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

Autoimmune uveitis is a leading cause of severe vision loss,and animal models provide unique opportunities for studying its pathogenesis and therapeutic strategies. Here we employ scRNA-seq,RNA-seq and various molecular and cellular approaches to characterize mouse models of classical experimental autoimmune uveitis (EAU),revealing that EAU causes broad retinal neuron degeneration and marker downregulation,and that Müller glia may act as antigen-presenting cells. Moreover,EAU immune response is primarily driven by Th1 cells,and results in dramatic upregulation of CC chemokines,especially CCL5,in the EAU retina. Accordingly,overexpression of CCR5,a CCL5 receptor,in mesenchymal stem cells (MSCs) enhances their homing capacity and improves their immunomodulatory outcomes in preventing EAU,by reducing infiltrating T cells and activated microglia and suppressing Nlrp3 inflammasome activation. Taken together,our data not only provide valuable insights into the molecular characteristics of EAU but also open an avenue for innovative MSC-based therapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-024-03134-3. View Publication

Previously,we reported that Lactobacillus paragasseri SBT2055 (LG2055) activates plasmacytoid dendritic cells (pDCs) and induces interferon alpha (IFN-α) in vitro. Our clinical trial suggested that LG2055 intake may enhance pDC activity,supporting immune maintenance and reducing subjective common cold symptoms. However,the precise mechanisms remain unclear. In this study,we investigated how LG2055 engages with pDCs to stimulate IFN-α production. We evaluated LG2055-induced pDC activation using flow cytometry,ELISA,and phagocytosis assays. Human peripheral blood mononuclear cells (PBMCs) were stimulated with LG2055 and its components to evaluate immune responses. An in vitro M cell model was used to examine LG2055 translocation. We found that DNA extracted from LG2055 activated pDCs and enhanced IFN-α production via Toll-like receptor 9 (TLR9). Phagocytosis assays demonstrated that LG2055 DNA was internalized by PBMC-derived pDCs,enabling TLR9-mediated signaling. Additionally,LG2055 translocated across M cells in vitro,suggesting potential transport into Peyer’s patches,where it may interact with pDCs. These findings demonstrate that intestinal LG2055 can translocate across M cells,interact with pDCs,and exert immune-stimulatory effects to enhance host antiviral immunity. This study provides mechanistic insight into how dietary components support immune health and could inform the development of novel functional foods or therapeutic strategies. View Publication

Induction of ferroptosis,an iron-dependent form of regulated cell death,holds promise as a strategy to overcome tumor resistance to conventional therapies and enhance immunotherapy responses. However,while the susceptibility of tumor cells to ferroptosis is extensively studied,limited data exists on the vulnerability of immune cells to disturbed iron balance and lipid peroxidation. Here,we found that T-cell stimulation rewires iron and redox homeostasis and by increasing levels of reactive oxygen species and labile iron promotes lipid peroxidation and T-cells’ ferroptosis. Upon stimulation,we detected changes in the balance of ferroptosis-suppressive proteins,including decrease of GPX4. Subsequently,we identified GPX4 as a master regulator orchestrating T/CAR-T-cells’ sensitivity to ferroptosis and observed that GPX4 inhibitors impair CAR-T cells’ antitumor functions. Our study demonstrated differential GPX4 expression and diverse susceptibility to ferroptosis between CD4⁺ and CD8⁺ T cells. Among analyzed subsets of naïve,central memory (CM),effector memory (EM),and terminally differentiated effector memory (TEMRA),CD8⁺ EM and CD8⁺ TEMRA cells exhibited the highest sensitivity to ferroptosis. We also showed that ferroptosis limited the anti-tumor efficacy of CAR-T cells,while ferroptosis inhibition improved their therapeutic effect,both in vitro and in vivo. Our findings are not only important to understand vulnerabilities of CAR-T cells but may also hold particular significance for their therapeutic development. In this context,future anticancer therapies should be carefully designed to selectively induce the ferroptosis of tumor cells without impeding cytotoxic cells’ antitumor efficacy. Additionally,we postulate that promoting less differentiated phenotype of CAR-T cells should be exploited therapeutically to create CAR-T products characterized by decreased sensitivity to ferroptosis within tumor microenvironment.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-025-04133-w. View Publication

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that affects communication,social interaction,and behavior. Calcium (Ca2+) signaling dysregulation has been frequently highlighted in genetic studies as a contributing factor to aberrant developmental processes in ASD. Herein,we used ASD and control induced pluripotent stem cells (iPSCs) to investigate transcriptomic and functional Ca2+ dynamics at various stages of differentiation to cortical neurons. Idiopathic ASD and control iPSC lines underwent the dual SMAD inhibition differentiation protocol to direct their fate toward cortical neurons. Samples from multiple time points along the course of differentiation were processed for bulk RNA sequencing,spanning the following sequential stages: the iPSC stage,neural induction (NI) stage,neurosphere (NSP) stage,and differentiated cortical neuron (Diff) stage. Our transcriptomic analyses suggested that the numbers of Ca2+ signaling-relevant differentially expressed genes between ASD and control samples were higher in the iPSC and Diff stages. Accordingly,samples from the iPSC and Diff stages were processed for Ca2+ imaging studies. Results revealed that iPSC-stage ASD samples displayed elevated maximum Ca2+ levels in response to ATP compared to controls. By contrast,in the Diff stage,ASD neurons showed reduced maximum Ca2+ levels in response to ATP but increased maximum Ca2+ levels in response to KCl and DHPG relative to controls. Considering the distinct functional signaling contexts of these stimuli,this differential profile of receptor- and ionophore-mediated Ca2+ response suggests that aberrant calcium homeostasis underlies the pathophysiology of ASD neurons. Our data provides functional evidence for Ca2+ signaling dysregulation during neurogenesis in idiopathic ASD. View Publication

Differentiation of embryonic stem cells and induced pluripotent stem cells (iPSCs) into endoderm derivatives,including thyroid,thymus,lungs,liver,and pancreas,has broad implications for disease modeling and therapy. We utilize and expand a model development approach previously outlined by the authors to construct a model for the directed differentiation of iPSCs into definitive endoderm (DE). Assuming discrete intermediate stages in the differentiation process with a homogeneous population in each stage,three lineage models with two,three,and four populations and three growth models are constructed. Additionally,three models for error distribution are defined,resulting in a total of 27 models. Experimental data obtained in vitro are used for model calibration,model selection,and final validation. Model selection suggests that no transitory state during differentiation expresses the DE biomarkers CD117 and CD184,a finding corroborated by existing literature. Additionally,space-limited growth models,such as logistic and Gompertz growth,outperform exponential growth. Validation of the inferred model with leave-out data results in prediction errors of 26.4%. Using the inferred model,it is predicted that the optimal differentiation period is between 1.9 and 2.4 days,plating populations closer to 300 000 cells per well result in the highest yield efficiency,and that iPSC differentiation outpaces the DE proliferation as the main driver of the population dynamics. We also demonstrate that the model can predict the effect of growth modulators on cell population dynamics. Our model serves as a valuable tool for optimizing differentiation protocols,providing insights into developmental biology. View Publication

Chimeric antigen receptor (CAR) T cells show suboptimal efficacy in acute myeloid leukemia (AML). We find that CAR T cells exposed to myeloid leukemia show impaired activation and cytolytic function,accompanied by impaired antigen receptor downstream calcium,ZAP70,ERK,and C-JUN signaling,compared to those exposed to B-cell leukemia. These defects are caused in part by the high expression of CD155 by AML. Overexpressing C-JUN,but not other antigen receptor downstream components,maximally restores anti-tumor function. C-JUN overexpression increases costimulatory molecules and cytokines through reinvigoration of ERK or transcriptional activation,independent of anti-exhaustion. We conduct an open-label,non-randomized,single-arm,phase I trial of C-JUN-overexpressing CAR-T in AML ( NCT04835519 ) with safety and efficacy as primary and secondary endpoints,respectively. Of the four patients treated,one has grade 4 (dose-limiting toxicity) and three have grade 1–2 cytokine release syndrome. Two patients have no detectable bone marrow blasts and one patient has blast reduction after treatment. Thus,overexpressing C-JUN endows CAR-T efficacy in AML. Subject terms: Translational research,Leukaemia View Publication

HOX genes,notably members of the HOXA cluster,and HOX cofactors have increasingly been linked to human leukemia. Intriguingly,HOXD13,a member of the HOXD cluster not normally expressed in hematopoietic cells,was recently identified as a partner of NUP98 in a t(2;11) translocation associated with t-AML/MDS. We have now tested directly the leukemogenic potential of the NUP98-HOXD13 t(2; 11) fusion gene in the murine hematopoietic model. NUP98-HOXD13 strongly promoted growth and impaired differentiation of early hematopoietic progenitor cells in vitro; this effect was dependent on the NUP98 portion and an intact HOXD13 homeodomain. Expression of the NUP98-HOXD13 fusion gene in vivo resulted in a partial impairment of lymphopoiesis but did not induce evident hematologic disease until late after transplantation (more than 5 months),when some mice developed a myeloproliferative-like disease. In contrast,mice transplanted with bone marrow (BM) cells cotransduced with NUP98-HOXD13 and the HOX cofactor Meis1 rapidly developed lethal and transplantable acute myeloid leukemia (AML),with a median disease onset of 75 days. In summary,this study demonstrates that NUP98-HOXD13 can be directly implicated in the molecular process leading to leukemic transformation,and it supports a model in which the transforming properties of NUP98-HOXD13 are mediated through HOX-dependent pathways. View Publication

T cell dysfunction is crucial to the pathogenesis of systemic lupus erythematosus (SLE); however,the molecular mechanisms involved in the deficient expression of the T cell receptor-associated CD3zeta chain in SLE are not clear. SLE T cells express abnormally increased levels of an alternatively spliced isoform of CD3zeta that lacks a 562-bp region in its 3'-untranslated region (UTR). We showed previously that two adenosine/uridine-rich elements (ARE) in this splice-deleted region of CD3zeta transcript are critical for the mRNA stability and protein expression of CD3zeta. In this study we show for the first time that the mRNA-stabilizing protein HuR binds to these two ARE bearing regions of CD3zeta 3'-UTR. Knockdown of HuR resulted in decreased expression of the CD3zeta chain,whereas overexpression led to the increase of CD3zeta chain levels. Additionally,overexpression of HuR in human T cells resulted in increased mRNA stability of CD3zeta. Our results identify the 3'-UTR of CD3zeta as a novel target for the mRNA-stabilizing protein HuR. Thus,the absence of two critical AREs in the alternatively spliced CD3zeta 3'-UTR found in SLE T cells may result in decreased HuR binding,representing a possible molecular mechanism contributing to the reduced stability and expression of CD3zeta in SLE. View Publication

Efficient differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to a variety of lineages requires step-wise approaches replicating the key commitment stages found during embryonic development. Here we show that expression of PdgfR-α segregates mouse ESC-derived Flk-1 mesoderm into Flk-1(+)PdgfR-α(+) cardiac and Flk-1(+)PdgfR-α(-) hematopoietic subpopulations. By monitoring Flk-1 and PdgfR-α expression,we found that specification of cardiac mesoderm and cardiomyocytes is determined by remarkably small changes in levels of Activin/Nodal and BMP signaling. Translation to human ESCs and iPSCs revealed that the emergence of cardiac mesoderm could also be monitored by coexpression of KDR and PDGFR-α and that this process was similarly dependent on optimal levels of Activin/Nodal and BMP signaling. Importantly,we found that individual mouse and human pluripotent stem cell lines require optimization of these signaling pathways for efficient cardiac differentiation,illustrating a principle that may well apply in other contexts. View Publication

Cell Research 23,122 (2013). doi:10.1038/cr.2012.119 View Publication

Interleukin (IL)-23 is thought to be critical in the pathogenesis of psoriasis,and anti-IL-23 monoclonal antibodies (mAbs) have been approved for the treatment of psoriasis. We speculated that an anti-IL-23 receptor mAb might have greater efficacy than an anti-IL-23 mAb in the treatment of local inflamed lesions with high IL-23 levels. We previously generated an anti-human IL-23 receptor mAb,AS2762900-00,which potently blocked IL-23-induced cell proliferation,regardless of the concentration of IL-23. Here,we evaluated the therapeutic potential of AS2762900-00 in the treatment of psoriasis. Compared with untreated control,AS2762900-00 significantly reduced the epidermal thickness of lesions in a clinically relevant psoriatic human skin xenograft model. The expression of inflammatory genes including genes downstream of IL-23 signaling in the lesion tended to be lower in the AS2762900-00 group than the untreated group,suggesting that the inhibitory effects of AS2762900-00 in the psoriatic human skin xenograft model might occur via blockade of IL-23 signaling pathways. Further,AS2762900-00 showed an inhibitory effect on signal transducer and activator of transcription 3 (STAT3) phosphorylation as a downstream signal of IL-23 receptor activation in whole blood from patients with psoriasis. We also confirmed that AS2762900-00 inhibited IL-23-induced STAT3 phosphorylation in a concentration-dependent manner using whole blood from healthy donors. These data suggest that AS2762900-00 is a promising drug candidate for the treatment of psoriasis. In addition,STAT3 phosphorylation in whole blood may be a useful biomarker for the evaluation of the pharmacodynamic effects of AS2762900-00 in healthy volunteers in clinical development. View Publication

Development of targeted cancer therapy requires a thorough understanding of mechanisms of tumorigenesis as well as mechanisms of action of therapeutics. This is challenging because by the time patients are diagnosed with cancer,early events of tumorigenesis have already taken place. Similarly,development of cancer immunotherapies is hampered by a lack of appropriate small animal models with autologous human tumor and immune system. In this article,we report the development of a mouse model of human acute myeloid leukemia (AML) with autologous immune system for studying early events of human leukemogenesis and testing the efficacy of immunotherapeutics. To develop such a model,human hematopoietic stem/progenitor cells (HSPC) are transduced with lentiviruses expressing a mutated form of nucleophosmin (NPM1),referred to as NPM1c. Following engraftment into immunodeficient mice,transduced HSPCs give rise to human myeloid leukemia,whereas untransduced HSPCs give rise to human immune cells in the same mice. The de novo AML,with CD123+ leukemic stem or initiating cells (LSC),resembles NPM1c+ AML from patients. Transcriptional analysis of LSC and leukemic cells confirms similarity of the de novo leukemia generated in mice with patient leukemia and suggests Myc as a co-operating factor in NPM1c-driven leukemogenesis. We show that a bispecific conjugate that binds both CD3 and CD123 eliminates CD123+ LSCs in a T cell-dependent manner both in vivo and in vitro. These results demonstrate the utility of the NPM1c+ AML model with an autologous immune system for studying early events of human leukemogenesis and for evaluating efficacy and mechanism of immunotherapeutics. View Publication