome engineering tools in Drosophila melanogaster.

Drosophila melanogaster has been used as a model organism for over a century. Mutant-based analyses have been used extensively to understand the genetic basis of different cellular processes, including development, neuronal function and diseases. Most of the earlier genetic mutants and specific tools were generated by random insertions and deletion strategies and then mapped to specific genomic loci. Since all genomic regions are not equally accessible to random mutations and insertions, many genes still remain uncharacterized. Low efficiency of targeted genomic manipulation approaches that rely on homologous recombination, and difficulty in generating resources for sequence-specific endonucleases, such as ZFNs (Zinc Finger Nucleases) and TALENs (Transcription Activator-Like Effector Nucleases), could not make these gene targeting techniques very popular. However, recently RNA directed DNA endonucleases, such as CRISPR-Cas, have transformed genome engineering owing to their comparative ease, versatility, and low expense. With the added advantage of preexisting genetic tools, CRISPR-Cas-based manipulations are being extensively used in Drosophila melanogaster and simultaneously being fine-tuned for specific experimental requirements. In this chapter, I will discuss various uses of CRISPR-Cas-based genetic engineering and specific design methods in Drosophila melanogaster. I will summarize various already available tools that are being utilized in conjunction with CRISPR-Cas technology to generate specific genetic manipulation and are being optimized to address specific questions. Finally, I will discuss the future directions of Drosophila genetics research and how CRISPR-Cas can be utilized to target specific questions, addressing which has not been possible thus far.

Genetic timestamping of plasma cells in vivo reveals tissue-specific homeostatic population turnover.

Plasma cells (PCs) are essential for protection from infection, and at the origin of incurable cancers. Current studies do not circumvent the limitations of removing PCs from their microenvironment and confound formation and maintenance. Also, the investigation of PC population dynamics has mostly relied on nucleotide analog incorporation that does not label quiescent cells, a property of most PCs. The main impediment is the lack of tools to perform specific genetic manipulation in vivo. Here we characterize a genetic tool (JchaincreERT2) in the mouse that permits first-ever specific genetic manipulation in PCs in vivo, across immunoglobulin isotypes. Using this tool, we found that splenic and bone marrow PC numbers remained constant over-time with the decay in genetically labeled PCs being compensated by unlabeled PCs, supporting homeostatic population turnover in these tissues. The JchaincreERT2 tool paves the way for an in-depth mechanistic understanding of PC biology and pathology in vivo, in their microenvironment.

Asymmetric dimethylarginine (ADMA) accelerates renal cell fibrosis under high glucose condition through NOX4/ROS/ERK signaling pathway

We previously reported that the circulatory level of Asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of nitric oxide synthase, was increased in diabetic kidney disease patients. However, the mechanism and the role of ADMA in diabetic kidney injury remain unclear. Hence, our principal aim is to investigate the causal role of ADMA in the progression of renal cell fibrosis under high glucose (HG) treatment and to delineate its signaling alterations in kidney cell injury. High Glucose/ADMA significantly increased fibrotic events including cell migration, invasion and proliferation along with fibrotic markers in the renal cells; whereas ADMA inhibition reversed the renal cell fibrosis. To delineate the central role of ADMA induced fibrotic signaling pathway and its downstream signaling, we analysed the expression levels of fibrotic markers, NOX4, ROS and ERK activity by using specific inhibitors and genetic manipulation techniques. ADMA stimulated the ROS generation along with a significant increase in NOX4 and ERK activity. Further, we observed that ADMA activated NOX-4 and ERK are involved in the extracellular matrix proteins accumulation. Also, we observed that ADMA induced ERK1/2 phosphorylation was decreased after NOX4 silencing. Our study mechanistically demonstrates that ADMA is involved in the progression of kidney cell injury under high glucose condition by targeting coordinated complex mechanisms involving the NOX4- ROS-ERK pathway.

Dysfunction of parvalbumin neurons in the cerebellar nuclei produces an action tremor.

Essential tremor is a common brain disorder affecting millions of people, yet the neuronal mechanisms underlying this prevalent disease remain elusive. Here, we showed that conditional deletion of synaptotagmin-2, the fastest Ca2+ sensor for synaptic neurotransmitter release, from parvalbumin neurons in mice caused an action tremor syndrome resembling the core symptom of essential tremor patients. Combining brain region-specific and cell type-specific genetic manipulation methods, we found that deletion of synaptotagmin-2 from excitatory parvalbumin-positive neurons in cerebellar nuclei was sufficient to generate an action tremor. The synaptotagmin-2 deletion converted synchronous into asynchronous neurotransmitter release in projections from cerebellar nuclei neurons onto gigantocellular reticular nucleus neurons, which might produce an action tremor by causing signal oscillations during movement. The tremor was rescued by completely blocking synaptic transmission with tetanus toxin in cerebellar nuclei, which also reversed the tremor phenotype in the traditional harmaline-induced essential tremor model. Using a promising animal model for action tremor, our results thus characterized a synaptic circuit mechanism that may underlie the prevalent essential tremor disorder.

T-Cell Derived Interferon Gamma Directly Targets Intestinal Epithelium to Induce Stem Cell Apoptosis in GI Gvhd

Crypt base intestinal stem cells (ISCs) marked by Lgr5 and Olfm4 maintain the intestinal epithelium, and Paneth cells (PCs) provide an epithelial niche for ISCs in the small bowel. ISCs are reduced during gastrointestinal (GI) GVHD, but the precise mechanisms including the role of niche injury are unknown. Additionally, the specific effects of Interferon-γ (IFNγ) on intestinal epithelium in GVHD remain ill-defined. We evaluated kinetics of ISC loss by histology after allogeneic (allo) BMT using Lgr5-LacZ reporter mice. In both MHC- and miHA-mismatched models (LP>B6 and B6>BDF1), ISC numbers quickly recovered from pretransplant TBI conditioning in recipients of T-cell-depleted (TCD) BMT by day +10, but ISCs failed to recover in recipients of allo T cells. T-cell-induced ISC reduction was functionally validated by genetic marking of stem cell progeny and by culturing intestinal organoids from crypts isolated post-BMT. Similar to the kinetics of ISC loss, ISC-dependent organoid-forming capacity was impaired in recipients of allo T cells compared with TCD BMT recipients on day +10 (p<0.05). Likewise, BMT into Olfm4 reporter mice showed significantly reduced lineage tracing from ISCs in recipients of allo T cells (Fig. 1). To better understand the potential for T cells to interact with the ISC compartment, we performed whole-mount 3-D microscopy to distinguish T cell localization within the intraepithelial and lamina propria compartments post-transplant. We found that donor T cells invading the small bowel after BMT were mostly in the crypt region, and infiltration within the lamina propria adjacent to the ISC compartment was much greater than invasion within the epithelium itself (Fig. 2). We next established an ex vivo co-culture system, to model interactions between intestinal epithelium and donor T cells and investigate mechanisms of T-cell-mediated ISC injury. Screening of effector pathways revealed no impact of perforin or FasL, but identified IFNγ as a principal mediator of ISC injury. Culture with allo T cells significantly reduced viable human and mouse intestinal organoid numbers, and this was inhibited by IFNγ neutralization. IFNγ receptor knockout (IFNγR-/-) organoids were resistant to T cells. IFNγ increased expression of Bak1 and decreased expression of Bcl2 in organoids, and induced ISC apoptosis defined by Annexin+Dapi-Lgr5+ phenotype. ISC killing was mediated by intraepithelial JAK/STAT signaling, as JAK1- and STAT1-deficient organoids were resistant, and it was inhibited by Ruxolitinib. Investigating the role of IFNγ in vivo, FACS analysis confirmed donor T cells to be the primary producers of IFNγ in crypt lamina propria, and BMT with IFNγ-/- donor T cells reduced crypt apoptosis, and preserved ISC frequencies. Moreover, BMT with recipients lacking IFNγR specifically in the intestinal epithelium significantly protected ISCs, reduced crypt apoptosis, and ameliorated GI GVHD pathology (Fig.3). Furthermore, ISCs were also protected by epithelial deletion of STAT1 and by Ruxolitinib treatment. As specific genetic manipulation of ISCs in vivo is not possible because genetic targeting of ISCs results in the same changes in their progeny, we utilized ex vivo models to determine if IFNγ kills ISCs by directly inducing their apoptosis or by damaging the PC niche. Manipulation of the niche by culturing wild-type ISCs with IFNγR-/- PCs was not protective to allo T cells. Using a niche-independent high-purity Lgr5+ ISC culture system based on combined GSK3β and HDAC inhibition, IFNγ directly induced cleaved-caspase-3+ ISC apoptosis and substantial ISC colony death, which were inhibited by Bak/Bax deficiency and by the pan-caspase inhibitor QVD. These results confirmed that IFNγ can directly induce ISC apoptosis independent of other cytotoxic effector molecules and independent of injury to the PC niche. In summary, T cells migrating to the GI tract primarily infiltrated the lamina propria adjacent to the ISC compartment, and T-cell-derived IFNγ directly targeted intestinal epithelium via JAK1/STAT1 signaling to induce ISC apoptosis in a PC-independent manner. ISC reduction and GI GVHD pathology were prevented by inhibiting the IFNγR/JAK1/STAT1 axis within the intestinal epithelium, indicating that in addition to their effects on T cells, JAK inhibitors may treat GVHD by inhibiting pathologic cytokine signaling within target organs and shielding them from allo T cells. Disclosures Hanash: Nexus Global Group LLC: Consultancy.

A 16q22.1 variant confers susceptibility to colorectal cancer as a distal regulator of ZFP90

Genome-wide association studies (GWASs) implicate 16q22.1 locus in risk for colorectal cancer (CRC). However, the underlying oncogenic mechanisms remain unknown. Here, through comprehensive filtration, we prioritized rs7198799, a common SNP in the second intron of the CDH1, as the putative causal variant. In addition, we found an association of CRC-risk allele C of rs7198799 with elevated transcript level of biological plausible candidate gene ZFP90 via expression quantitative trait loci analysis. Mechanistically, causal variant rs7198799 resides in an enhancer element and remotely regulate ZFP90 expression by targeting the transcription factor NFATC2. Remarkably, CRISPR/Cas9-guided single-nucleotide editing demonstrated the direct effect of rs7198799 on ZFP90 expression and CRC cellular malignant phenotype. Furthermore, ZFP90 affects several oncogenic pathways, including BMP4, and promotes carcinogenesis in patients and in animal models with ZFP90 specific genetic manipulation. Taken together, these findings reveal a risk SNP-mediated long-range regulation on the NFATC2-ZFP90-BMP4 pathway underlying the initiation of CRC.

Generation of Knockout Human Primary Keratinocytes by CRISPR/Cas9.

The culture of epidermal human primary keratinocytes (HPKs) represents a well-established model in biological and dermatological research. In addition, HPKs are used in three-dimensional organotypic cultures (OTCs), and gene therapeutic approaches have been reported for the treatment of patients suffering from epidermolysis bullosa, a severe blistering disease that can result in postnatal lethality. Therefore, there is a strong need for the development of techniques for the stable and specific genetic manipulation of HPKs, for example, by genome editing via the CRISPR/Cas9 approach. However, the main disadvantage of working with HPKs is the fact that these cells are prone to terminal differentiation and proliferate only for few passages in monoculture. As it is well known that the co-culture of HPKs with fibroblasts strongly increases the lifetime of the epidermal cells, we developed a protocol for the stable modification of HPKs by CRISPR/Cas9 via lentiviral transduction in the presence of 3T3-J2 fibroblasts as feeder cells. Selection of transduced HPKs is achieved with antibiotics in co-culture with antibiotic-resistant feeder cells. Modified HPKs generated by our protocol have the potential to generate epidermis-like structures in OTCs.

Arabidopsis response to the spider mite Tetranychus urticae depends on the regulation of reactive oxygen species homeostasis

Reactive oxygen species (ROS) are molecules that play a prominent role in plant response to numerous stresses, including plant interactions with herbivores. Previous findings indicate that Arabidopsis plants showed an increase in H2O2 accumulation after Tetranychus urticae infestation. Despite its importance, no information has been reported on the relationships between ROS-metabolizing systems and the spider mite-triggered plant-induced responses. In this work, four ROS-related genes that were differentially expressed between the resistant Bla-2 and the susceptible Kon Arabidopsis accessions were selected for the analysis. These genes encode proteins putatively involved in the generation (BBE22) and degradation (GPX7 and GSTU4) of H2O2, and in the degradation of ascorbate (AO). Overexpressing BBE22 and silencing GPX7, GSTU4 and AO resulted in higher leaf damage and better mite performance relative to the wild-type plants. Minor effects on H2O2 accumulation obscure major effects on the expression of genes related to ROS-metabolism and JA and SA signaling pathways, and on ROS-related enzymatic activities. In conclusion, the integration of ROS and ROS-related compounds and enzymes in the response of Arabidopsis to the spider mite T. urticae was confirmed. However, the complex network involved in ROS signaling makes difficult to predict the impact of a specific genetic manipulation.

Glia in brain energy metabolism: A perspective.

Early views of glia as relatively inert, housekeeping cells have evolved, and glia are now recognized as dynamic cells that not only respond to neuronal activity but also sense metabolic changes and regulate neuronal metabolism. This evolution has been aided in part by technical advances permitting progressively better spatial and temporal resolution. Recent advances in cell-type specific genetic manipulation and sub-cellular metabolic probes promise to further this evolution by enabling study of metabolic interactions between intertwined fine neuronal and glial processes in vivo. Views of glia in disease processes have also evolved. Long considered purely reactive, glia and particularly microglia are now seen to play active roles in both promoting and limiting brain injury. At the same time, established concepts of glial energetics are now being linked to areas such as learning and neural network function, topics previously considered far removed from glial biology.

An Introduction to Plant Tissue Culture: Advances and Perspectives.

Plant tissue culture techniques are the most frequently used biotechnological tools for basic and applied purposes ranging from investigation on plant developmental processes, functional gene studies, commercial plant micropropagation, generation of transgenic plants with specific industrial and agronomical traits, plant breeding and crop improvement, virus elimination from infected materials to render high-quality healthy plant material, preservation and conservation of germplasm of vegetative propagated plant crops, and rescue of threatened or endangered plant species. Additionally, plant cell and organ cultures are of interest for the production of secondary metabolites of industrial and pharmaceutical interest. New technologies, such as the genome editing ones combined with tissue culture and Agrobacterium tumefaciens infection, are currently promising alternatives for the highly specific genetic manipulation of interesting agronomical or industrial traits in crop plants. Application of omics (genomics, transcriptomics, and proteomics) to plant tissue culture will certainly help to unravel complex developmental processes such as organogenesis and somatic embryogenesis, which will probably enable to improve the efficiency of regeneration protocols for recalcitrant species. Additionally, metabolomics applied to tissue culture will facilitate the extraction and characterization of complex mixtures of natural plant products of industrial interest. General and specific aspects and applications of plant tissue culture and the advances and perspectives are described in this edition.

Inhibition of ROS induces apoptosis and increases sensitivity to chemotherapy of pancreatic cancer cells

s / Pancreatology 15 (2015) S1eS141 S21 time-specific Cre-activation a Flp-dependent, Tamoxifen-inducible CreERT2 was generated. The influence of mast cells in PDAC development and progression was investigated using mast cell-deficient mice. Results: We could show that transgenic Pdx1-Flp;FSF-KrasG12D mice developed PanIN lesions and PDAC similar to well-established Pdx1Cre;LSL-KrasG12D model. Flp-mediated recombination results in CreERT2 expression selectively in KrasG12D positive pancreatic cells. Tamoxifen treatment allows sequential and site specific genetic manipulation of recombined as shown by conditional activation of reporter genes (eGFP). Depletion of mast cells showed no effect on ADMs and PanIN formation and PDAC development. Implantation experiments also indicated that mast cell deficiency as no effect on tumor formation and its progression. Conclusion: Using this novel dual recombination model we are able to sequentially activate or inactivate genes in the pancreas and to target stromal cells in PDAC formation. By this we show that mast cells are dispensable for PDAC development.

Wnt Signaling in Mammary Glands: Plastic Cell Fates and Combinatorial Signaling

The mouse mammary gland is an outstanding developmental model that exemplifies the activities of many of the effector pathways known to organize mammalian morphogenesis; furthermore, there are well-characterized methods for the specific genetic manipulation of various mammary epithelial cell components. Among these signaling pathways, Wnt signaling has been shown to generate plasticity of fate determination, expanding the genetic programs available to cells in the mammary lineage. It is responsible first for the appearance of the mammary fate in embryonic ectoderm and then for maintaining bi-potential basal stem cells in adult mammary ductal trees. Recent technical developments have led to the separate analysis of various mammary epithelial cell subpopulations, spurring the investigation of Wnt-dependent interactions. Although Wnt signaling was shown to be oncogenic for mouse mammary epithelium even before being identified as the principle oncogenic driver for gut epithelium, conclusive data implicating this pathway as a tumor driver for breast cancer lag behind, and we examine potential reasons.

Consensus in silico computational modelling of the p22phox subunit of the NADPH oxidase

The p22phox protein is an essential subunit of the cytochrome b558 of the NADPH oxidase (Nox) complex which by generating reactive oxygen species (ROS) plays important role in regulating cellular function. p22phox stabilises the Nox enzyme, assists in catalytic core maturation and in the meantime provides an anchoring site for cytosolic regulatory subunits to bind. However, the protein structure of the p22phox is still uncertain. In this study we use an in silico computational bioinformatic approach to produce a consensus 3-dimensional model of the p22phox. Based on published protein sequence data of human p22phox and by using transmembrane specific protein prediction algorithms, we found that p22phox consists of two domains: an N-terminal transmembrane domain (124 a.a.) and a C-terminal cytoplasmic domain (71 a.a.). In its predicted most stable form, p22phox contains three transmembrane helices leading to an extracellular N-terminus and an extensive (39 a.a.) extracellular loop between helices 2 and 3. Furthermore, we locate the cytosolic domain phosphorylation site at threonine147 which literature shows is capable of priming the p22phox, in order to accept its binding partners. Our results are consistent with the biological characterisation of p22phox derived from experiments using specific antibody or genetic manipulation. Our 3-D protein model provides insights into the biological function of p22phox and cytochrome b558, and can be used as tool to investigate the regulatory mechanism of Nox isoforms.

Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci

The strength of recombinant gene expression is a key property of cell lines for biopharmaceutical protein production. In most stable cell lines the expression vector is stably introduced into the host chromosomal DNA. Apart from the copy number and the used expression control elements the performance of recombinant expression vectors is modulated by genetic and epigenetic features provided by flanking host elements. Since targeted integration is very difficult cell clones with high expression of a recombinant vector are created by random integration and large scale screening for gene expression. This allows the isolation of those rare recombinant cells in which gene expression is optimal. This is usually due to locus-specific influences of the chromosomal surroundings. We have developed an efficient methodology for targeting expression cassettes to specific chromosomal sites [1,2]]. The method (Flp recombinase mediated cassette exchange - RMCE) allows the repeated use of defined loci by targeting constructs for expression of proteins and viruses, thereby allowing to exploit the positive features of a given integration site [3,4]]. Thereby, a systematic evaluation of the performance of a set of expression vectors in various chromosomal sites becomes feasible. In this study we screened for high performance integration sites in HEK293 and CHO-K1 cells supporting expression cassettes driven by a potent promoter. As a read out, production of antibodies and recombinant retroviral vectors were used. Thereby, we could show that high level expression of a given promoter is restricted to defined integration sites, while other sites show only moderate expression (data not shown). An important new finding was that a given chromosomal site is not flexible with respect to the integrated cassette but requires the integration of specific promoters. As illustrated in figure ​figure1,1, an integration site, identified for supporting high level expression of an SV40 promoter driven cassette fails to adequately support expression of MPSV and adenoviral major late promoter (AdmlP). Vice versa, another site, initially screened for supporting MSCV promoter expression, could restore expression of the highly homologous MPSV promoter while the SV40 promoter and the AdmlP promoter give only moderate expression. Figure 1 Mode of tagging defines the optimal targeting cassette. Two highly potent chromosomal integration sites were screened upon random integration of an expression cassette driven by the SV40 promoter and the MSCV promoter, respectively. By means of Flp recombinase ... Finally, we tested the impact of the orientation of the cassette in a specific chromosomal site. We found that some integration sites are flexible with respect to the orientation of the expression cassettes while others support expression only in one direction (data not shown). While classical enhancer elements are known to activate promoters largely independent from the relative position this finding suggests that other cis acting elements affect the incoming cassettes in an orientation dependent manner. Together, this shows that not the nature of integration site and the design of the vector as such define the performance of a producer cell clone. Rather, the interplay between these components defines the level and stability of expression. Since these interactions cannot be predicted, the performance of a vector in a given site has to be evaluated empirically. In order to exploit favourable sets of chromosomal sites and vectors we made use of bacterial artificial chromosome (BAC) vectors. By recombineering, expression cassettes were integrated into pre-selected chromosomal sites as encoded by BAC vectors. These vectors were randomly integrated into cells by standard transfection protocols. Clones were isolated and evaluated for expression. As expected, highly reproducible expression characteristics were found in individual clones (data not shown). In conclusion, the definition of favorable combinations of specific integration sites and vector design allow the rational exploitation of given chromosomal sites. For this purpose, technologies for site specific genetic manipulation of mammalian cells are essential. This concerns both targeted integration of expression cassettes into defined loci (such as RMCE or site specific nuclease induced homologous recombination) or by transduction of large chromosomal domains (as provided by BAC vectors). These technologies pave the way for predictable and high expression of biotechnologically relevant products such as antibodies and recombinant viral vectors.

Origin of Thymic Adipocytes in Aging: Incidental to Thymopoiesis or Instigator of Immunosenescence? (132.22)

Abstract Vishwa Deep Dixit, Yun-Hee Youm, Hyunwon Yang, Christo Venkov, Nancy R Manley, Eric Nielson, Todd Leff, Bolormaa Vandanmagsar By 50 years of age most of human thymus is replaced with adipocytes of unknown provenance and uncertain function. We investigated the lineage and mechanism of thymic adipocyte formation during aging and asked whether manipulating these pathways regulates thymic function. Using.We demonstrate that indelibly LacZ marked FoxN1 thymic epithelial cells transition to give rise to local tissue fibroblasts via the epithelial-mesenchymal transition(EMT)process. Importantly, we found an age-related increase in pro-EMT regulator FSP1 and pro-adipogenic transcription factor PPARγ in thymus. Consistent with these findings, a subset of FoxN1LacZ+ cells are FSP1+ with large unilocular lipid droplet, typical of adipocyte phenotype. Furthermore, aged FSP1.GFP reporter mice revealed that the sorted FSP1+ thymic fibroblasts lack TEC signatures and express PPARγ, indicative of adipogenic lineage commitment. Notably, overexpression of constitutive-active PPARγ in FSP1+ adipogenic lineage cells led to increased thymic adipogenesis and reduced thymopoiesis. Conversely, inhibition of EMT and thymoadipogenesis via anti-aging intervention, caloric restriction prevented thymic aging and immunosenescence. Future longevity studies using specific genetic manipulation of these pathways may provide a definitive answer to the puzzle of age-related thymic adiposity and involution.

Involvement of adipokines in rimonabant-mediated insulin sensitivity in ob/ob mice

Abstract Objectives It has been recently reported that blockade of type 1 cannabinoid (CB1) receptors by specific antagonists or genetic manipulation alleviates dyslipidaemia, hyperglycaemia and insulin resistance in animal models of obesity and type 2 diabetes. However, the precise role of adipokines in the insulin-sensitising effects of the CB1 antagonist rimonabant is not clear. Methods ob/ob mice were treated with different doses of rimonabant and then subjected to an oral glucose tolerance test. The expression of different adipokines in white adipose tissue was analysed by quantitative real-time PCR. Key findings Rimonabant (30 mg/kg) significantly inhibited body weight and fat pad weight gain (P < 0.05) and improved glucose tolerance. Gene expression analysis indicated that tumour necrosis factor-α, visfatin and retinol binding protein-4 were downregulated in the adipose tissue of ob/ob mice treated with rimonabant compared with controls, whereas adiponectin was significantly upregulated. Conclusions Rimonabant-mediated alteration of adipokines in white adipose tissues may play a role in improving insulin sensitivity in obese animals.

Involvement of adipokines in rimonabant-mediated insulin sensitivity in <I>ob/ob</I> mice

It has been recently reported that blockade of type 1 cannabinoid (CB1) receptors by specific antagonists or genetic manipulation alleviates dyslipidaemia, hyperglycaemia and insulin resistance in animal models of obesity and type 2 diabetes. However, the precise role of adipokines in the insulin-sensitising effects of the CB1 antagonist rimonabant is not clear. ob/ob mice were treated with different doses of rimonabant and then subjected to an oral glucose tolerance test. The expression of different adipokines in white adipose tissue was analysed by quantitative real-time PCR. Rimonabant (30 mg/kg) significantly inhibited body weight and fat pad weight gain (P < 0.05) and improved glucose tolerance. Gene expression analysis indicated that tumour necrosis factor-alpha, visfatin and retinol binding protein-4 were downregulated in the adipose tissue of ob/ob mice treated with rimonabant compared with controls, whereas adiponectin was significantly upregulated. Rimonabant-mediated alteration of adipokines in white adipose tissues may play a role in improving insulin sensitivity in obese animals.

Regulation of Primate Trophoblast Lineage Differentiation&amp;mdash;Insights Learned from Human Embryonic Stem Cells

Trophectoderm formation is the first cell lineage segregation during early embryo development. Further differentiation of trophectoderm to various types of trophoblasts is the key event of placentation process, which is crucial for embryonic implantation and successful pregnancy. The anatomic structure of human placenta is different from that of rodents which have provided the best model to study molecular and cellular mechanisms of trophoblast development. However, due to ethical and practical restrictions, the regulatory mechanisms of human trophoblast development are poorly explored and understood. Recent evidences demonstrate that human embryonic stem cells (ESCs) are capable of differentiation towards trophoblasts in culture, after induction by extracellular signals or specific genetic manipulation of master regulatory factors. This review summarizes recent advances in deriving human trophoblasts from human ESCs, and suggests novel mechanisms that regulate the human trophectoderm commitment.