搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence,which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here,we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity,simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity. textcopyright 2013 Elsevier Inc. View Publication

The anisotropic alignment of cardiomyocytes in native myocardium tissue is a functional feature that is absent in traditional in vitro cardiac cell culture. Microenvironmental factors cue structural organization of the myocardium,which promotes the mechanical contractile properties and electrophysiological patterns seen in mature cardiomyocytes. Current nano- and microfabrication techniques,such as photolithography,generate simplified cell culture topographies that are not truly representative of the multifaceted and multi-scale fibrils of the cardiac extracellular matrix. In addition,such technologies are costly and require a clean room for fabrication. This chapter offers an easy,fast,robust,and inexpensive fabrication of biomimetic multi-scale wrinkled surfaces through the process of plasma treating and shrinking prestressed thermoplastic. Additionally,this chapter includes techniques for culturing stem cells and their cardiac derivatives on these substrates. Importantly,this wrinkled cell culture platform is compatible with both fluorescence and bright-field imaging; real-time physiological monitoring of CM action potential propagation and contraction properties can elucidate cardiotoxicity drug effects. View Publication

Human sensory neurons are inaccessible for functional examination,and thus little is known about the mechanisms mediating touch sensation in humans. Here we demonstrate that the mechanosensitivity of human embryonic stem (hES) cell-derived touch receptors depends on PIEZO2. To recapitulate sensory neuron development in vitro,we established a multistep differentiation protocol and generated sensory neurons via the intermediate production of neural crest cells derived from hES cells or human induced pluripotent stem (hiPS) cells. The generated neurons express a distinct set of touch receptor-specific genes and convert mechanical stimuli into electrical signals,their most salient characteristic in vivo. Strikingly,mechanosensitivity is lost after CRISPR/Cas9-mediated PIEZO2 gene deletion. Our work establishes a model system that resembles human touch receptors,which may facilitate mechanistic analysis of other sensory subtypes and provide insight into developmental programs underlying sensory neuron diversity. View Publication

Thrombopoietin (TPO) is a potent regulator of megakaryopoiesis and stimulates megakaryocyte (MK) progenitor expansion and MK differentiation. In this study,we show that TPO induces activation of the mammalian target of rapamycin (mTOR) signaling pathway,which plays a central role in translational regulation and is required for proliferation of MO7e cells and primary human MK progenitors. Treatment of MO7e cells,human CD34+,and primary MK cells with the mTOR inhibitor rapamycin inhibits TPO-induced cell cycling by reducing cells in S phase and blocking cells in G0/G1. Rapamycin markedly inhibits the clonogenic growth of MK progenitors with high proliferative capacity but does not reduce the formation of small MK colonies. Addition of rapamycin to MK suspension cultures reduces the number of MK cells,but inhibition of mTOR does not significantly affect expression of glycoproteins IIb/IIIa (CD41) and glycoprotein Ib (CD42),nuclear polyploidization levels,cell size,or cell survival. The downstream effectors of mTOR,p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1),are phosphorylated by TPO in a rapamycin- and LY294002-sensitive manner. Part of the effect of the phosphatidyl inositol 3-kinase pathway in regulating megakaryopoiesis may be mediated by the mTOR/S6K/4E-BP1 pathway. In conclusion,these data demonstrate that the mTOR pathway is activated by TPO and plays a critical role in regulating proliferation of MK progenitors,without affecting differentiation or cell survival. View Publication

The mechanisms by which megakaryocytes (MKs) differentiate and release platelets into the circulation are not well understood. However,growing evidence indicates that a complex regulatory mechanism involving MK-matrix interactions may contribute to the quiescent or permissive microenvironment related to platelet release within bone marrow. To address this hypothesis,in this study we demonstrate that human MKs express and synthesize cellular fibronectin (cFN) and transglutaminase factor XIII-A (FXIII-A). We proposed that these 2 molecules are involved in a new regulatory mechanism of MK-type I collagen interaction in the osteoblastic niche. In particular,we demonstrate that MK adhesion to type I collagen promotes MK spreading and inhibits pro-platelet formation through the release and relocation to the plasma membrane of cFN. This regulatory mechanism is dependent on the engagement of FN receptors at the MK plasma membrane and on transglutaminase FXIII-A activity. Consistently,the same mechanism regulated the assembly of plasma FN (pFN) by adherent MKs to type I collagen. In conclusion,our data extend the knowledge of the mechanisms that regulate MK-matrix interactions within the bone marrow environment and could serve as an important step for inquiring into the origins of diseases such as myelofibrosis and congenital thrombocytopenias that are still poorly understood. View Publication

Next-generation mass spectrometric (MS) techniques such as SWATH-MS have substantially increased the throughput and reproducibility of proteomic analysis,but ensuring consistent quantification of thousands of peptide analytes across multiple liquid chromatography-tandem MS (LC-MS/MS) runs remains a challenging and laborious manual process. To produce highly consistent and quantitatively accurate proteomics data matrices in an automated fashion,we developed TRIC (http://proteomics.ethz.ch/tric/),a software tool that utilizes fragment-ion data to perform cross-run alignment,consistent peak-picking and quantification for high-throughput targeted proteomics. TRIC reduced the identification error compared to a state-of-the-art SWATH-MS analysis without alignment by more than threefold at constant recall while correcting for highly nonlinear chromatographic effects. On a pulsed-SILAC experiment performed on human induced pluripotent stem cells,TRIC was able to automatically align and quantify thousands of light and heavy isotopic peak groups. Thus,TRIC fills a gap in the pipeline for automated analysis of massively parallel targeted proteomics data sets. View Publication

Human pluripotent stem cells (PSCs) are critical in vitro tools forbackslashnunderstanding mechanisms that regulate lineage differentiation inbackslashnthe human embryo as well as a potentially unlimited supply of stembackslashncells for regenerative medicine. Pluripotent human and mouse embryonicbackslashnstem cells (ESCs) derived from the inner cell mass of blastocystsbackslashnshare a similar transcription factor network to maintain pluripotencybackslashnand self-renewal,yet there are considerable molecular differencesbackslashnreflecting the diverse environments in which mouse and human ESCsbackslashnare derived. In the current study we evaluated the role of Proteinbackslashnarginine methyltransferase 5 (PRMT5) in human ESC (hESC) self-renewalbackslashnand pluripotency given its critical role in safeguarding mouse ESCbackslashnpluripotency. Unlike the mouse,we discovered that PRMT5 has no rolebackslashnin hESC pluripotency. Using microarray analysis we discovered thatbackslashna significant depletion in PRMT5 RNA and protein from hESCs changedbackslashnthe expression of only 78 genes,with the majority being repressed.backslashnFunctionally,we discovered that depletion of PRMT5 had no effectbackslashnon expression of OCT4,NANOG or SOX2,and did not prevent teratomabackslashnformation. Instead,we show that PRMT5 functions in hESCs to regulatebackslashnproliferation in the self-renewing state by regulating the fractionbackslashnof cells in Gap 1 (G1) of the cell cycle and increasing expressionbackslashnof the G1 cell cycle inhibitor P57. Taken together our data unveilsbackslashna distinct role for PRMT5 in hESCs and identifies P57 as new target. View Publication

The development of mature blood cells from hematopoietic stem cells requires coordinated activities of transcriptional networks. Transcriptional repressor growth factor independence 1 (Gfi-1) is required for the development of B cells,T cells,neutrophils,and for the maintenance of hematopoietic stem cell function. However,the mechanisms by which Gfi-1 regulates hematopoiesis and how Gfi-1 integrates into transcriptional networks remain unclear. Here,we provide evidence that Id2 is a transcriptional target of Gfi-1,and repression of Id2 by Gfi-1 is required for B-cell and myeloid development. Gfi-1 binds to 3 conserved regions in the Id2 promoter and represses Id2 promoter activity in transient reporter assays. Increased Id2 expression was observed in multipotent progenitors,myeloid progenitors,T-cell progenitors,and B-cell progenitors in Gfi-1(-/-) mice. Knockdown of Id2 expression or heterozygosity at the Id2 locus partially rescues the B-cell and myeloid development but not the T-cell development in Gfi-1(-/-) mice. These studies demonstrate a role of Id2 in mediating Gfi-1 functions in B-cell and myeloid development and provide a direct link between Gfi-1 and the B-cell transcriptional network by its ability to repress Id2 expression. View Publication

The platelet glycoprotein Ib-IX-V complex (GPIb-IX-IV) is the receptor for VWF and is responsible for VWF-mediated platelet activation and aggregation. Loss of the GPIb-IX-V complex is pathogenic for Bernard-soulier Syndrome (BSS),which is characterized by macrothrombocytopenia and impaired platelet function. It remains unclear how the GPIb-IX-V complex is assembled and whether there is a role for a specific molecular chaperone in the process. In the present study,we report that the assembly of the GPIb-IX-V complex depends critically on a molecular chaperone in the endoplasmic reticulum (ER): gp96 (also known as grp94 and HSP90b1). gp96/grp94 deletion in the murine hematopoietic system results in thrombocytopenia,prolonged bleeding time,and giant platelets that are clinically indistinguishable from human BSS. Loss of gp96/grp94 in vivo and in vitro leads to the concomitant reduction in GPIb-IX complex expression due to ER-associated degradation. We further demonstrate that gp96/grp94 binds selectively to the GPIX subunit,but not to gpIbα or gpIbβ. Therefore,we identify the platelet GPIX subunit of the GPIb-IX-V complex as an obligate and novel client of gp96/grp94. View Publication

The HOX family of homeobox genes plays an important role in normal and malignant hematopoiesis. Dysregulated HOX gene expression profoundly effects the proliferation and differentiation of hematopoietic stem cells (HSCs) and committed progenitors,and aberrant activation of HOX genes is a common event in human myeloid leukemia. HOXB6 is frequently overexpressed in human acute myeloid leukemia (AML). To gain further insight into the role of HOXB6 in hematopoiesis,we overexpressed HOXB6 in murine bone marrow using retrovirus-mediated gene transfer. We also explored structure-function relationships using mutant HOXB6 proteins unable to bind to DNA or a key HOX-binding partner,pre-B-cell leukemia transcription factor-1 (PBX1). Additionally,we investigated the potential cooperative interaction with myeloid ecotropic viral integration site 1 homolog (MEIS1). In vivo,HOXB6 expanded HSCs and myeloid precursors while inhibiting erythropoiesis and lymphopoiesis. Overexpression of HOXB6 resulted in AML with a median latency of 223 days. Coexpression of MEIS1 dramatically shortened the onset of AML. Cytogenetic analysis of a subset of HOXB6-induced AMLs revealed recurrent deletions of chromosome bands 2D-E4,a region frequently deleted in HOXA9-induced AMLs. In vitro,HOXB6 immortalized a factor-dependent myelomonocytic precursor capable of granulocytic and monocytic differentiation. These biologic effects of HOXB6 were largely dependent on DNA binding but independent of direct interaction with PBX1. View Publication

Hematologic stem cell rescue after high-dose cytotoxic therapy is extensively used for the treatment of many hematopoietic and solid cancers. Gene marking studies suggest that occult tumor cells within the autograft may contribute to clinical relapse. To date purging of autografts contaminated with cancer cells has been unsuccessful. The selective oncolytic property of reovirus against myriad malignant histologies in in vitro,in vivo,and ex vivo systems has been previously demonstrated. In the present study we have shown that reovirus can successfully purge cancer cells within autografts. Human monocytic and myeloma cell lines as well as enriched ex vivo lymphoma,myeloma,and Waldenström macroglobulinemia patient tumor specimens were used in an experimental purging model. Viability of the cell lines or purified ex vivo tumor cells of diffuse large B-cell lymphoma,chronic lymphocytic leukemia,Waldenström macroglobulinemia,and small lymphocytic lymphoma was significantly reduced after reovirus treatment. Further,[35S]-methionine labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of cellular proteins demonstrated reovirus protein synthesis and disruption of host cell protein synthesis as early as 24 hours. Admixtures of apheresis product with the abovementioned tumor cells and cell lines treated with reovirus showed complete purging of disease. In contrast,reovirus purging of enriched ex vivo multiple myeloma,Burkitt lymphoma,and follicular lymphoma was incomplete. The oncolytic action of reovirus did not affect CD34+ stem cells or their long-term colony-forming assays even after granulocyte colony-stimulating factor (G-CSF) stimulation. Our results indicate the ex vivo use of an unattenuated oncolytic virus as an attractive purging strategy for autologous stem cell transplantations. View Publication