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

BACKGROUND Hypoxia and inflammation tumor microenvironment (TME) play a crucial role in tumor development and progression. Although increased understanding of TME contributed to gastric cancer (GC) progression and prognosis,the direct interaction between macrophage and GC cells was not fully understood. METHODS Hypoxia and normoxia macrophage microarrays of GEO database was analyzed. The peripheral blood mononuclear cell acquired from the healthy volunteers. The expression of C-X-C Motif Chemokine Ligand 8 (CXCL8) in GC tissues and cell lines was detected by quantitative reverse transcription PCR (qRT-PCR),western-blot,Elisa and immunofluorescence. Cell proliferation,migration,and invasion were evaluated by cell counting kit 8 (CCK8),colony formation,real-time imaging of cell migration and transwell. Flow Cytometers was applied to identify the source of cytokines. Luciferase reporter assays and chromatin immunoprecipitation were used to identify the interaction between transcription factor and target gene. Especially,a series of truncated and mutation reporter genes were applied to identify precise binding sites. The corresponding functions were verified in the complementation test and in vivo animal experiment. RESULTS Our results revealed that hypoxia triggered macrophage secreted CXCL8,which induced GC invasion and proliferation. This macrophage-induced GC progression was CXCL8 activated C-X-C Motif Chemokine Receptor 1/2 (CXCR1/2) on the GC cell membrane subsequently hyperactivated Janus kinase 1/ Signal transducer and activator of transcription 1 (JAK/STAT1) signaling pathway. Then,the transcription factor STAT1 directly led to the overexpression and secretion of Interleukin 10 (IL-10). Correspondingly,IL-10 induced the M2-type polarization of macrophages and continued to increase the expression and secretion of CXCL8. It suggested a positive feedback loop between macrophage and GC. In clinical GC samples,increased CXCL8 predicted a patient's pessimistic outcome. CONCLUSION Our work identified a positive feedback loop governing cancer cells and macrophage in GC that contributed to tumor progression and patient outcome. View Publication

BACKGROUND Human pancreata contain many types of cells,such as endocrine islets,acinar,ductal,fat,and mesenchymal stromal cells (MSCs). MSCs are important and shown to have a promising therapeutic potential to treat various disease conditions. METHODS We investigated intra-pancreatic tissue-derived (IPTD) MSCs isolated from tissue fractions that are routinely discarded during pancreatic islet isolation of human cadaveric donors. Furthermore,whether pro-angiogenic and anti-inflammatory properties of these cells could be enhanced was investigated. RESULTS IPTD-MSCs were expanded in GMP-compatible CMRL-1066 medium supplemented with 5{\%} human platelet lysate (hPL). IPTD-MSCs were found to be highly pure,with {\textgreater} 95{\%} positive for CD90,CD105,and CD73,and negative for CD45,CD34,CD14,and HLA-DR. Immunofluorescence staining of pancreas tissue demonstrated the presence of CD105+ cells in the vicinity of islets. IPTD-MSCs were capable of differentiation into adipocytes,chondrocytes,and osteoblasts in vitro,underscoring their multipotent features. When these cells were cultured in the presence of a low dose of TNF-$\alpha$,gene expression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) was significantly increased,compared to control. In contrast,treating cells with dimethyloxallyl glycine (DMOG) (a prolyl 4-hydroxylase inhibitor) enhanced mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor (VEGF). Interestingly,a combination of TNF-$\alpha$ and DMOG stimulated the optimal expression of all three genes in IPTD-MSCs. Conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-$\alpha$ contained higher levels of pro-angiogenic (VEGF,IL-6,and IL-8) compared to controls,promoting angiogenesis of human endothelial cells in vitro. In contrast,levels of MCP-1,a pro-inflammatory cytokine,were reduced in the conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-$\alpha$. CONCLUSIONS The results demonstrate that IPTD-MSCs reside within the pancreas and can be separated as part of a standard islet-isolation protocol. These IPTD-MSCs can be expanded and potentiated ex vivo to enhance their anti-inflammatory and pro-angiogenic profiles. The fact that IPTD-MSCs are generated in a GMP-compatible procedure implicates a direct clinical application. View Publication

mRNA applications have undergone unprecedented applications—from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production. NK cells can be engineered with either viral vectors or electroporation,involving high costs,risks,and toxicity,emphasizing the need for alternative way as mRNA technology. We successfully developed,screened,and optimized novel lipid-based platforms based on imidazole lipids. Formulations are produced by microfluidic mixing and exhibit a size of approximately 100 nm with a polydispersity index of less than 0.2. They are able to transfect NK-92 cells,KHYG-1 cells,and primary NK cells with high efficiency without cytotoxicity,while Lipofectamine Messenger Max and D-Lin-MC3 lipid nanoparticle-based formulations do not. Moreover,the translation of non-modified mRNA was higher and more stable in time compared with a modified one. Remarkably,the delivery of therapeutically relevant interleukin 2 mRNA resulted in extended viability together with preserved activation markers and cytotoxic ability of both NK cell lines and primary NK cells. Altogether,our platforms feature all prerequisites needed for the successful deployment of NK-based therapeutic strategies. Graphical abstract Pichon and colleagues developed imidazole lipids-based mRNA platforms very efficient to transfect both NK-92 cells,KHYG-1 cells and primary NK cells without cytotoxicity. They succeeded to replace IL-2 protein by IL-2 mRNA transfection and obtained NK cells with extended viability with preserved biomarkers and full functionalities to kill target cells. View Publication

Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these embryonically derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic endothelial-macrophage (EndoMac) progenitor cells in the adventitia of embryonic and postnatal mouse aorta,that are independent of Flt3-mediated bone marrow hematopoiesis and derive from an early embryonic CX 3 CR1 + and CSF1R + source. These bipotent progenitors are proliferative and vasculogenic,contributing to adventitial neovascularization and formation of perfused blood vessels after transfer into ischemic tissue. We establish a regulatory role for angiotensin II,which enhances their clonogenic and differentiation properties and rapidly stimulates their proliferative expansion in vivo. Our findings demonstrate that embryonically derived EndoMac progenitors participate in local vasculogenic responses in the aortic wall by contributing to the expansion of endothelial cells and macrophages postnatally. Subject terms: Angiogenesis,Myelopoiesis,Haematopoietic stem cells View Publication

Understanding metabolic pathways that regulate Th17 development is important to broaden therapeutic options for Th17-mediated autoimmunity. Here,we report a pivotal role of mitochondrial oxidative phosphorylation (OXPHOS) for lineage specification toward pathogenic Th17 differentiation. Th17 cells rapidly increase mitochondrial respiration during development,and this is necessary for metabolic reprogramming following T cell activation. Surprisingly,specific inhibition of mitochondrial ATP synthase ablates Th17 pathogenicity in a mouse model of autoimmunity by preventing Th17 pathogenic signature gene expression. Notably,cells activated under OXPHOS-inhibited Th17 conditions preferentially express Foxp3,rather than Th17 genes,and become suppressive Treg cells. Mechanistically,OXPHOS promotes the Th17 pioneer transcription factor,BATF,and facilitates T cell receptor (TCR) and mTOR signaling. Correspondingly,overexpression of BATF rescues Th17 development when ATP synthase activity is restricted. Together,our data reveal a regulatory role of mitochondrial OXPHOS in dictating the fate decision between Th17 and Treg cells by supporting early molecular events necessary for Th17 commitment. View Publication

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in COL7A1. Patient-derived induced pluripotent stem cells (iPSCs) enable the personalized study of RDEB pathogenesis and potential therapies. However,current skin cell differentiation protocols via 2D culture perform suboptimally when applied to engineered 3D skin constructs (ESC). Here,we present an approach to source fibroblasts (iFBs) and keratinocytes (iKCs) from iPSC-derived skin organoids using an optimized differentiation protocol,and utilize them to engineer ESCs modeling wild-type and RDEB phenotypes. The resulting iPSC-derived skin cells display marker expression consistent with primary counterparts and produce ESCs exhibiting significant extracellular matrix remodeling,protein deposition,and epidermal differentiation. RDEB constructs recapitulated hallmark disease features,including absence of collagen VII and reduced iFB proliferation. This work establishes a robust and scalable strategy for generating physiologically-relevant,iPSC-derived skin constructs,offering a powerful model for studying RDEB mechanisms and advancing personalized regenerative medicine. View Publication

We report a considerable number of cells in the ventricular and the subventricular zones (SVZ) of newborn mice to stain positive for the PDGF beta-receptor (PDGFRB). Many of them also stained for nestin and/or GFAP but less frequently for the neuroblast marker doublecortin and for the mitotic marker Ki-67. The SVZ of mice with nestin-Cre conditional deletion of PDGFRB expressed the receptor only on blood vessels and was devoid of any morphological abnormality. PDGFRB(-/-) neurospheres showed a higher rate of apoptosis without any significant decrease in proliferation. They demonstrated reduced capacities of migration and neuronal differentiation in response to not only PDGF-BB but also bFGF. Furthermore,the PDGFR kinase inhibitor STI571 blocked the effects of bFGF in control neurosphere cultures. bFGF increased the activity of the PDGFRB promoter as well as the expression and phosphorylation of PDGFRB. These results suggest the presence of the signaling convergence between PDGF and FGF. PDGFRB is needed for survival,and the effects of bFGF in migration and neural differentiation of the cells may be potentiated by induction of PDGFRB. View Publication

The generation of pluripotent stem cells from an individual patient would enable the large-scale production of the cell types affected by that patient's disease. These cells could in turn be used for disease modeling,drug discovery,and eventually autologous cell replacement therapies. Although recent studies have demonstrated the reprogramming of human fibroblasts to a pluripotent state,it remains unclear whether these induced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease. We have generated iPS cells from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS). These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiate into motor neurons,the cell type destroyed in ALS. View Publication

The abnormal trafficking of CD34+ cells is a unique characteristic of primary myelofibrosis (PMF). We have further studied the behavior of PMF CD34+ cells by examining their homing to the marrow and the spleens of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Following the infusion of PMF and normal granulocyte colony-stimulating factor-mobilized peripheral blood (mPB) CD34+ cells into NOD/SCID mice,reduced numbers of PMF CD34+ cells and granulocyte-macrophage colony-forming unit (CFU-GM) compared with mPB were detected in the marrow of these mice,whereas similar numbers of PMF and mPB CD34+ cells and CFU-GM homed to their spleens. The abnormal homing of PMF CD34+ cells was associated with reduced expression of CXCR4,but was not related to the presence of JAK2V617F. The sequential treatment of PMF CD34+ cells with the chromatin-modifying agents 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA),but not treatment with small molecule inhibitors of JAK2,resulted in the generation of increased numbers of CD34+CXCR4+ cells,which was accompanied by enhanced homing of PMF CD34+ cells to the marrow but not the spleens of NOD/SCID mice. Following 5azaD/TSA treatment,JAK2V617F-negative PMF hematopoietic progenitor cells preferentially homed to the marrow but not the spleens of recipient mice. Our data suggest that PMF CD34+ cells are characterized by a reduced ability to home to the marrow but not the spleens of NOD/SCID mice and that this homing defect can be corrected by sequential treatment with chromatin-modifying agents. View Publication

The recent development of HIV-1 lentiviral vectors is especially useful for gene transfer because they achieve efficient integration into nondividing cell genomes and successful long-term expression of the transgene. These attributes make the vector useful for gene delivery,mutagenesis,and other applications in mammalian systems. Here we describe two HIV-1-based lentiviral vector derivatives,pZR-1 and pZR-2,that can be used in gene-trap experiments in mammalian cells in vitro and in vivo. Each lentiviral gene-trap vector contains a reporter gene,either beta-lactamase or enhanced green fluorescent protein (EGFP),that is inserted into the U3 region of the 3' long terminal repeat. Both of the trap vectors readily integrate into the host genome by using a convenient infection technique. Appropriate insertion of the vector into genes causes EGFP or beta-lactamase expression. This technique should facilitate the rapid enrichment and cloning of the trapped cells and provides an opportunity to select subpopulations of trapped cells based on the subcellular localization of reporter genes. Our findings suggest that the reporter gene is driven by an upstream,cell-specific promoter during cell culture and cell differentiation,which further supports the usefulness of lentivirus-based gene-trap vectors. Lentiviral gene-trap vectors appear to offer a wealth of possibilities for the study of cell differentiation and lineage commitment,as well as for the discovery of new genes. View Publication

p40,a Lactobacillus rhamnosus GG (LGG)-derived protein,transactivates epidermal growth factor receptor (EGFR) in intestinal epithelial cells,leading to amelioration of intestinal injury and inflammation. To elucidate mechanisms by which p40 regulates mucosal immunity to prevent inflammation,this study aimed to determine the effects and mechanisms of p40 on regulation of a proliferation-inducing ligand (APRIL) expression in intestinal epithelial cells for promoting immunoglobulin A (IgA) production. p40 upregulated April gene expression and protein production in mouse small intestine epithelial (MSIE) cells,which were inhibited by blocking EGFR expression and kinase activity. Enteroids from Egfr(fl/fl),but not Egfr(fl/fl)-Vil-Cre mice with EGFR specifically deleted in intestinal epithelial cells,exhibited increased April gene expression by p40 treatment. p40-conditioned media from MSIE cells increased B-cell class switching to IgA(+) cells and IgA production,which was suppressed by APRIL receptor-neutralizing antibodies. Treatment of B cells with p40 did not show any effects on IgA production. p40 treatment increased April gene expression and protein production in small intestinal epithelial cells,fecal IgA levels,IgA(+)B220(+),IgA(+)CD19(+),and IgA(+) plasma cells in lamina propria of Egfr(fl/fl),but not of Egfr(fl/fl)-Vil-Cre,mice. Thus p40 upregulates EGFR-dependent APRIL production in intestinal epithelial cells,which may contribute to promoting IgA production. View Publication

CD4(+) T cells develop distinct and often contrasting helper,regulatory,or cytotoxic activities. Typically a property of CD8(+) T cells,granzyme-mediated cytotoxic T cell (CTL) potential is also exerted by CD4(+) T cells. However,the conditions that induce CD4(+) CTLs are not entirely understood. Using single-cell transcriptional profiling,we uncover a unique signature of Granzyme B (GzmB)(+) CD4(+) CTLs,which distinguishes them from other CD4(+) T helper (Th) cells,including Th1 cells,and strongly contrasts with the follicular helper T (Tfh) cell signature. The balance between CD4(+) CTL and Tfh differentiation heavily depends on the class of infecting virus and is jointly regulated by the Tfh-related transcription factors Bcl6 and Tcf7 (encoding TCF-1) and by the expression of the inhibitory receptors PD-1 and LAG3. This unique profile of CD4(+) CTLs offers targets for their study,and its antagonism by the Tfh program separates CD4(+) T cells with either helper or killer functions. View Publication