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

IntroductionThe role of miRNAs in regulating variable molecular functions has been sought by scientists for its promising utility in regulating the immune response and,hence,in treating various diseases. In hepatocellular carcinoma (HCC) specifically,a reduction in the number and efficiency of circulating and intrahepatic natural killer (NK) cells has been reported. Our project aims to investigate the role of miR-216a-3p in the regulation of NK cell cytotoxicity,especially since it plays a tumor suppressor role in the context of HCC.MethodsTo achieve our aim,we isolated NK cells from the whole blood of 86 patients with HCC and 23 healthy controls. We assessed the expression profile of miR-216a-3p in NK cells of patients and controls. Furthermore,we induced the expression of miR-216a-3p in NK cells isolated from healthy controls,followed by measuring the release of interferon-gamma (IFN-γ),tumor necrosis factor-alpha (TNF-α),perforins (PRF) and granzyme B (GrB) using ELISA as well as NK cells cytolytic activity against Huh7 cells using lactate dehydrogenase (LDH) cytotoxicity assay. After that,we performed an in silico analysis to understand the mechanistic regulation imposed by miR-216a-3p on NK cells to study its impact on one of its potential downstream targets.ResultsOur results have indicated that miR-216a-3p has higher expression in NK cells of patients with HCC,and simulating this elevated expression pattern via forcing miR-216a-3p expression in normal NK cells has negatively impacted the release of TNF- α,IFN- γ,GrB,and PRF. Consequently,a decrease in cell cytolysis was observed. Our in silico analysis revealed that the predicted downstream targets of miR-216a-3p are enriched in the FOXO-signaling pathway. Among those targets is FOXO-1,which has been reported to play a role in NK cell maturation. Thus,we evaluated FOXO-1 expression upon mimicking miR-216a-3p in control NK cells that showed significant downregulation of FOXO-1 on both RNA and protein levels.ConclusionIn conclusion,we report miR-216-3p as a negative regulator of NK cell cytotoxicity. View Publication

T cells have been reported to play critical roles in preventing of microsporidia dissemination. However,there roles and functions of each subset remain unclear. Here in the study,we performed a thorough analysis of murine splenic T-cell response analysis via single-cell RNA sequencing during microsporidia E. cuniculi infection. We demonstrated that Type I T helper (Th1) cells,T follicular helper (Tfh) cells,effector CD8 + T cells and proliferating CD8 + T cells were activated and expanded after infection. Activated Th1 cells and Tfh cells presented significantly upregulated gene expression of Ifng and Il21,respectively. A subcluster of Th1 cells with high Csf1 expression was detected after infection. Subsets of activated CD8 + T cells were markedly enriched with high expression of cytotoxic-function related genes such as Gzma and Gzmb,whereas some active CD8 T cells were enriched with proliferation-function related genes Mki67 and Stmn1. Other subsets of T cells including NK T cells,Myb+ T cells,γδ T cells and Cxcr6+ T cells,were also analyzed in this study yet no expansion was observed. In summary,our findings provide in-depth and comprehensive insights into T-cell responses during microsporidia infection,which will be valuable for further investigations. This study provides a comprehensive landscape of mouse T cells responses during microsporidia infection at a single-cell resolution reporting that Th1,Tfh,effector and proliferating CD8 + T cell subsets were activated and expanded upon infection. View Publication

Embryonic stem cells (ESC) are pluripotent,with the potential to differentiate into multiple cell types,making them a valuable tool for regenerative medicine and disease therapy. However,common culture methods face challenges,including strict operating procedures and high costs. Currently,Leukemia inhibitory factor (LIF),an indispensable bioactive protein for ESC culture,is typically applied to maintain self-renewal and pluripotency,but its instability and high cost limit its effectiveness in stable culture conditions. Hence,we have developed an innovative strategy using a soluble nanomaterial,metal-organic polyhedra (MOPs),to effectively maintain the self-renewal and pluripotency of ESC. The selected amino-modified vanadium-based MOP not only exhibits excellent biocompatibility and high stability but also possesses similar or even superior biological functions compared to commercial LIF. Due to the precise structure of MOPs,the active site responsible for maintaining ESC pluripotency has been identified and regulated at the molecular level. The new ESC culture method significantly reduces costs,simplifies preparation,and enhances the practicality of biopharmaceutical preparation and storage. This represents the first case of using MOPs to maintain self-renewal of ECS,opening an avenue for introducing advanced materials into the development of innovative ESC culture methods. Subject terms: Biomaterials - cells,Chemical biology View Publication

The developmental outcomes of preimplantation mammalian embryos are regulated directly by the surrounding microenvironment,and inappropriate concentrations of amino acids,or the loss of amino acid-sensing mechanisms,can be detrimental and impact further development. A specific role for l-proline in the differentiation of embryonic stem (ES) cells,a cell population derived from the blastocyst,has been shown in culture. l-proline acts as a signalling molecule,exerting its effects through cell uptake and subsequent metabolism. Uptake in ES cells occurs predominantly through the sodium-coupled neutral amino acid transporter 2,Slc38a2 (SNAT2). Dynamic expression of amino acid transporters has been shown in the early mammalian embryo,reflecting functional roles for amino acids in embryogenesis. The expression of SNAT2 and family member Slc38a1 (SNAT1) was determined in mouse embryos from the 2-cell stage through to the early post-implantation pre-gastrulation embryo. Key changes in expression were validated in cell culture models of development. Both transporters showed temporal dynamic expression patterns and changes in intracellular localisation as differentiation progressed. Changes in transporter expression likely reflect different amino acid requirements during development. Findings include the differential expression of SNAT1 in the inner and outer cells of the compacted morula and nuclear localisation of SNAT2 in the trophectoderm and placental lineages. Furthermore,SNAT2 expression was up-regulated in the epiblast prior to primitive ectoderm formation,an expression pattern consistent with a role for the transporter in later developmental decisions within the pluripotent lineage. We propose that the differential expression of SNAT2 in the epiblast provides evidence for an l-proline-mediated mechanism contributing to the regulation of embryonic development. View Publication

Patterning of bioactive enzymes with subcellular resolution is achieved by dispensing droplets of dextran (DEX) onto polyethylene glycol (PEG)-covered cells though a glass capillary needle connected to a pneumatic pump. This technique is applied to purify colonies of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblast (MEF) feeder cultures and inefficiently induced iPSC colonies by selectively dissociating the iPSCs with proteases. View Publication

Infusion of T cells modified with a chimeric antigen receptor (CAR) targeting CD19 has achieved exceptional responses in patients with non-Hodgkin’s lymphoma (NHL),which led to the approval of CAR targeting CD19 (CART19) (Axi-cel and Liso-cel) as second line of treatment for adult patients with relapsed/refractory NHL. Unfortunately,60% of patients still relapse after CART19 due to either a loss of expression of the target antigen (CD19) in the tumor cell,observed in 27% of relapsed patients,a limited CAR-T persistence,and additional mechanisms,including the suppression of the tumor microenvironment. Clinic strategies to prevent target antigen loss include sequential treatment with CARs directed at CD20 or CD22,which have caused loss of the second antigen,suggesting targeting other antigens less prone to disappear. CD79b,expressed in NHL,is a target in patients treated with antibody-drug conjugates (ADC). However,the limited efficacy of ADC suggests that a CAR therapy targeting CD79b might improve results. We designed three new CARs against CD79b termed CAR for Lymphoma (CARLY)1,2 and 3. We compared their efficacy,phenotype,and inflammatory profiles with CART19 (ARI0001) and CARTBCMA (ARI0002h),which can treat NHL. We also analyzed the target antigen’s expression loss (CD79b,CD19,and B-cell maturation antigen(BCMA)). We found that CARLY2 and CARLY3 had high affinity and specificity towards CD79b on B cells. In vitro,all CAR-T cells had similar anti-NHL efficacy,which was retained in an NHL model of CD19 − relapse. In vivo,CARLY3 showed the highest efficacy. Analysis of the loss of the target antigen demonstrated that CARLY cells induced CD79b and CD19 downregulation on NHL cells with concomitant trogocytosis of these antigens to T cells,being most notorious in CARLY2,which had the highest affinity towards CD79b and CD19,and supporting the selection of CARLY3 to design a new treatment for patients with NHL. Finally,we created a CAR treatment based on dual targeting of CD79b and BCMA to avoid losing the target antigen. This treatment showed the highest efficacy and did not cause loss of the target antigen. Based on specificity,efficacy,and loss of the target antigen,CARLY3 represents a potential novel CAR treatment for NHL. View Publication

A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here,human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ωtextperiodcenteredcm(2). By assessing the permeabilities of several known drugs,a benchmarking system to evaluate brain permeability of drugs was established. Furthermore,relationships between TEER and permeability to both small and large molecules were established,demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype,and the benchmarks established here are useful for assessing functionality of other in vitro BBB models. View Publication

PURPOSE Demonstrate a novel manufacturing method to generate extracellular matrix scaffolds from cardiac fibroblasts (CF-ECM) as a therapeutic mesenchymal stem cell-transfer device. MATERIALS AND METHODS Rat CF were cultured at high-density (˜1.6×10(5)/cm(2)) for 10-14 days. Cell sheets were removed from the culture dish by incubation with EDTA and decellularized with water and peracetic acid. CF-ECM was characterized by mass spectrometry,immunofluorescence and scanning electron microscopy. CF-ECM seeded with human embryonic stem cell derived mesenchymal stromal cells (hEMSCs) were transferred into a mouse myocardial infarction model. 48 hours later,mouse hearts were excised and examined for CF-ECM scaffold retention and cell transfer. RESULTS CF-ECM scaffolds are composed of fibronectin (82%),collagens type I (13%),type III (3.4%),type V (0.2%),type II (0.1%) elastin (1.3%) and 18 non-structural bioactive molecules. Scaffolds remained intact on the mouse heart for 48 hours without the use of sutures or glue. Identified hEMSCs were distributed from the epicardium to the endocardium. CONCLUSIONS High density cardiac fibroblast culture can be used to generate CF-ECM scaffolds. CF-ECM scaffolds seeded with hEMSCs can be maintained on the heart without suture or glue. hEMSC are successfully delivered throughout the myocardium. View Publication