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

Acute myelogenous leukemia is driven by leukemic stem cells (LSCs) generated by mutations that confer (or maintain) self-renewal potential coupled to an aberrant differentiation program. Using retroviral mutagenesis,we identified genes that generate LSCs in collaboration with genetic disruption of the gene encoding interferon response factor 8 (Irf8),which induces a myeloproliferation in vivo. Among the targeted genes,we identified Mef2c,encoding a MCM1-agamous-deficiens-serum response factor transcription factor,and confirmed that overexpression induced a myelomonocytic leukemia in cooperation with Irf8 deficiency. Strikingly,several of the genes identified in our screen have been reported to be up-regulated in the mixed-lineage leukemia (MLL) subtype. High MEF2C expression levels were confirmed in acute myelogenous leukemia patient samples with MLL gene disruptions,prompting an investigation of the causal interplay. Using a conditional mouse strain,we demonstrated that Mef2c deficiency does not impair the establishment or maintenance of LSCs generated in vitro by MLL/ENL fusion proteins; however,its loss led to compromised homing and invasiveness of the tumor cells. Mef2c-dependent targets included several genes encoding matrix metalloproteinases and chemokine ligands and receptors,providing a mechanistic link to increased homing and motility. Thus,MEF2C up-regulation may be responsible for the aggressive nature of this leukemia subtype. View Publication

Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomib's inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts,thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy,we identified GRP-78,a chaperone protein of the unfolded protein response,as being responsible for bortezomib resistance. Indeed,a variety of bortezomib-resistant solid tumor cell lines (PC-3,HRT-18),but not myeloma cell lines (U266,OPM-2),were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal-related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data,we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment,thus demonstrating a hitherto unknown mechanism of resistance to bortezomib. View Publication

Synthetic materials that promote the growth or differentiation of cells have advanced the fields of tissue engineering and regenerative medicine. Most functional biomaterials are based on a handful of peptide sequences derived from protein ligands for cell surface receptors. Because few proteins possess short peptide sequences that alone can engage cell surface receptors,the repertoire of receptors that can be targeted with this approach is limited. Materials that bind diverse classes of receptors,however,may be needed to guide cell growth and differentiation. To provide access to such new materials,we utilized phage display to identify novel peptides that bind to the surface of pluripotent cells. Using human embryonal carcinoma (EC) cells as bait,approximately 3 x 10(4) potential cell-binding phage clones were isolated. The pool was narrowed using an enzyme-linked immunoassay: 370 clones were tested,and seven cell-binding peptides were identified. Of these,six sequences possess EC cell-binding ability. Specifically,when displayed by self-assembled monolayers (SAMs) of alkanethiols on gold,they mediate cell adhesion. The corresponding soluble peptides block this adhesion,indicating that the identified peptide sequences are specific. They also are functional. Synthetic surfaces displaying phage-derived peptides support growth of undifferentiated human embryonic stem (ES) cells. When these cells were cultured on SAMs presenting the sequence TVKHRPDALHPQ or LTTAPKLPKVTR in a chemically defined medium (mTeSR),they expressed markers of pluripotency at levels similar to those of cells cultured on Matrigel. Our results indicate that this screening strategy is a productive avenue for the generation of materials that control the growth and differentiation of cells. View Publication

It is well known that hepatic stellate cells (HSC) develop into cells,which are thought to contribute to liver fibrogenesis. Recent data suggest that HSC are progenitor cells with the capacity to differentiate into cells of endothelial and hepatocyte lineages. The present study shows that beta-catenin-dependent canonical Wnt signaling is active in freshly isolated HSC of rats. Mimicking of the canonical Wnt pathway in cultured HSC by TWS119,an inhibitor of the glycogen synthase kinase 3beta,led to reduced beta-catenin phosphorylation,induced nuclear translocation of beta-catenin,elevated glutamine synthetase production,impeded synthesis of alpha-smooth muscle actin and Wnt5a,but promoted the expression of glial fibrillary acidic protein,Wnt10b,and paired-like homeodomain transcription factor 2c. In addition,canonical Wnt signaling lowered DNA synthesis and hindered HSC from entering the cell cycle. The findings demonstrate that beta-catenin-dependent Wnt signaling maintains the quiescent state of HSC and,similar to stem and progenitor cells,influences their developmental fate. View Publication

Neurogenesis persists in the adult brain subventricular zone where neural stem/progenitor cells (NSPCs) lie close to brain endothelial cells (BECs). We show in mouse that BECs produce bone morphogenetic proteins (BMPs). Coculture of embryonic and adult NSPCs with BECs activated the canonical BMP/Smad pathway and reduced their proliferation. We demonstrate that coculture with BECs in the presence of EGF and FGF2 induced a reversible cell cycle exit of NSPCs (LeX+) and an increase in the amount of GFAP/LeX-expressing progenitors thought to be stem cells. Levels of the phosphatidylinositol phosphatase PTEN were upregulated in NSPCs after coculture with BECs,or treatment with recombinant BMP4,with a concomitant reduction in Akt phosphorylation. Silencing Smad5 with siRNA or treatment with Noggin,a BMP antagonist,demonstrated that upregulation of PTEN in NSPCs required BMP/Smad signaling and that this pathway regulated cell cycle exit of NSPCs. Therefore,BECs may provide a feedback mechanism to control the proliferation of NSPCs. View Publication

BACKGROUND AND AIMS Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However,the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated. APPROACH AND RESULTS We analyzed the immune landscapes of hypoxia-low and hypoxia-high tumor regions using cytometry by time of light,immunohistochemistry,and transcriptomic analyses. The mechanisms of immunosuppression in immune subsets of interest were further explored using in vitro hypoxia assays. Regulatory T cells (Tregs) and a number of immunosuppressive myeloid subsets,including M2 macrophages and human leukocyte antigen-DR isotype (HLA-DRlo ) type 2 conventional dendritic cell (cDC2),were found to be significantly enriched in hypoxia-high tumor regions. On the other hand,the abundance of active granzyme Bhi PD-1lo CD8+ T cells in hypoxia-low tumor regions implied a relatively active immune landscape compared with hypoxia-high regions. The up-regulation of cancer-associated genes in the tumor tissues and immunosuppressive genes in the tumor-infiltrating leukocytes supported a highly pro-tumorigenic network in hypoxic HCC. Chemokine genes such as CCL20 (C-C motif chemokine ligand 20) and CXCL5 (C-X-C motif chemokine ligand 5) were associated with recruitment of both Tregs and HLA-DRlo cDC2 to hypoxia-high microenvironments. The interaction between Tregs and cDC2 under a hypoxic TME resulted in a loss of antigen-presenting HLA-DR on cDC2. CONCLUSIONS We uncovered the unique immunosuppressive landscapes and identified key immune subsets enriched in hypoxic HCC. In particular,we identified a potential Treg-mediated immunosuppression through interaction with a cDC2 subset in HCC that could be exploited for immunotherapies. View Publication

Chronic decreases in the second messenger cyclic AMP (cAMP) occur in numerous settings,but how cells compensate for such decreases is unknown. We have used a unique system-murine dendritic cells (DCs) with a DC-selective depletion of the heterotrimeric GTP binding protein G$\alpha$s-to address this issue. These mice spontaneously develop Th2-allergic asthma and their DCs have persistently lower cAMP levels. We found that phosphodiesterase 4B (PDE4B) is the primary phosphodiesterase expressed in DCs and that its expression is preferentially decreased in G$\alpha$s-depleted DCs. PDE4B expression is dynamic,falling and rising in a protein kinase A-dependent manner with decreased and increased cAMP concentrations,respectively. Treatment of DCs that drive enhanced Th2 immunity with a PDE4B inhibitor ameliorated DC-induced helper T cell response. We conclude that PDE4B is a homeostatic regulator of cellular cAMP concentrations in DCs and may be a target for treating Th2-allergic asthma and other settings with low cellular cAMP concentrations. View Publication

Type 1 diabetes is an autoimmune disease with no cure,where clinical translation of promising therapeutics has been hampered by the reproducibility crisis. Here,short-term administration of an antagonist to the receptor for advanced glycation end products (sRAGE) protected against murine diabetes at two independent research centers. Treatment with sRAGE increased regulatory T cells (Tregs) within the islets,pancreatic lymph nodes,and spleen,increasing islet insulin expression and function. Diabetes protection was abrogated by Treg depletion and shown to be dependent on antagonizing RAGE with use of knockout mice. Human Tregs treated with a RAGE ligand downregulated genes for suppression,migration,and Treg homeostasis (FOXP3,IL7R,TIGIT,JAK1,STAT3,STAT5b,CCR4). Loss of suppressive function was reversed by sRAGE,where Tregs increased proliferation and suppressed conventional T-cell division,confirming that sRAGE expands functional human Tregs. These results highlight sRAGE as an attractive treatment to prevent diabetes,showing efficacy and reproducibility at multiple research centers and in human T cells. View Publication

Circulating tumor cells (CTC) play important roles in various cancers; however,few studies have assessed their clinical utility in neuroendocrine tumors. This study aimed to prospectively evaluate the prognostic value of CTC counts in Asian patients with neuroendocrine tumors before and during anti-cancer therapy. Patients who were diagnosed with unresectable histological neuroendocrine tumors between September 2011 and September 2017 were enrolled. CTC testing was performed before and during anti-cancer therapy using a negative selection protocol. Chromogranin A levels were also assessed. Univariate and multivariate Cox's proportional hazard model with forward LR model was performed to investigate the impact of independent factors on overall survival and progression-free survival. Kaplan-Meier method with log-rank tests were used to determine the difference among different clinicopathological signatures and CTC cutoff. The baseline CTC detection rate was 94.3{\%} (33/35). CTC counts were associated with cancer stages (I-III vs. IV,P = 0.015),liver metastasis (P = 0.026),and neuroendocrine tumor grading (P = 0.03). The median progression-free survival and overall survivals were 12.3 and 30.4 months,respectively. In multivariate Cox regression model,neuroendocrine tumors grading and baseline CTC counts were both independent prognostic factors for progression-free survival (PFS,P = 0.005 and 0.015,respectively) and overall survival (OS,P = 0.018 and 0.023,respectively). In Kaplan-Meier analysis,lower baseline chromogranin A levels were associated with longer PFS (P = 0.024). Baseline CTC counts are associated with the clinicopathologic features of neuroendocrine tumors and are an independent prognostic factor for this malignancy. View Publication

A variety of factors produced by stromal fibroblasts,including Flt3-ligand (FL),interleukin-11 (IL-11),Steel factor (SF),and IL-7,have been implicated in stimulating the production of pre-B cells and myeloid cells from primitive hematopoietic precursors. To investigate their relative roles in this process,either as single-acting or synergistic agents,we compared the yield and types of cells produced after 2 weeks from small numbers of Sca-1+ Lin- (i.e.,B220-,Ly-1-,Gr-1-,and Ter-119-) day 14.5 murine fetal liver cells placed in stromal cell-free cultures containing all possible combinations of FL,SF,IL-7,and IL-11. None of these factors alone supported the production (or survival) of any cells beyond 1 week: only pairs of factors consisting of either FL or SF plus either IL-11 or IL-7 were effective in this regard,with FL plus IL-11 being the most potent pair (approximately 7 x 10(4) cells obtained per 100 Sca-1+ Lin- input cells). The maximum numbers of cells were produced in the presence of FL,IL-11,and IL-7: these included both B220+ and Mac-1+/Gr-1+ cells (approximately 10(6) and approximately 2 x 10(5),respectively,per 100 Sca-1+ Lin- input cells). Both of these lineages were also obtained with each of the other possible three-factor combinations,albeit with variable effectiveness. Omission of either FL or IL-7 caused the greatest reduction in the yield of B220+ cells (approximately 130-fold and approximately 80-fold,respectively). Omission of IL-11 and,to a lesser extent,FL caused the greatest reduction in the yield of Mac-1+/Gr-1+ cells (approximately 90-fold and approximately 3-fold,respectively). When fetal calf serum was replaced with a defined serum substitute,the out put of B220+ cells remained the same but myelopoiesis was consistently enhanced (approximately 5- to 20-fold). These findings support a model involving factor redundancy in the extracellular signals required to stimulate the production and amplification of both lymphoid and myeloid cells from early Sca-1+ Lin- cells. They also reveal quantitative differences in the abilities of different competent factor combinations to promote this process,which may be further modulated by the presence of undefined serum components. View Publication

A series of hydroxamate based HDAC inhibitors containing a phenylisoxazole as the CAP group has been synthesized using nitrile oxide cycloaddition chemistry. An HDAC6 selective inhibitor having a potency of approximately 2 picomolar was identified. Some of the compounds were examined for their ability to block pancreatic cancer cell growth and found to be about 10-fold more potent than SAHA. This research provides valuable,new molecular probes for use in exploring HDAC biology. View Publication