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

In mammals,early embryonic gastrulation process is high energy demanding. Previous studies showed that,unlike endoderm and mesoderm cells,neuroectoderm differentiated from human embryonic stem cells relied on aerobic glycolysis as the major energy metabolic process,which generates lactate as the final product. Here we explored the function of intracellular lactate during neuroectoderm differentiation. Our results revealed that the intracellular lactate level was elevated in neuroectoderm and exogenous lactate could further promote hESCs differentiation towards neuroectoderm. Changing intracellular lactate levels by sodium lactate or LDHA inhibitors had no obvious effect on BMP or WNT/?-catenin signaling during neuroectoderm differentiation. Notably,histone lactylation,especially H3K18 lactylation was significant upregulated during this process. We further performed CUT&Tag experiments and the results showed that H3K18la is highly enriched at gene promoter regions. By analyzing data from CUT&Tag and RNA-seq experiments,we further identified that four genes,including PAX6,were transcriptionally upregulated by lactate during neuroectoderm differentiation. A H3K18la modification site at PAX6 promoter was verified and exogenous lactate could also rescue the level of PAX6 after shPAX6 inhibition.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-024-05510-x. View Publication

To characterize mechanisms of CTL inhibition within an ocular tumor microenvironment,tumor-specific CTLs were transferred into mice with tumors developing within the anterior chamber of the eye or skin. Ocular tumors were resistant to CTL transfer therapy whereas skin tumors were sensitive. CTLs infiltrated ocular tumors at higher CTL/tumor ratios than in skin tumors and demonstrated comparable ex vivo effector function to CTLs within skin tumors indicating that ocular tumor progression was not due to decreased CTL accumulation or inhibited CTL function within the eye. CD11b(+)Gr-1(+)F4/80(-) cells predominated within ocular tumors,whereas skin tumors were primarily infiltrated by CD11b(+)Gr-1(-)F4/80(+) macrophages (Ms),suggesting that myeloid derived suppressor cells may contribute to ocular tumor growth. However,CD11b(+) myeloid cells isolated from either tumor site suppressed CTL activity in vitro via NO production. Paradoxically,the regression of skin tumors by CTL transfer therapy required NO production by intratumoral Ms indicating that NO-producing intratumoral myeloid cells did not suppress the effector phase of CTL. Upon CTL transfer,tumoricidal concentrations of NO were only produced by skin tumor-associated Ms though ocular tumor-associated Ms demonstrated comparable expression of inducible NO synthase protein suggesting that NO synthase enzymatic activity was compromised within the eye. Correspondingly,in vitro-activated Ms limited tumor growth when co-injected with tumor cells in the skin but not in the eye. In conclusion,the decreased capacity of Ms to produce NO within the ocular microenvironment limits CTL tumoricidal activity allowing ocular tumors to progress. View Publication

BACKGROUND Adoptive T-cell transfer has become an attractive therapeutic approach for hematological malignancies but shows poor activity against large and heterogeneous solid tumors. Interleukin-12 (IL-12) exhibits potent antitumor efficacy against solid tumors,but its clinical application has been stalled because of toxicity. Here,we aimed to develop a safe approach to IL-12 T-cell therapy for eliminating large solid tumors. METHODS We generated a cell membrane-anchored IL-12 (aIL12),a tumor-targeted IL-12 (ttIL12),and a cell membrane-anchored and ttIL-12 (attIL12) and a cell membrane-anchored and tumor-targeted ttIL-12 (attIL12) armed T cells,chimeric antigen receptor-T cells,and T cell receptor-T (TCR-T) cells with each. We compared the safety and efficacy of these armed T cells in treating osteosarcoma patient-derived xenograft tumors and mouse melanoma tumors after intravenous infusions of the armed T cells. RESULTS attIL12-T cell infusion showed remarkable antitumor efficacy in human and mouse large solid tumor models. Mechanistically,attIL12-T cells targeted tumor cells expressing cell-surface vimentin,enriching effector T cell and interferon $\gamma$ production in tumors,which in turn stimulates dendritic cell maturation for activating secondary T-cell responses and tumor antigen spreading. Both attIL12- and aIL12-T-cell transfer eliminated peripheral cytokine release and the associated toxic effects. CONCLUSIONS This novel approach sheds light on the safe application of IL-12-based T-cell therapy for large and heterogeneous solid tumors. View Publication

Small non‐coding microRNAs (miRNAs) play essential roles in Alzheimer's disease (AD) pathogenesis. Repressor element 1‐silencing transcription factor (REST) is involved in AD,though its regulation remains unclear. We performed real‐time quantitative polymerase chain reaction (qPCR) in autopsied brain tissues to determine miR‐153‐3p and AD associations. A reporter‐based assay measured the activity of REST mRNA 3′‐untranslated region (3′‐UTR). Induced pluripotent stem cells (iPSC)‐derived neurons and human cell lines were applied to determine miR‐153‐3p regulation of endogenous proteins. Elevation of miR‐153‐3p is associated with a reduced probability of AD,while elevated REST is associated with a greater probability of AD. The 3′‐UTR functional assay pinpointed the miR‐153‐3p binding sites. miR‐153‐3p treatment reduced REST,amyloid precursor protein (APP),and α‐synuclein (SNCA) 3′‐UTR activities and protein levels. miR‐153‐3p treatment altered REST and neuronal differentiation in iPSC‐derived neuronal stem cells. RNA‐sequencing and proteomics revealed miR‐153‐3p‐associated networks. miR‐153‐3p reduces REST,APP,and SNCA expression,pointing toward its therapeutic and biomarker potential in neurodegenerative diseases. With the increased emphasis on comorbidities of Alzheimer's disease (AD) and other neurodegenerative diseases,we identified that miR‐153‐3p,as a master regulator,reduced a group of neurodegeneration related proteins: REST,amyloid precursor protein (APP) and α‐synuclein (SNCA) levels. The elevation of miR‐153‐3p levels is associated with reduced probability of AD in posterior cingulate cortex (PCC),while REST,by contrast,is associated with a greater probability of AD. miR‐153‐3p treatment alters REST protein levels and neuronal differentiation in induced pluripotent stem cells (iPSC) derived neuronal cells. RNA sequencing proteomics and interactome analysis revealed the role of miR‐153‐3p in axonal guidance. View Publication

5-Ethynyl-2′-deoxyuridine (EdU) has revolutionized DNA replication and cell cycle analyses through fast,efficient click chemistry detection. However,commercial EdU kits suffer from high costs,proprietary formulations,limited antibody multiplexing capabilities,and difficulties with larger biological specimens. Here,we present OpenEMMU (Open-source EdU Multiplexing Methodology for Understanding DNA replication dynamics),an optimized,affordable,and user-friendly click chemistry platform utilizing off-the-shelf reagents. OpenEMMU enhances efficiency,brightness,and multiplexing capabilities of EdU staining with both non-conjugated and conjugated antibodies across diverse cell types,including T cell activation and proliferation assays. We validated its effectiveness for the fluorescent imaging of nascent DNA synthesis in developing embryos and organs,including embryonic heart,forelimbs,and 3D hiPSC-derived cardiac organoids. OpenEMMU also enabled the deep-tissue 3D imaging of DNA synthesis in zebrafish larvae and under replication stress in embryos at high spatial resolution. This approach opens new avenues for understanding organismal development,cell proliferation,and DNA replication dynamics with unprecedented precision and flexibility. Subject areas: Biochemistry,Cell biology,Developmental biology,Computational bioinformatics View Publication

Ovarian cancer (OV) has the highest mortality rate among gynecological cancers. As OV progresses,tumor cells spread outside the ovaries to the peritoneal and abdominal cavities,forming cell clusters that float in the ascitic fluid caused by peritonitis carcinomatosa,leading to further dissemination and metastasis. These cell clusters are enriched with cancer stem cells (CSCs) which are responsible for treatment resistance,recurrence,and metastasis. Therefore,targeting CSCs is a potentially effective approach for treating OV. However,understanding how CSCs acquire treatment resistance and identifying targets against CSCs remains challenging. In this study,we demonstrate that 3D-spheroids of OV cell lines exhibit higher stemness than conventional adherent cells. Metabolomics profiling studies have revealed that 3D-spheroids maintain a high-energy state through increased glucose utilization in the citric acid cycle (TCA),efficient nucleotide phosphorylation,and elevated phosphocreatine as an energy buffer. We also found that nicotinamide phosphoribosyltransferase (NAMPT),the rate-limiting enzyme for NAD + production,is highly expressed in OV. Furthermore,the approach based on NAMPT dependence rather than histology found NAMPT to be a potential therapeutic target against CSCs,while also serving as a prognostic indicator in OV. Moreover,we identified a previously unrecognized anti-tumor mechanism whereby disulfiram,an aldehyde dehydrogenase (ALDH) inhibitor,synergistically inhibited mitochondrial function when combined with NAMPT inhibitors - leading to cell cycle arrest in G2/M. Finally,the combination of a NAMPT inhibitor and disulfiram showed significant anti-tumor effects and extended survival in an animal model. Our findings demonstrate the potential of spheroids as a preclinical model for targeting OV CSCs and also indicate that the combination of NAMPT inhibitors and disulfiram is a promising therapeutic strategy to overcome recurrent OV. Subject terms: Ovarian cancer,Metabolomics,Apoptosis,Cancer stem cells View Publication

The receptor tyrosine kinase c-Kit plays a critical role in hematopoiesis,and gain-of-function mutations of the receptor are frequently seen in several malignancies,including acute myeloid leukemia,gastrointestinal stromal tumors,and testicular carcinoma. The most common mutation of c-Kit in these disorders is a substitution of the aspartic acid residue in position 816 to a valine (D816V),leading to constitutive activation of the receptor. In this study,we aimed to investigate the role of Src family kinases in c-Kit/D816V signaling. Src family kinases are necessary for the phosphorylation of wild-type c-Kit as well as of activation of downstream signaling pathways including receptor ubiquitination and the Ras/Mek/Erk pathway. Our data demonstrate that,unlike wild-type c-Kit,the phosphorylation of c-Kit/D816V is not dependent on Src family kinases. In addition,we found that neither receptor ubiquitination nor Erk activation by c-Kit/D816V required activation of Src family kinases. In vitro kinase assay using synthetic peptides revealed that c-Kit/D816V had an altered substrate specificity resembling Src and Abl tyrosine kinases. We further present evidence that,in contrast to wild-type c-Kit,Src family kinases are dispensable for c-Kit/D816V cell survival,proliferation,and colony formation. Taken together,we demonstrate that the signal transduction pathways mediated by c-Kit/D816V are markedly different from those activated by wild-type c-Kit and that altered substrate specificity of c-Kit circumvents a need for Src family kinases in signaling of growth and survival,thereby contributing to the transforming potential of c-Kit/D816V. View Publication

Cell type-specific surface markers offer a powerful tool for purifying defined cell types for restorative therapies and drug screenings. Midbrain dopaminergic neurons (mesDA) are the nerve cells preferentially lost in the brains of Parkinson's disease patients. Clinical trials of transplantation of fetal neural precursors suggest that cell therapy may offer a cure for this devastating neurological disease. Many lines of preclinical studies demonstrate that neural progenitors committed to dopaminergic fate survive and integrate better than postmitotic DA neurons. We show that the folate-receptor 1 (FolR1),a GPI-anchored cell surface molecule,specifically marks mesDA neural progenitors and immature mesDA neurons. FolR1 expression superimposes with Lmx1a,a bona-fide mesDA lineage marker,during the active phase of mesDA neurogenesis from E9.5 to E14.5 during mouse development,as well as in ESC-derived mesDA lineage. FolR1(+) neural progenitors can be isolated by FACS or magnetic sorting (MAC) which give rise to dopamine neurons expressing TH and Pitx3,whilst FolR1 negative cells generate non-dopaminergic neurons and glia cells. This study identifies FolR1 as a new cell surface marker selectively expressed in mesDA progenitors in vivo and in vitro and that can be used to enrich in vitro differentiated TH neurons. View Publication

When transmitted through the oral route,Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication,as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However,due to a lack of tractable infection models,we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However,the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here,we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular,we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization,and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with,and support replication of,T. gondii. Using quantitative label-free mass spectrometry,we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism,extracellular exosomes,intermicrovillar adhesion,and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses,indicating that de novo synthesis may support,but is not required for,parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection. View Publication