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

Mast cells are unique hematopoietic cells that are richly distributed in the skin and mucosal surfaces of the respiratory and gastrointestinal tract. They play a key role in allergic inflammation by releasing a cocktail of granular constituents,including histamine,serine proteases,and various eicosanoids and cytokines. As such,a number of drugs target either inhibition of mast cell degranulation or the products of degranulation. To identify potential novel drugs and mechanisms in mast cell biology,assays were developed to identify inhibitors of mast cell degranulation and activation in a phenotypic screen. Due to the challenges associated with obtaining primary mast cells,cord blood-derived mononuclear cells were reproducibly differentiated to mast cells and assays developed to monitor tryptase release and prostaglandin D2 generation. The tryptase assay was particularly sensitive,requiring only 500 cells per data point,which permitted a set of approximately 12,000 compounds to be screened robustly and cost-effectively. Active compounds were tested for concomitant inhibition of prostaglandin D2 generation. This study demonstrates the robustness and effectiveness of this approach in the identification of potential novel compounds and mechanisms targeting mast cell-driven inflammation,to enable innovative drug discovery efforts to be prosecuted. View Publication

Efficient approaches for the precise genetic engineering of stem cells can enhance both basic and applied stem cell research. Adeno-associated virus (AAV) vectors have demonstrated high-efficiency gene delivery and gene targeting to numerous cell types,and AAV vectors developed specifically for gene delivery to stem cells have further increased gene targeting frequency compared to plasmid construct techniques. This chapter details the production and purification techniques necessary to generate adeno-associated viral vectors for use in high-efficiency gene targeting of adult or pluripotent stem cell applications. Culture conditions used to achieve high gene targeting frequencies in rat neural stem cells and human pluripotent stem cells are also described. View Publication

Cancer stem cells (CSCs) have been identified in a variety of cancers and emerged as a new target for cancer therapy. CSCs are resistant to many current cancer treatments,including chemotherapy and radiation therapy. Therefore,eradication of this cell population is a primary objective in cancer therapy. Here,we report gold nanorods (AuNRs) mediated photothermal treatment can selectively eliminate CSCs in MCF-7 breast cancer cells. It significantly reduced the aldehyde dehydrogenase positive (ALDH(+)) cells subpopulation and the mammosphere formation ability of treated cells. Also,the gene expression of stem cell markers was decreased. Cellular uptake assay revealed that polyelectrolyte conjugated AuNRs could be internalized by CSCs much more and faster than non cancer stem cells (NCSCs),which might be the main reason for the selective elimination of CSCs. We further loaded salinomycin (SA),a CSCs inhibitor with polyelectrolyte conjugated AuNRs to get a synergistic CSCs inhibition. Enhanced inhibition of CSCs was obtained by NIR light triggered drug release and hyperthermia. This CSCs-targeted thermo-chemotherapy platform provides a new combinatorial strategy for efficient inhibition of CSCs,which is promising to improve cancer treatment and may overcome the chemoresistance and recurrence of cancer. View Publication

Recent studies suggest that highly activated,polyfunctional CD4+ T cells are incredibly effective in strengthening and sustaining overall host antitumor immunity,promoting tumor-specific CD4+ T-cell responses and effectively enhancing antitumor immunity by immunotherapy. Previously,we developed a novel cryo-thermal therapy for local tumor ablation and achieved long-term survival rates in several tumor models. It was discovered that cryo-thermal therapy remodeled the tumor microenvironment and induced an antigen-specific CD4+ T-cell response,which mediated stronger antitumor immunity in vivo. In this study,the phenotype of bulk T cells in spleen was analyzed by flow cytometry after cryo-thermal therapy and both CD4+ Th1 and CD8+ CTL were activated. In addition,by using T-cell depletion,isolation,and adoptive T-cell therapy,it was found that cryo-thermal therapy induced Th1-dominant CD4+ T cells that directly inhibited the growth of tumor cells,promoted the maturation of MDSCs via CD4+ T-cell-derived IFN-? and enhanced the cytotoxic effector function of NK cells and CD8+ T cells,and promoted the maturation of APCs via cell-cell contact and CD4+ T-cell-derived IFN-?. Considering the multiple roles of cryo-thermal-induced Th1-dominant CD4+ T cells in augmenting antitumor immune memory,we suggest that local cryo-thermal therapy is an attractive thermo-immunotherapy strategy to harness host antitumor immunity and has great potential for clinical application. View Publication

Human pancreatic islets regulate organ development and metabolic homeostasis,with dysfunction leading to diabetes. Human pluripotent stem cells (hPSCs) provide a potential alternative source to cadaveric human pancreatic islets for replacement therapy in diabetes. However,human islet-like organoids (HILOs) generated from hPSCs in vitro often exhibit heterogeneous immature phenotypes such as aberrant gene expression and inadequate insulin secretion in response to glucose. Here we show that FXYD Domain Containing Ion Transport Regulator 2 (FXYD2) marks and regulates functional maturation and heterogeneity of generated HILOs,by controlling the ? cell transcriptome necessary for glucose-stimulated insulin secretion (GSIS). Despite its presence in mature ? cells,FXYD2 is diminished in hPSC-derived ?-like cells. Mechanistically,we find that FXYD2 physically interacts with SRC proto-oncogene,non-receptor tyrosine kinase (SRC) protein to regulate FXYD2-SRC-TEAD1 signaling to modulate ? cell transcriptome. We demonstrate that FXYD2High HILOs significantly outperform FXYD2Low counterparts to improve hyperglycemia in STZ-induced diabetic immune deficient mice. These results suggest that FXYD2 marks and regulates human ? cell maturation via channel-sensing signal transduction and that it can be used as a selection marker for functional heterogeneity of stem cell derived human islet organoids. Tacto et al. uncover a key marker that enables the selection of functional,transplantable human islets derived from stem cells,potentially paving the way for more precise and effective diabetes cell therapy. View Publication

BackgroundCNS stromal cells,especially fibroblasts and endothelial cells,support leukocyte accumulation through upregulation of adhesion molecules and lymphoid chemokines. While chronically activated fibroblast networks can drive pathogenic immune cell aggregates known as tertiary lymphoid structures (TLS),early stromal cell activation during CNS infection can support anti-viral T cells. However,the cell types and factors driving early stromal cell activation is poorly explored.AimsA neurotropic murine coronavirus (mCoV) infection model was used to better characterize signals that promote fibroblast networks supporting accumulation of antiviral lymphocytes. Based on the early appearance of IgD+ B cells with unknown functions during several CNS infections,we probed their potential to activate stromal cells through lymphotoxin β (LTβ),a molecule critical in maintaining fibroblast-networks in lymphoid tissues as well as promoting TLS in autoimmunity and cancers.ResultsKinetic analysis of stromal cell activation in olfactory bulbs and brains revealed that upregulation of adhesion molecules and lymphoid chemokines Ccl19,Ccl21 and Cxcl13 closely tracked viral replication. Immunohistochemistry revealed that upregulation of the fibroblast marker podoplanin (PDPN) at meningeal and perivascular sites mirrored kinetics of RNA expression. Moreover,both B cells and T cells colocalized to areas of PDPN reactivity,supporting a potential role in regulating stromal cell activation. However,specific depletion of LTβ from B cells using Mb1-creERT2 x Ltβfl/fl mice had no effect on T or B cell recruitment or viral replication. B cell depletion by anti-CD20 antibody also had no adverse effects. Surprisingly,LTβR agonism reduced viral control and parenchymal T cell localization despite increasing stromal cell lymphoid chemokines and PDPN. Additional assessment of direct stromal cell activation by the viral RNA mimic poly I:C showed induction of Pdpn and Ccl19 preceding Ltb.ConclusionsNeither B cell-derived LTβ or B cells are primary drivers of stromal cell activation networks in the CNS following mCoV infection. Although supplementary agonist mediated LTβR engagement confirmed a role for LTβ in enhancing PDPN and lymphoid chemokine expression,it impeded T cell migration to the CNS parenchyma and viral control. Our data overall indicate that stromal cells can integrate LTβR signals to tune their activation,but that LTβ is not necessarily essential and can even dysregulate protective antiviral T cell functions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03491-7. View Publication

Growing evidence shows that the reprogramming of fatty acid (FA) metabolism plays a key role in HER2-positive (HER2 +) breast cancer (BC) aggressiveness,therapy resistance and cancer stemness. In particular,HER2 + BC has been defined as a "lipogenic disease" due to the functional and bi-directional crosstalk occurring between HER2-mediated oncogenic signaling and FA biosynthesis via FA synthase activity. In this context,the functional role exerted by the reprogramming of CD36-mediated FA uptake in HER2 + BC poor prognosis and therapy resistance remains unclear. In this study,we aimed to elucidate whether enhanced CD36 in mesenchymal HER2 + cancer stem cells (CSCs) is directly involved in anti-HER2 treatment refractoriness in HER2 + BC and to design future metabolism-based approaches targeting both FA reprogramming and the “root” of cancer. Molecular,biological and functional characterization of CD36-mediated FA uptake was investigated in HER2 + BC patients,cell lines,epithelial and mesenchymal CSCs. Cell proliferation was analyzed by SRB assay upon treatment with lapatinib,CD36 inhibitor,or Wnt antagonist/agonist. Engineered cell models were generated via lentivirus infection and transient silencing. CSC-like properties and tumorigenesis of HER2 + BC cells with or without CD36 depletion were examined by mammosphere forming efficiency assay,flow cytometry,cell sorting,ALDH activity assay and xenograft mouse model. FA uptake was examined by flow cytometry with FA BODIPY FL C16. Intratumor expression of CSC subsets was evaluated via multiplex immunostaining and immunolocalization analysis. Molecular data demonstrated that CD36 is significantly upmodulated on treatment in therapy resistant HER2 + BC patients and its expression levels in BC cells is correlated with FA uptake. We provided evidence of a consistent enrichment of CD36 in HER2 + epithelial-mesenchymal transition (EMT)-like CSCs from all tested resistant cell models that mechanistically occurs via Wnt signaling pathway activation. Consistently,both in vitro and in vivo dual blockade of CD36 and HER2 increased the anti-CSC efficacy of anti-HER2 drugs favoring the transition of the therapy resistant mesenchymal CSCs into therapy-sensitive mesenchymal-epithelial transition (MET)-like epithelial state. In addition,expression of CD36 in intratumor HER2 + mesenchymal CSCs is significantly associated with resistance to trastuzumab in HER2 + BC patients. These results support the metabolo-oncogenic nature of CD36-mediated FA uptake in HER2 + therapy-refractory BC. Our study provides evidence that targeting CD36 might be an effective metabolic therapeutic strategy in the treatment of this malignancy. The online version contains supplementary material available at 10.1186/s13046-025-03276-z. View Publication

The manufacturing of allogeneic cell therapeutics based on human-induced pluripotent stem cells (hiPSCs) holds considerable potential to revolutionize the accessibility and affordability of modern healthcare. However,achieving the cell yields necessary to ensure robust production hinges on identifying suitable and scalable single-use (SU) bioreactor systems. While specific stirred SU bioreactor types have demonstrated proficiency in supporting hiPSC expansion at L -scale,others,notably instrumented SU multiplate and fixed-bed bioreactors,remain relatively unexplored. By characterizing these bioreactors using both computational fluid dynamics and experimental bioengineering methods,operating ranges were identified for the Xpansion ® 10 and Ascent™ 1 m 2 bioreactors in which satisfactory hiPSC expansion under serum-free conditions was achieved. These operating ranges were shown not only to effectively limit cell exposure to wall shear stress but also facilitated sufficient oxygen transfer and mixing. Through their application,almost 5 × 10 9 viable cells could be produced within 5 days,achieving expansion factors of up to 35 without discernable impact on cell viability,identity,or differentiation potential. Key Points • Bioengineering characterizations allowed the identification of operating ranges that supported satisfactory hiPSC expansion • Both the Xpansion ® 10 multiplate and Ascent™ 1 m 2 fixed-bed reactor accommodated the production of almost 5 × 10 9 viable cells within 5 days • Exposing the hiPSCs to a median wall shear stress of up to 8.2 × 10 −5 N cm −2 did not impair quality The online version contains supplementary material available at 10.1007/s00253-024-13373-2. View Publication

Aim of work was to locate a simple,reproducible protocol for uniform seeding and optimal cellularization of biodegradable patch minimizing the risk of structural damages of patch and its contamination in long-term culture. Two seeding procedures are exploited,namely static seeding procedures on biodegradable and biocompatible patches incubated as free floating (floating conditions) or supported by CellCrownTM insert (fixed conditions) and engineered by porcine bone marrow MSCs (p-MSCs). Scaffold prototypes having specific structural features with regard to pore size,pore orientation,porosity,and pore distribution were produced using two different techniques,such as temperature-induced precipitation method and electrospinning technology. The investigation on different prototypes allowed achieving several implementations in terms of cell distribution uniformity,seeding efficiency,and cellularization timing. The cell seeding protocol in stating conditions demonstrated to be the most suitable method,as these conditions successfully improved the cellularization of polymeric patches. Furthermore,the investigation provided interesting information on patches' stability in physiological simulating experimental conditions. Considering the in vitro results,it can be stated that the in vitro protocol proposed for patches cellularization is suitable to achieve homogeneous and complete cellularizations of patch. Moreover,the protocol turned out to be simple,repeatable,and reproducible. View Publication

Constitutive activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway in human cancers is often associated with mutational activation of BRAF or RAS. MAPK/ERK kinase 1/2 kinases lie downstream of RAS and BRAF and are the only acknowledged activators of ERK1/2,making them attractive targets for therapeutic intervention. AZD6244 (ARRY-142886) is a potent,selective,and ATP-uncompetitive inhibitor of MAPK/ERK kinase 1/2. In vitro cell viability inhibition screening of a tumor cell line panel found that lines harboring BRAF or RAS mutations were more likely to be sensitive to AZD6244. The in vivo mechanisms by which AZD6244 inhibits tumor growth were investigated. Chronic dosing with 25 mg/kg AZD6244 bd resulted in suppression of growth of Colo-205,Calu-6,and SW-620 xenografts,whereas an acute dose resulted in significant inhibition of ERK1/2 phosphorylation. Increased cleaved caspase-3,a marker of apoptosis,was detected in Colo-205 and Calu-6 but not in SW-620 tumors where a significant decrease in cell proliferation was detected. Chronic dosing of AZD6244 induced a morphologic change in SW-620 tumors to a more differentiated phenotype. The potential of AZD6244 in combination with cytotoxic drugs was evaluated in mice bearing SW-620 xenografts. Treatment with tolerated doses of AZD6244 and either irinotecan or docetaxel resulted in significantly enhanced antitumor efficacy relative to that of either agent alone. These results indicate that AZD6244 has potential to inhibit proliferation and induce apoptosis and differentiation,but the response varies between different xenografts. Moreover,enhanced antitumor efficacy can be obtained by combining AZD6244 with the cytotoxic drugs irinotecan or docetaxel. View Publication

The study of human erythropoiesis in health and disease requires a robust culture system that consistently and reliably generates large numbers of immature erythroblasts that can be induced to differentiate synchronously. We describe a culture method modified from Leberbauer et al. (2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34(+) cells. This pure population of immature erythroblasts can be expanded to obtain 4x10(8) erythroblasts from 1x10(8) PBMC after 13-14 days in culture. Upon synchronized differentiation,high levels of enucleation (80-90%) and low levels of cell death (textless10%) are achieved. We compared the yield of erythroblasts obtained from PBMC,CD34(+) cells or PBMC depleted of CD34(+) cells and show that CD34(-) cells represent the most significant early erythroid progenitor population. This culture system may be particularly useful for investigating the pathophysiology of anemic patients where only small blood volumes are available. View Publication

OBJECTIVE The p15INK4B tumor suppressor is frequently silenced by promoter hypermethylation in myelodysplastic syndrome and acute myeloid leukemia (AML). Clinically approved DNA methylation inhibitors,such as 5-aza-2'-deoxycytidine,can reverse p15INK4B promoter methylation,but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. The cytidine analog zebularine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. We evaluated zebularine effects on p15INK4B reactivation and cell growth in vitro to investigate a potential role for zebularine in treating myeloid malignancies. METHODS We examined the specific effects of zebularine on reexpression of transcriptionally silenced p15INK4B and its global effects on cell cycle and apoptosis in AML cell lines and primary patient samples. RESULTS Zebularine treatment of AML193,which has a densely methylated p15INK4B promoter,results in a dose-dependent increase in p15INK4B expression that correlates with CpG island promoter demethylation and enrichment of local histone acetylation. We observed enhanced p15INK4B induction following co-treatment with zebularine and the histone deacetylase inhibitor Trichostatin A. Zebularine inhibits cell proliferation,arrests cells at G(2)/M,and induces apoptosis at dosages that effectively demethylate the p15INK4B promoter. Zebularine treatment of KG-1 cells and AML patient blasts with hypermethylated p15INK4B promoters also reactivates p15INK4B reexpression and induces apoptosis. CONCLUSION Zebularine is an effective inhibitor of p15INK4B methylation and cell growth in human AML in vitro. Our results extend the spectrum of zebularine effects to nonepithelial malignancies and provide a strong rationale for evaluating its clinical utility in the treatment of myeloid malignancies. View Publication