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

Reversal of promoter DNA hypermethylation and associated gene silencing is an attractive cancer therapy approach. The DNA methylation inhibitors decitabine and azacitidine are efficacious for hematological neoplasms at lower,less toxic,doses. Experimentally,high doses induce rapid DNA damage and cytotoxicity,which do not explain the prolonged time to response observed in patients. We show that transient exposure of cultured and primary leukemic and epithelial tumor cells to clinically relevant nanomolar doses,without causing immediate cytotoxicity,produce an antitumor memory" response View Publication

In this study,we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic kinase that ultimately drives the transition from an epithelial to a highly invasive mesenchymal phenotype in estrogen receptor $$-positive (ER$$(+)) breast cancer cells. The transition from an epithelial- to a mesenchymal-like phenotype was characterized by reduced expression of ER$$,HER-2/Neu overexpression and loss of CD24 surface receptor (CD24(-/low)). Importantly,expression of key epithelial-to-mesenchymal transition (EMT) markers and upregulation of the stemness gene SOX2 was linked to acquisition of stem cell-like properties such as the ability to form mammospheres in vitro and tumor self-renewal in vivo. Moreover,aberrant Aurora-A kinase activity induced phosphorylation and nuclear translocation of SMAD5,indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in the development of EMT,stemness and ultimately tumor progression. Importantly,pharmacological and molecular inhibition of Aurora-A kinase activity restored a CD24(+) epithelial phenotype that was coupled to ER$$ expression,downregulation of HER-2/Neu,inhibition of EMT and impaired self-renewal ability,resulting in the suppression of distant metastases. Taken together,our findings show for the first time the causal role of Aurora-A kinase in the activation of EMT pathway responsible for the development of distant metastases in ER$$(+) breast cancer cells. Moreover,this study has important translational implications because it highlights the mitotic kinase Aurora-A as a novel promising therapeutic target to selectively eliminate highly invasive cancer cells and improve the disease-free and overall survival of ER$$(+) breast cancer patients resistant to conventional endocrine therapy. View Publication

Studies of repertoires of mouse monoclonal CD4(+) T cells have revealed several mechanisms of self-tolerance; however,which mechanisms operate in normal repertoires is unclear. Here we studied polyclonal CD4(+) T cells specific for green fluorescent protein expressed in various organs,which allowed us to determine the effects of specific expression patterns on the same epitope-specific T cells. Peptides presented uniformly by thymic antigen-presenting cells were tolerated by clonal deletion,whereas peptides excluded from the thymus were ignored. Peptides with limited thymic expression induced partial clonal deletion and impaired effector T cell potential but enhanced regulatory T cell potential. These mechanisms were also active for T cell populations specific for endogenously expressed self antigens. Thus,the immunotolerance of polyclonal CD4(+) T cells was maintained by distinct mechanisms,according to self-peptide expression patterns. View Publication

The intestinal epithelial repair after injury is coordinated by intestinal stem cells (ISCs). Fucosylation catalyzed by fucosyltransferase 2 (FUT2) of the intestinal epithelium is beneficial to mucosal healing but poorly defined is the influence on ISCs. The dextran sulfate sodium (DSS) and lipopolysaccharide (LPS) model were used to assess the role of FUT2 on ISCs after injury. The apoptosis,function,and stemness of ISCs were analyzed using intestinal organoids from WT and Fut2?ISC (ISC-specific Fut2 knockout) mice incubated with LPS and fucose. N-glycoproteomics,UEA-1 chromatography,and site-directed mutagenesis were monitored to dissect the regulatory mechanism,identify the target fucosylated protein and the corresponding modification site. Fucose could alleviate intestinal epithelial damage via upregulating FUT2 and ?-1,2-fucosylation of ISCs. Oxidative stress,mitochondrial dysfunction,and cell apoptosis were impeded by fucose. Meanwhile,fucose sustained the growth and proliferation capacity of intestinal organoids treated with LPS. Contrarily,FUT2 depletion in ISCs aggravated the epithelial damage and disrupted the growth and proliferation capacity of ISCs via escalating LPS-induced endoplasmic reticulum (ER) stress and initiating the IRE1/TRAF2/ASK1/JNK branch of unfolded protein response (UPR). Fucosylation of the chaperone protein HYOU1 at the N-glycosylation site of asparagine (Asn) 862 mediated by FUT2 was identified to facilitate ISCs survival and self-renewal,and improve ISCs resistance to ER stress and inflammatory injury. Our study highlights a fucosylation-dependent protective mechanism of ISCs against inflammation,which may provide a fascinating strategy for treating intestinal injury disorders. View Publication

Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity,we engineered induced pluripotent stem cell-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity,non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity. In addition,we introduced a membrane-bound IL-15/IL-15R fusion protein to promote in vivo persistence. These engineered cells,termed iDuo NK cells,displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models,iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore,iDuo NK cells effectively eliminated both CD19+ and CD19- lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19,features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represent a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies. View Publication

SummaryKCNQ1/Kv7,a low-voltage-gated K+ channel,regulates cardiac rhythm and glucose homeostasis. While KCNQ1 mutations are associated with long-QT syndrome and type2 diabetes,its function in human pancreatic cells remains controversial. We identified a homozygous KCNQ1 mutation (R397W) in an individual with permanent neonatal diabetes melitus (PNDM) without cardiovascular symptoms. To decipher the potential mechanism(s),we introduced the mutation into human embryonic stem cells and generated islet-like organoids (SC-islets) using CRISPR-mediated homology-repair. The mutation did not affect pancreatic differentiation,but affected channel function by increasing spike frequency and Ca2+ flux,leading to insulin hypersecretion. With prolonged culturing,the mutant islets decreased their secretion and gradually deteriorated,modeling a diabetic state,which accelerated by high glucose levels. The molecular basis was the downregulated expression of voltage-activated Ca2+ channels and oxidative phosphorylation. Our study provides a better understanding of the role of KCNQ1 in regulating insulin secretion and ?-cell survival in hereditary diabetes pathology. Graphical abstract Highlights•A permanent neonatal diabetes melitus patient carries a homozygous KCNQ1 mutation•KCNQ1R397W is loss of function and shows atypical electrophysiology in hESC-islets•Under high glucose,elevated Ca2+ flux leads to insulin hypersecretion•Mutant cells gradually switch phenotype,deteriorate,accelerated by high glucose Biological sciences; Endocrinology; Endocrinology; Health sciences; Internal medicine; Medical specialty; Medicine; Natural sciences; Physiology View Publication

IntroductionCytokine release syndrome (CRS) is one of the leading causes of mortality in patients with COVID-19 caused by the SARS-CoV-2 coronavirus. However,the mechanism of CRS induced by SARS-CoV-2 is vague.MethodsUsing spike protein combined with IL-2,IFN-γ,and TNF-α to stimulate human peripheral blood mononuclear cells (PBMCs) to secrete CRS-related cytokines,the content of cytokines in the supernatant was detected,and the effects of NK,T,and monocytes were analyzed.ResultsThis study shows that dendritic cells loaded with spike protein of SARS-CoV-2 stimulate T cells to release much more interleukin-2 (IL-2,) which subsequently cooperates with spike protein to facilitate PBMCs to release IL-1β,IL-6,and IL-8. These effects are achieved via IL-2 stimulation of NK cells to release tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ),as well as T cells to release IFN-γ Mechanistically,IFN-γ and TNF-α enhance the transcription of CD40,and the interaction of CD40 and its ligand stabilizes the membrane expression of toll-like receptor 4 (TLR4) that serves as a receptor of spike protein on the surface of monocytes. As a result,there is a constant interaction between spike protein and TLR4,leading to continuous activation of nuclear factor-κ-gene binding (NF-κB). Furthermore,TNF-α also activates NF-κB signaling in monocytes,which further cooperates with IFN-γ and spike protein to modulate NF-κB–dependent transcription of CRS-related inflammatory cytokines.DiscussionTargeting TNF-α/IFN-γ in combination with TLR4 may represent a promising therapeutic approach for alleviating CRS in individuals with COVID-19. View Publication

The ability to generate natural killer (NK) cells from induced pluripotent stem cells (iPSCs) has given rise to new possibilities for the large-scale production of homogeneous immunotherapeutic cellular products and opened new avenues towards the creation of “off-the-shelf” cancer immunotherapies. However,the differentiation of NK cells from iPSCs remains poorly understood,particularly regarding the ontogenic landscape of iPSC-derived NK (iNK) cells produced in vitro and the influence that the differentiation strategy employed may have on the iNK profile. To investigate this question,we conducted a comparative analysis of two sets of iNK cells generated from the same iPSC line using two different protocols: (i) a short-term,clinically compatible feeder-free protocol corresponding to primitive hematopoiesis,and (ii) a lymphoid-based protocol representing the definitive hematopoietic step. Our work demonstrated that both protocols are capable of producing functional iNK cells. However,the two sets of resulting iNKs exhibited distinct phenotypes and transcriptomic profiles. The lymphoid-based differentiation approach generated iNKs with a more mature and activated profile,which demonstrated higher cytotoxicity against cancer cell lines compared to iNK cells produced under short-term feeder-free conditions suggesting that the differentiation strategy must be considered when designing iNK cell–based adoptive immunotherapies. View Publication

Aim To analyse the ability of mesenchymal stem cells (MSCs) to regulate interleukin 6 (IL-6) and transforming growth factor (TGF-$\beta$) expression in vitro under co-culture conditions in human systemic lupus erythematosus (SLE). Method This study used a post-test group design that used peripheral blood mononuclear cells (PBMCs) from SLE patients at Kariadi Hospital,Semarang,Indonesia,and MSCs from a human umbilical cord. The cells were divided into two groups. The control group of PBMCs was treated with a standard medium,and the treatment group was co-cultured with the MSCs at a 1:40 ratio. Following 24 h incubation,the levels of IL-6 and TGF-$\beta$ released in the culture medium were measured using a specific ELISA assay. Results This study showed a significant decrease in IL-6 level (p{\textless}0.05) and a significant increase in TGF-$\beta$ level (p{\textless}0.001) following 24 h of co-culture incubation of human SLE PBMCs cells and MSCs. Conclusion The PBMCs-to-MSCs ratio of 1:40 can regulate the IL-6 and TGF-$\beta$ levels in human SLE PBMCs. View Publication

Dendritic cell (DC) maturation state is a key parameter for the issue of DC-T cell cognate interaction,which determines the outcome of T cell activation. Indeed,immature DCs induce tolerance while fully mature DCs generate immunity. Here we show that,in the absence of any deliberate activation signal,DCs freshly isolated from mouse spleen spontaneously produce IL-12 and tumor necrosis factor-alpha and up-regulate co-stimulation molecules,even when directly re-injected into their natural environment. Furthermore,after their isolation,these cells acquire the capacity to induce specific T(h)1 responses in vivo. These results demonstrate that the sole isolation of spleen DCs leads to the full maturation of these cells,which therefore cannot be considered as immature DCs. Moreover,we also show that the kinetics of DC activation do not influence the polarization of T(h) response in vivo challenging the idea that exhausted DCs induce preferentially T(h)2 response. Altogether,these observations should be taken into account in all experiments based on the transfer of ex vivo purified DCs. View Publication

BACKGROUND Pluripotent embryonic stem (ES) cells,which have the capacity to give rise to all tissue types in the body,show great promise as a versatile source of cells for regenerative therapy. However,the basic mechanisms of lineage specification of pluripotent stem cells are largely unknown,and generating sufficient quantities of desired cell types remains a formidable challenge. Small molecules,particularly those that modulate key developmental pathways like the bone morphogenetic protein (BMP) signaling cascade,hold promise as tools to study in vitro lineage specification and to direct differentiation of stem cells toward particular cell types. METHODOLOGY/ PRINCIPAL FINDINGS We describe the use of dorsomorphin,a selective small molecule inhibitor of BMP signaling,to induce myocardial differentiation in mouse ES cells. Cardiac induction is very robust,increasing the yield of spontaneously beating cardiomyocytes by at least 20 fold. Dorsomorphin,unlike the endogenous BMP antagonist Noggin,robustly induces cardiomyogenesis when treatment is limited to the initial 24-hours of ES cell differentiation. Quantitative-PCR analyses of differentiating ES cells indicate that pharmacological inhibition of BMP signaling during the early critical stage promotes the development of the cardiomyocyte lineage,but reduces the differentiation of endothelial,smooth muscle,and hematopoietic cells. CONCLUSIONS/ SIGNIFICANCE Administration of a selective small molecule BMP inhibitor during the initial stages of ES cell differentiation substantially promotes the differentiation of primitive pluripotent cells toward the cardiomyocytic lineage,apparently at the expense of other mesodermal lineages. Small molecule modulators of developmental pathways like dorsomorphin could become versatile pharmacological tools for stem cell research and regenerative medicine. View Publication

Interactions between developmental signaling pathways govern the formation and function of stem cells. Prostaglandin (PG) E2 regulates vertebrate hematopoietic stem cells (HSC). Similarly,the Wnt signaling pathway controls HSC self-renewal and bone marrow repopulation. Here,we show that wnt reporter activity in zebrafish HSCs is responsive to PGE2 modulation,demonstrating a direct interaction in vivo. Inhibition of PGE2 synthesis blocked wnt-induced alterations in HSC formation. PGE2 modified the wnt signaling cascade at the level of beta-catenin degradation through cAMP/PKA-mediated stabilizing phosphorylation events. The PGE2/Wnt interaction regulated murine stem and progenitor populations in vitro in hematopoietic ES cell assays and in vivo following transplantation. The relationship between PGE2 and Wnt was also conserved during regeneration of other organ systems. Our work provides in vivo evidence that Wnt activation in stem cells requires PGE2,and suggests the PGE2/Wnt interaction is a master regulator of vertebrate regeneration and recovery. View Publication