131 research outputs found

    Utility of CRISPR/Cas9 systems in hematology research

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    Since the end of the 20th century, the development of novel approaches have emerged to manipulate experimental models of hematological disorders, so they would more accurately mirror what is observed in the clinic. Despite these technological advances, the characterization of crucial genes for benign or malignant hematological disorders remains challenging, mainly because of the dynamic nature of the hematopoietic system and the genetic heterogeneity of these disorders. To overcome this limitation, genome editing technologies have been developed to specifically manipulate the genome via deletion, insertion or modification of targeted loci. These technologies have swiftly progressed, allowing their common use to investigate genetic function in experimental hematology. Amongst them, homologous recombination (HR)-mediated targeting technologies have facilitated the manipulation of specific loci by generating knockout and knock-in models. Despite promoting significant advances in the understanding of the molecular mechanisms involved in hematology, these inefficient, time-consuming and labor-intensive approaches did not permit the development of cellular or animal models recapitulating the complexity of hematological disorders. In October 2016, Dr. Ben Ebert and Dr. Chad Cowan shared their knowledge and experiences with the utilization of CRISPR for models of myeloid malignancy, disease, and novel therapeutics. Here we provide an overview of the topics they covered including insights into the novel applications of the technique as well as its strengths and limitations

    Induced pluripotent stem (iPS) cells: an up-to-the-minute review

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    Recent advances in nuclear reprogramming technology allow the transformation of terminally differentiated, adult cells into induced pluripotent stem cells whose phenotype is indistinguishable from that of embryonic stem cells. This leap forward enables the creation of patient-specific pluripotent cell lines that carry disease genotypes. These cell lines could be used both as in vitro models for the study of disease and as potential sources of material for cell replacement therapy. Ultimately, a greater understanding of the process by which cellular identity is shaped and altered may allow the generation of particular cell types for the treatment of degenerative disease

    Ephrin-B2 reverse signaling is required for axon pathfinding and cardiac valve formation but not early vascular development

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    AbstractVascular development begins with the formation of a primary vascular plexus that is rapidly remodeled by angiogenesis into the interconnected branched patterns characteristic of mature vasculature. Several receptor tyrosine kinases and their ligands have been implicated to control early development of the vascular system. These include the vascular endothelial growth factor receptors (VEGFR-1 and VEGFR-2) that bind VEGF, the Tie-1 and Tie-2 receptors that bind the angiopoietins, and the EphB4 receptor that binds the membrane-anchored ligand ephrin-B2. Targeted mutations in the mouse germline have revealed essential functions for these molecules in vascular development. In particular, protein-null mutations that delete either EphB4 or ephrin-B2 from the mouse have been shown to result in early embryonic lethality due to failed angiogenic remodeling. The venous expression of EphB4 and arterial expression of ephrin-B2 has lead to the speculation that the interaction of these two molecules leads to bidirectional signaling into both the receptor-expressing cell and the ligand-expressing cell, and that both forward and reverse signals are required for proper development of blood vessels in the embryo. Indeed, targeted removal of the ephrin-B2 carboxy-terminal cytoplasmic tail by another group was shown to perturb vascular development and result in the same early embryonic lethality as the null mutation, leading the authors to propose that ephrin-B2 reverse signaling directs early angiogenic remodeling of the primary vascular plexus [Cell 104 (2001) 57]. However, we show here that the carboxy-terminal cytoplasmic domain of ephrin-B2, and hence reverse signaling, is not required during early vascular development, but it is necessary for neonatal survival and functions later in cardiovascular development in the maturation of cardiac valve leaflets. We further show that ephrin-B2 reverse signaling is required for the pathfinding of axons that form the posterior tract of the anterior commissure. Our results thus indicate that ephrin-B2 functions in the early embryo as a typical instructive ligand to stimulate EphB4 receptor forward signaling during angiogenic remodeling and that later in embryonic development ephrin-B2 functions as a receptor to transduce reverse signals involved in cardiac valve maturation and axon pathfinding

    Efficient Culturing and Genetic Manipulation of Human Pluripotent Stem Cells

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    Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions, and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs

    Pattern Specification and Immune Response Transcriptional Signatures of Pericardial and Subcutaneous Adipose Tissue

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    Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality in the United States. Recent studies suggest that pericardial adipose tissue (PCAT) secretes inflammatory factors that contribute to the development of CVD. To better characterize the role of PCAT in the pathogenesis of disease, we performed a large-scale unbiased analysis of the transcriptional differences between PCAT and subcutaneous adipose tissue, analysing 53 microarrays across 19 individuals. As it was unknown whether PCAT-secreted factors are produced by adipocytes or cells in the supporting stromal fraction, we also sought to identify differentially expressed genes in isolated pericardial adipocytes vs. isolated subcutaneous adipocytes. Using microarray analysis, we found that: 1) pericardial adipose tissue and isolated pericardial adipocytes both overexpress atherosclerosis-promoting chemokines and 2) pericardial and subcutaneous fat depots, as well as isolated pericardial adipocytes and subcutaneous adipocytes, express specific patterns of homeobox genes. In contrast, a core set of lipid processing genes showed no significant overlap with differentially expressed transcripts. These depot-specific homeobox signatures and transcriptional profiles strongly suggest different functional roles for the pericardial and subcutaneous adipose depots. Further characterization of these inter-depot differences should be a research priority

    A distant trophoblast-specific enhancer controls HLA-G expression at the maternal–fetal interface

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    HLA-G, a nonclassical HLA molecule uniquely expressed in the placenta, is a central component of fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Here, systematic interrogation of the HLA-G locus using massively parallel reporter assay (MPRA) uncovered a previously unidentified cis-regulatory element 12 kb upstream of HLA-G with enhancer activity, Enhancer L. Strikingly, clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of this enhancer resulted in ablation of HLA-G expression in JEG3 cells and in primary human trophoblasts isolated from placenta. RNA-seq analysis demonstrated that Enhancer L specifically controls HLA-G expression. Moreover, DNase-seq and chromatin conformation capture (3C) defined Enhancer L as a cell type-specific enhancer that loops into the HLA-G promoter. Interestingly, MPRA-based saturation mutagenesis of Enhancer L identified motifs for transcription factors of the CEBP and GATA families essential for placentation. These factors associate with Enhancer L and regulate HLA-G expression. Our findings identify long-range chromatin looping mediated by core trophoblast transcription factors as the mechanism controlling tissue-specific HLA-G expression at the maternal–fetal interface. More broadly, these results establish the combination of MPRA and CRISPR/Cas9 deletion as a powerful strategy to investigate human immune gene regulation
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