No abstract

Key Points LMO2 is deacetylated by the NAMPT/SIRT2 pathway. LMO2 deacetylation is essential for LIM domain binding 1 binding and TAL1 complex activation during hematopoiesis and T-ALL leukemogenesis.

Viral vectors are valuable tools to deliver genetic materials into cells. Vectors derived from human immunodeficiency virus type 1 are being widely used for gene delivery, mainly because they are able to transduce both dividing and non-dividing cells which leads to stable and long term gene expression. In addition, these types of vectors are safe, with low toxicity, high stability and cell type specificity. Therefore, this work was aimed to produce lentivirus-based vector using a three-plasmid system. To produce this system, the eGFP marker gene was cloned into the plasmid pWPXLd. Subsequently, this vector plasmid, along with packaging plasmids, psPAX2 and envelope plasmid, pMD2.G, was co-transfected into packaging cell line (293T) using calcium phosphate method. 48 h post transfection, the constructed viral vector was harvested, purified and concentrated and stored at -80°C for next experiments. The titration of the vector was carried out, using ELISA, flowcytometry, and fluorescent microscopy. Finally, transduction of HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell lines was carried out with indicated cell numbers and multiplicities of infections of the vector in the presence of polybrene. Using this system, high titer lentivirus at titers of up to 2 9 10 8 transducing units/ml (TU/ml) was successfully generated and its transduction efficacy was improved by seven to over 20-fold in various cell types. We demonstrate the applicability of this vector for the efficient transduction of dividing and nondividing cells, including HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell line. Transduction efficiency yielded titers of (6.3 ± 1.2) 10 5 TU/ml. Furthermore, lentivirus transferred transgene was expressed at high level in the target cells and expression was followed until 90 days after transduction. Thus, the vector generated in this work, might be able to deliver the transgene into a wide range of mammalian cells.

Aims  Beyond classical roles in thrombosis and haemostasis, it becomes increasingly clear that platelets contribute as key players to inflammatory processes. The involvement of platelets in these processes is often mediated through a variety of platelet-derived chemokines which are released upon activation and act as paracrine and autocrine factors. In this study, we investigate CXCL14, a newly described platelet chemokine and its role in thrombus formation as well as monocyte and platelet migration. In addition, we examine the chemokine receptor CXCR4 as a possible receptor for CXCL14 on platelets. Furthermore, with the use of artificially generated platelets derived from induced pluripotent stem cells (iPSC), we investigate the importance of CXCR4 for CXCL14-mediated platelet functions. Methods and results  In this study, we showed that CXCL14 deficient platelets reveal reduced thrombus formation under flow compared with wild-type platelets using a standardized flow chamber. Addition of recombinant CXCL14 normalized platelet-dependent thrombus formation on collagen. Furthermore, we found that CXCL14 is a chemoattractant for platelets and mediates migration via CXCR4. CXCL14 promotes platelet migration of platelets through the receptor CXCR4 as evidenced by murine CXCR4-deficient platelets and human iPSC-derived cultured platelets deficient in CXCR4. We found that CXCL14 directly interacts with the CXCR4 as verified by immunoprecipitation and confocal microscopy. Conclusions  Our results reveal CXCL14 as a novel platelet-derived chemokine that is involved in thrombus formation and platelet migration. Furthermore, we identified CXCR4 as principal receptor for CXCL14, an interaction promoting platelet migration.

CRISPR/Cas9-mediated gene editing of stem cells and primary cell types has several limitations for clinical applications. The direct delivery of ribonucleoprotein (RNP) complexes consisting of Cas9 nuclease and guide RNA (gRNA) has improved DNA- and virus-free gene modifications, but it does not enable the essential enrichment of the gene-edited cells. Here, we established a protocol for the fluorescent labeling and delivery of CRISPR/Cas9–gRNA RNP in primary human hematopoietic stem and progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). As a proof of principle for genes with low-abundance transcripts and context-dependent inducible expression, we successfully deleted growth arrest and DNA-damage-inducible β (GADD45B). We found that GADD45B is indispensable for DNA damage protection and survival in stem cells. Thus, we describe an easy and efficient protocol of DNA-free gene editing of hard-to-target transcripts and enrichment of gene-modified cells that are generally difficult to transfect.