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

Although FOXP3+ regulatory T cells (Treg) depend on IL-2 produced by other cells for their survival and function,the levels of IL-2 in inflamed tissue are low,making it unclear how Treg access this critical resource. Here,we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo,including in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely,endowing monoclonal antibody-directed chimeric antigen receptor (mAbCAR) Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their ability to suppress neuroinflammation in vivo. Together,these data identify a role for HPSE and the ECM in immune tolerance,providing new avenues for improving Treg-based therapy of autoimmunity. Regulatory T cell (Treg) maintenance and function require IL-2,yet this cytokine is only present in low levels in vivo. In this study,the authors demonstrate that that Treg use heparanase to access IL-2 bound to heparan sulfate proteoglycans in the extracellular matrix of inflamed brain tissue in mice. View Publication

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterized by desmoplastic stroma,immunosuppressive tumor microenvironment (TME),and resistance to standard therapies. Natural killer (NK) cell-based immunotherapies have shown limited efficacy due to impaired persistence,infiltration,and function in PDAC. Methods: We established a direct reprogramming strategy to generate cytotoxic NK cells (1 F-NKs) by targeting BCL11B,a transcription factor essential for T cell lineage commitment,using shRNA or CRISPR/Cas9 in peripheral blood mononuclear cells (PBMCs). A genome-wide CRISPR/Cas9 screen identified tumor-intrinsic modulators of NK resistance. Functional and in vivo studies assesses the efficacy of 1 F-NKs alone and in combination with mesothelin (MSLN)-CAR engineering and PKMYT1 inhibition. Results: BCL11B depletion enabled the generation of CD56brightCD16bright 1 F-NKs with potent cytotoxicity and elevated NKG2D and CX3CR1 expression. Site-specific integration of a mesothelin (MSLN)-CAR into BCL11B locus generated MSLN-1 F-NKs with stable antigen specific activity. A genome-wide screen identified PKMYT1 as a modulator of tumor resistance to NK cell-mediated killing; its inhibition by RP6306 upregulated NKG2D ligands (MICA/B) and CX3CL1,sensitizing PDACs to 1 F-NK cytotoxicity. In PDAC xenograft models,1 F-NKs alone or combined with CAR engineering and RP6306 significantly reduced tumor growth and prolonged survival. Notably,this triple combination elicited a synergistic antitumor effect,outperforming each monotherapy or dual combination. Conclusions: This study presents a synergistic immunotherapy platform that integrates NK reprogramming,CAR engineering,and tumor sensitization. The combinatorial approach significantly enhances antitumor efficacy in PDAC and offers a promising strategy for overcoming immune resistance in solid tumors. View Publication

Cellular senescence and the senescence-associated secretory phenotype (SASP) are implicated in aging and age-related disease,and SASP-related inflammation is thought to contribute to tissue dysfunction in aging and diseased animals. However,whether and how SASP factors influence the regenerative capacity of tissues remains unclear. Here,using intestinal organoids as a model of tissue regeneration,we show that SASP factors released by senescent fibroblasts deregulate stem cell activity and differentiation and ultimately impair crypt formation. We identify the secreted N-terminal domain of Ptk7 as a key component of the SASP that activates non-canonical Wnt / Ca2+ signaling through FZD7 in intestinal stem cells (ISCs). Changes in cytosolic [Ca2+] elicited by Ptk7 promote nuclear translocation of YAP and induce expression of YAP/TEAD target genes,impairing symmetry breaking and stem cell differentiation. Our study discovers secreted Ptk7 as a factor released by senescent cells and provides insight into the mechanism by which cellular senescence contributes to tissue dysfunction in aging and disease. View Publication

We have cloned the FP receptor from rat corpus luteum and human uterus cDNA libraries,respectively. The coding DNA sequence in the rat cDNA is 1101 bp and is similar to the mouse cDNA coding for a receptor protein of 366 amino acids. The human sequence shows a 5 bp deficiency in the 3' region,truncating the coding sequence to 359 amino acids. Northern blot analysis indicates highest expression in the ovary. Cell lines have been established giving stable expression of the FP receptor. Activation of the cloned FP receptor gave an increase in intracellular calcium,indicating signaling via phospholipase C-mediated phosphoinositide turnover. Using [3H]PGF2 alpha,binding of PGs showed the rank order of fluprostenol textgreater PhXA70 textgreater PGF2 alpha textgreater or = PhXA85 textgreater PGD2 textgreater PGE2. View Publication

Human embryonic stem cells (hESCs) require specific niches for adhesion,expansion,and lineage-specific differentiation. In this study,we showed that a membrane substrate offers better tissue niches for hESC attachment,spreading,proliferation,and differentiation. The cell doubling time was shortened from 46.3±5.7 h for hESCs grown on solid substrates to 25.6±2.6 h for those on polyester (PE) membrane substrates with pore size of 0.4 μm. In addition,we observed an increase of approximately five- to ninefold of definitive endoderm marker gene expression in hESCs differentiated on PE or polyethylene terephthalate membrane substrates. Global gene expression analysis revealed upregulated expressions of a number of extracellular matrix and cell adhesion molecules in hESCs grown on membrane substrates. Further,an enhanced nuclear translocation of β-catenin was detected in these cells. These observations suggested the augmentation of Wnt signaling in hESCs grown on membrane substrates. These results also demonstrated that a membrane substrate can offer better physicochemical cues for enhancing in vitro hESC attachment,proliferation,and differentiation. View Publication

Early studies in exercise immunology suggested acute bouts of exercise had an immunosuppressive effect in human subjects. However,recent data,show acute bouts of combined aerobic and resistance training increase both lymphocyte activation and proliferation. We quantified resistance exercise-induced changes in the activation state of CD4+ T lymphocytes via surface protein expression and using a medically relevant model of infection (HIV-1). Using a randomized cross-over design,10 untrained subjects completed a control and exercise session. The control session consisted of 30-min seated rest while the exercise session entailed 3 sets × 10 repetitions of back squat,leg press,and leg extensions at 70{\%} 1-RM with 2-min rest between each set. Venous blood samples were obtained pre/post each session. CD4+ T lymphocytes were isolated from whole blood by negative selection. Expression of activation markers (CD69 {\&} CD25) in both nonstimulated and stimulated (costimulation through CD3+ CD28) cells were assessed by flow cytometry. Resistance exercised-induced effects on intracellular activation was further evaluated via in vitro infection with HIV-1. Nonstimulated CD4+ T lymphocytes obtained postexercise exhibited elevated CD25 expression following 24 h in culture. Enhanced HIV-1 replication was observed in cells obtained postexercise. Our results demonstrate that an acute bout of resistance exercise increases the activation state of CD4+ T lymphocytes and results in a greater susceptibility to HIV-1 infection in vitro. These findings offer further evidence that exercise induces activation of T lymphocytes and provides a foundation for the use of medically relevant pathogens as indirect measures of intracellular activation. View Publication

Peripheral blood was collected from a 39-year-old unaffected female carrier of an X-linked recessive mutation of Iduronate 2-sulfatase gene (NM000202.7(IDS):c.85CtextgreaterT) causing MPS II (OMIM 309900). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC showed normal karyotype. The line offers a good platform to study MPS II pathophysiology,for drug testing,early biomarker discovery and gene therapy studies. View Publication

BACKGROUND The in vitro generation of neurons from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages attempt to replicate,in different ways,the multistep process of embryonic neural development. However,to achieve this aim in an efficient and reproducible way,a better knowledge of the cellular and molecular events that are involved in the process,from the initial specification of neuroepithelial progenitors to their terminal differentiation into neurons and glial cells,is required. METHODOLOGY/PRINCIPAL FINDINGS In this work,we characterize the main stages and transitions that occur when ES cells are driven into a neural fate,using an adherent monolayer culture system. We established improved conditions to routinely produce highly homogeneous cultures of neuroepithelial progenitors,which organize into neural tube-like rosettes when they acquire competence for neuronal production. Within rosettes,neuroepithelial progenitors display morphological and functional characteristics of their embryonic counterparts,namely,apico-basal polarity,active Notch signalling,and proper timing of production of neurons and glia. In order to characterize the global gene activity correlated with each particular stage of neural development,the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes,we define five gene expression signatures that correlate with successive stages in the path from ES cells to neurons. These include a gene signature for a primitive ectoderm-like stage that appears after ES cells enter differentiation,and three gene signatures for subsequent stages of neural progenitor development,from an early stage that follows neural induction to a final stage preceding terminal differentiation. CONCLUSIONS/SIGNIFICANCE Overall,our work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development,revealing in addition novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons. View Publication

T cell antigen-presenting cell (APC) interactions early during chronic viral infection are crucial for determining viral set point and disease outcome,but how and when different APC subtypes contribute to these outcomes is unclear. The TNF receptor superfamily (TNFRSF) member GITR is important for CD4+ T cell accumulation and control of chronic lymphocytic choriomeningitis virus (LCMV). We found that type I interferon (IFN-I) induced TNFSF ligands GITRL,4-1BBL,OX40L,and CD70 predominantly on monocyte-derived APCs and CD80 and CD86 predominantly on classical dendritic cells (cDCs). Mice with hypofunctional GITRL in Lyz2+ cells had decreased LCMV-specific CD4+ T cell accumulation and increased viral load. GITR signals in CD4+ T cells occurred after priming to upregulate OX40,CD25,and chemokine receptor CX3CR1. Thus IFN-I (signal 3) induced a post-priming checkpoint (signal 4) for CD4+ T cell accumulation,revealing a division of labor between cDCs and monocyte-derived APCs in regulating T cell expansion. View Publication

Murine megakaryocytes (MKs) are defined by CD41/CD61 expression and acetylcholinesterase (AChE) activity; however,their stages of differentiation in bone marrow (BM) have not been fully elucidated. In murine lineage-negative (Lin(-))/CD45(+) BM cells,we found CD41(+) MKs without AChE activity (AChE(-)) except for CD41(++) MKs with AChE activity (AChE(+)),in which CD61 expression was similar to their CD41 level. Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs could differentiate into AChE(+),with an accompanying increase in CD41/CD61 during in vitro culture. Both proplatelet formation (PPF) and platelet (PLT) production for Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs were observed later than for Lin(-)/CD41(++)/CD45(+)/AChE(+) MKs,whereas MK progenitors were scarcely detected in both subpopulations. GeneChip and semiquantitative polymerase chain reaction analyses revealed that the Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs are assigned at the stage between the progenitor and PPF preparation phases in respect to the many MK/PLT-specific gene expressions,including beta1-tubulin. In normal mice,the number of Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs was 100 times higher than that of AChE(+) MKs in BM. When MK destruction and consequent thrombocytopenia were caused by an antitumor agent,mitomycin-C,Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs led to an increase in AChE(+) MKs and subsequent PLT recovery with interleukin-11 administration. It was concluded that MKs in murine BM at least in part consist of immature Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs and more differentiated Lin(-)/CD41(++)/CD45(+)/AChE(+) MKs. Immature Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs are a major MK population compared with AChE(+) MKs in BM and play an important role in rapid PLT recovery in vivo. View Publication

Innate lymphoid cells (ILCs) are a new family of immune cells that play important roles in innate immunity in mucosal tissues,and in the maintenance of tissue and metabolic homeostasis. Recently,group 2 ILCs (ILC2s) were found to promote the development and effector functions of Th2-type CD4(+) T cells by interacting directly with T cells or by activating dendritic cells,suggesting a role for ILC2s in regulating adaptive immunity. However,our current knowledge on the role of ILCs in humoral immunity is limited. In this study,we found that ILC2s isolated from the lungs of naive BALB/c mice enhanced the proliferation of B1- as well as B2-type B cells and promoted the production of IgM,IgG1,IgA,and IgE by these cells in vitro. Soluble factors secreted by ILC2s were sufficient to enhance B cell Ig production. By using blocking Abs and ILC2s isolated from IL-5-deficient mice,we found that ILC2-derived IL-5 is critically involved in the enhanced production of IgM. Furthermore,when adoptively transferred to Il7r(-/-) mice,which lack ILC2s and mature T cells,lung ILC2s promoted the production of IgM Abs to a polysaccharide Ag,4-hydroxy-3-nitrophenylacetyl Ficoll,within 7 d of airway exposure in vivo. These findings add to the growing body of literature regarding the regulatory functions of ILCs in adaptive immunity,and suggest that lung ILC2s promote B cell production of early Abs to a respiratory Ag even in the absence of T cells. View Publication

Reliance on volunteer blood donors can lead to transfusion product shortages,and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time,known as 'the storage lesion'. Thus,there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh,transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However,it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity,viability,deformability,and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo,we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel,chronically anemic,SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data,stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs,the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product. View Publication