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

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2–6,suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes,promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function,memory potential and metabolic fitness in settings of chronic stimulation,and exhibited enhanced persistence and tumour control in vivo. By contrast,overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably,FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes,underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells,and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states. The transcription factor FOXO1 has a key role in human T cell memory,and manipulating FOXO1 expression could provide a way to enhance CAR T cell therapies by increasing CAR T cell persistence and antitumour activity. View Publication

Poor graft function (PGF),characterized by myelosuppression,represents a significant challenge following allogeneic hematopoietic stem cell transplantation (allo-HSCT) with human cytomegalovirus (HCMV) being established as a risk factor for PGF. However,the underlying mechanism remains unclear. Bone marrow endothelial progenitor cells (BM-EPCs) play an important role in supporting hematopoiesis and their dysfunction contributes to PGF development. We aim to explore the effects of CMV on BM-EPCs and its underlying mechanism. We investigated the compromised functionality of EPCs derived from individuals diagnosed with HCMV viremia accompanied by PGF,as well as after infected by HCMV AD 169 strain in vitro,characterized by decreased cell proliferation,tube formation,migration and hematopoietic support,and increased apoptosis and secretion of TGF-β1. We demonstrated that HCMV-induced TGF-β1 secretion by BM-EPCs played a dominant role in hematopoiesis suppression in vitro experiment. Moreover,HCMV down-regulates Vitamin D receptor (VDR) and subsequently activates p38 MAPK pathway to promote TGF-β1 secretion by BM-EPCs. HCMV could infect BM-EPCs and lead to their dysfunction. The secretion of TGF-β1 by BM-EPCs is enhanced by CMV through the activation of p38 MAPK via a VDR-dependent mechanism,ultimately leading to compromised support for hematopoietic progenitors by BM EPCs,which May significantly contribute to the pathogenesis of PGF following allo-HSCT and provide innovative therapeutic strategies targeting PGF. View Publication

Prime Editing can rewrite genes in living cells by allowing point mutations,deletions,or insertion of small DNA sequences with high precision. However,its safe and efficient delivery into human stem cells remains a technical challenge. In this report,we engineer Nanoscribes,virus-like particles that encapsidate ribonucleoprotein complexes of the Prime Editing system and allow their delivery into recipient cells. We identify key features that unlock the potential of Nanoscribes,including the use of multiple fusogens,the improvement of pegRNAs structures,their encoding by a Pol II system and the optimization of Prime-Editors. Nanoscribes edit HEK293T with an efficiency of 68% at the HEK3 locus with increased fidelity over DNA-transfection and support pegRNA-multiplexing. Importantly,Nanoscribes permit editing of myoblasts,hiPSCs and hiPSCs-derived hematopoietic stem cells with an editing efficiency up to 25%. Nanoscribes is an asset for development of next generation genome editing approaches using VLPs. Subject terms: CRISPR-Cas9 genome editing,Genetic vectors,Nanoparticles View Publication

This protocol describes detailed procedures for the fluorescent high-throughput screening of small molecules that induce neurogenesis in cultures of skeletal muscle cells. The detection of neurogenesis relies on a fluorescent dye,FM 1-43,which is used to study the neuronal property of depolarization-induced synaptic vesicle recycling. Thus,small molecules with neurogenesis-inducing activity in skeletal muscle cells can be rapidly identified by measuring the fluorescence intensity of the treated cells using a fluorescent microplate reader. This protocol uses murine myoblast C2C12 cells for screening,which are readily available and relatively easy to culture. Neurogenesis of PC12 cells induced by nerve growth factor is employed as a positive control for this screening. The screening time for this protocol is 8 d,which also includes the procedure to detect depolarization-induced synaptic vesicle recycling using FM 1-43. View Publication

Genetic evidence indicates that Wnt signaling is critically involved in bone homeostasis. In this study,we investigated the functions of canonical Wnts on differentiation of adult multipotent human mesenchymal stem cells (hMSCs) in vitro and in vivo. We observe differential sensitivities of hMSCs to Wnt inhibition of osteogenesis versus adipogenesis,which favors osteoblastic commitment under binary in vitro differentiation conditions. Wnt inhibition of osteogenesis is associated with decreased expression of osteoblastic transcription factors and inhibition of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation,which are involved in osteogenic differentiation. An hMSC subpopulation exhibits high endogenous Wnt signaling,the inhibition of which enhances osteogenic and adipogenic differentiation in vitro. In an in vivo bone formation model,high levels of Wnt signaling inhibit de novo bone formation by hMSCs. However,hMSCs with exogenous expression of Wnt1 but not stabilized beta-catenin markedly stimulate bone formation by naive hMSCs,arguing for an important role of a canonical Wnt gradient in hMSC osteogenesis in vivo. View Publication

Differentiation of embryonic and adult myogenic progenitors undergoes a complex series of cell rearrangements and specification events which are controlled by distinct gene regulatory networks. Delineation of the molecular mechanisms that regulate skeletal muscle specification and formation should be important for understanding congenital myopathies and muscular degenerative diseases. Retinoic acid (RA) signaling plays an important role in development. However,the role of RA signaling in adult myogenic progenitors is poorly understood. Here,we investigate the role of RA signaling in regulating myogenic differentiation of myoblastic progenitor cells. Using the mouse myoblast progenitor C2C12 line as a model,we have found that the endogenous expression of most RAR and RXR isotypes is readily detected. While the nuclear receptor co-repressors are highly expressed,two of the three nuclear receptor co-activators and the enzymes involved in RA synthesis are expressed at low level or undetectable,suggesting that the RA signaling pathway may be repressed in myogenic progenitors. Using the alpha-myosin heavy chain promoter-driven reporter (MyHC-GLuc),we have demonstrated that either ATRA or 9CRA is able to effectively induce myogenic differentiation,which can be synergistically enhanced when both ATRA and 9CRA are used. Upon ATRA and 9CRA treatment of C2C12 cells the expression of late myogenic markers significantly increases. We have further shown that adenovirus-mediated exogenous expression of RARalpha and/or RXRalpha is able to effectively induce myogenic differentiation in a ligand-independent fashion. Morphologically,ATRA- and 9CRA-treated C2C12 cells exhibit elongated cell body and become multi-nucleated myoblasts,and even form myoblast fusion. Ultrastructural analysis under transmission electron microscope reveals that RA-treated myogenic progenitor cells exhibit an abundant presence of muscle fibers. Therefore,our results strongly suggest that RA signaling may play an important role in regulating myogenic differentiation. View Publication

To date,studies on T cells in acute myeloid leukemia (AML) have been limited to flow cytometric analysis of whole peripheral blood mononuclear cell (PBMC) specimens or functional work looking at the impact of AML myeloblasts on normal or remission T cells. This lack of information on T cells at the time of presentation with disease is due in part to the difficulty in isolating sufficiently pure T cells from these specimens for further study. Negative immunomagnetic selection has been the method of choice for isolating immune cells for functional studies due to concerns that binding antibodies to the cell surface may induce cellular activation,block ligand-receptor interactions or result in immune clearance. In order specifically to study T cells in presentation AML specimens,we set out to develop a method of isolating highly pure CD4 and CD8 T cells by negative selection from the peripheral blood (PB) of newly diagnosed AML patients. This technique,unlike T cell selection from PB from normal individuals or from patients with chronic lymphocytic leukaemia,was extremely problematic due to properties of the leukaemic myeloblasts. A successful method was eventually optimized requiring the use of a custom antibody cocktail consisting of CD33,CD34,CD123,CD11c and CD36,to deplete myeloblasts. View Publication