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

mRNA applications have undergone unprecedented applications—from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production. NK cells can be engineered with either viral vectors or electroporation,involving high costs,risks,and toxicity,emphasizing the need for alternative way as mRNA technology. We successfully developed,screened,and optimized novel lipid-based platforms based on imidazole lipids. Formulations are produced by microfluidic mixing and exhibit a size of approximately 100 nm with a polydispersity index of less than 0.2. They are able to transfect NK-92 cells,KHYG-1 cells,and primary NK cells with high efficiency without cytotoxicity,while Lipofectamine Messenger Max and D-Lin-MC3 lipid nanoparticle-based formulations do not. Moreover,the translation of non-modified mRNA was higher and more stable in time compared with a modified one. Remarkably,the delivery of therapeutically relevant interleukin 2 mRNA resulted in extended viability together with preserved activation markers and cytotoxic ability of both NK cell lines and primary NK cells. Altogether,our platforms feature all prerequisites needed for the successful deployment of NK-based therapeutic strategies. Graphical abstract Pichon and colleagues developed imidazole lipids-based mRNA platforms very efficient to transfect both NK-92 cells,KHYG-1 cells and primary NK cells without cytotoxicity. They succeeded to replace IL-2 protein by IL-2 mRNA transfection and obtained NK cells with extended viability with preserved biomarkers and full functionalities to kill target cells. View Publication

Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these embryonically derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic endothelial-macrophage (EndoMac) progenitor cells in the adventitia of embryonic and postnatal mouse aorta,that are independent of Flt3-mediated bone marrow hematopoiesis and derive from an early embryonic CX 3 CR1 + and CSF1R + source. These bipotent progenitors are proliferative and vasculogenic,contributing to adventitial neovascularization and formation of perfused blood vessels after transfer into ischemic tissue. We establish a regulatory role for angiotensin II,which enhances their clonogenic and differentiation properties and rapidly stimulates their proliferative expansion in vivo. Our findings demonstrate that embryonically derived EndoMac progenitors participate in local vasculogenic responses in the aortic wall by contributing to the expansion of endothelial cells and macrophages postnatally. Subject terms: Angiogenesis,Myelopoiesis,Haematopoietic stem cells View Publication

Understanding metabolic pathways that regulate Th17 development is important to broaden therapeutic options for Th17-mediated autoimmunity. Here,we report a pivotal role of mitochondrial oxidative phosphorylation (OXPHOS) for lineage specification toward pathogenic Th17 differentiation. Th17 cells rapidly increase mitochondrial respiration during development,and this is necessary for metabolic reprogramming following T cell activation. Surprisingly,specific inhibition of mitochondrial ATP synthase ablates Th17 pathogenicity in a mouse model of autoimmunity by preventing Th17 pathogenic signature gene expression. Notably,cells activated under OXPHOS-inhibited Th17 conditions preferentially express Foxp3,rather than Th17 genes,and become suppressive Treg cells. Mechanistically,OXPHOS promotes the Th17 pioneer transcription factor,BATF,and facilitates T cell receptor (TCR) and mTOR signaling. Correspondingly,overexpression of BATF rescues Th17 development when ATP synthase activity is restricted. Together,our data reveal a regulatory role of mitochondrial OXPHOS in dictating the fate decision between Th17 and Treg cells by supporting early molecular events necessary for Th17 commitment. View Publication

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in COL7A1. Patient-derived induced pluripotent stem cells (iPSCs) enable the personalized study of RDEB pathogenesis and potential therapies. However,current skin cell differentiation protocols via 2D culture perform suboptimally when applied to engineered 3D skin constructs (ESC). Here,we present an approach to source fibroblasts (iFBs) and keratinocytes (iKCs) from iPSC-derived skin organoids using an optimized differentiation protocol,and utilize them to engineer ESCs modeling wild-type and RDEB phenotypes. The resulting iPSC-derived skin cells display marker expression consistent with primary counterparts and produce ESCs exhibiting significant extracellular matrix remodeling,protein deposition,and epidermal differentiation. RDEB constructs recapitulated hallmark disease features,including absence of collagen VII and reduced iFB proliferation. This work establishes a robust and scalable strategy for generating physiologically-relevant,iPSC-derived skin constructs,offering a powerful model for studying RDEB mechanisms and advancing personalized regenerative medicine. View Publication

Antiserum to epithelial membrane antigen and three monoclonal antibodies (MAb) to milk-fat globule membranes immunocytochemically stain only epithelial cells,whereas a fourth reacts also with myoepithelial cells in inter- and intralobular ducts of human breast. Staining with peanut lectin shows a gradual increase for epithelial cells,from little or no staining in ducts through variable staining in ductules to intense staining in secretory alveoli. Antisera and MAb to vimentin,smooth-muscle actin,MAb to the common acute lymphoblastic leukemia antigen and to a glycoprotein of 135 KD stain myoepithelial cells in main ducts,but this staining is reduced in inter- and intralobular ducts and ductules. MAb to epithelial-specific keratin 18 stain a minor population of ductal epithelial cells,the major population of epithelial cells in interlobular (ILD) and extralobular terminal ducts (ETD),and epithelial cells in a minority of ductules. In lactating glands most epithelial cells in ductules are stained,but the alveolar and myoepithelial cells are unstained. Keratin MAb PKK2 and LP34 strongly stain myoepithelial cells,but only a minor population of epithelial cells in main ducts. However,these MAb stain principally the epithelial cells in ILD,ETD,and a minority of ductules. In lactating glands most epithelial cells are stained in ductules,but the myoepithelial and not the alveolar cells are stained intensely in secretory lobules. It is suggested that the unusual staining pattern of cells found principally in the ILD,ETD,and some ductules may represent regions of growth and/or subpopulation(s) of cells intermediate between epithelial and myoepithelial cells. View Publication

Human iPSC line L749.1 was generated from fibroblasts of a patient with Leigh syndrome associated with a heteroplasmic mutation in the MT-ATP6 gene. Reprogramming factors OCT4,SOX2,CMYC and KLF4 were delivered using retroviruses. View Publication

1. The effects of the non-selective phosphodiesterase (PDE) inhibitor theophylline and the selective PDE4 inhibitor rolipram on leukotriene C4 (LTC4) synthesis and chemotaxis of complement 5a (C5a)- and platelet-activating factor (PAF)-stimulated human eosinophils obtained from normal and atopic donors were investigated. 2. Eosinophils were purified from peripheral venous blood of normal and atopic subjects by an immunomagnetic procedure to a purity textgreater 99%. Eosinophils were stimulated with PAF (0.1 microM) or C5a 0.1 microM for 15 min and LTC4 was measured by radioimmunoassay (RIA). Eosinophil chemotaxis in response to PAF and C5a was assessed with 48-well microchambers (Boyden). 3. Under these conditions substantial amounts of LTC4 (about 300-1000 pg per 10(6) cells) were only detectable in the presence of indomethacin (0.1-10 microM). To explain this finding it was hypothesized that indomethacin reversed the inhibition of LTC4 synthesis by endogenously synthesized prostaglandins,in particular prostaglandin E2 (PGE2). In fact,eosinophils release 23 pg PGE2 per 10(6) cells following PAF stimulation; this PGE2 synthesis was completely inhibited by indomethacin and readdition of PGE2 inhibited eosinophil LTC4 synthesis (IC50 = 3 nM). The following experiments were performed in the presence of 10 microM indomethacin. 4. Theophylline (IC50 approximately 50 microM) and rolipram (IC50 approximately 0.03-0.2 microM) suppressed PAF- and C5a-stimulated LTC4 synthesis. This PDE inhibitor-induced suppression of LTC4 generation is mediated by activation of protein kinase A,since it was reversed by the protein kinase A inhibitor Rp-8-Br-cyclic AMPS. In addition,exogenous arachidonic acid concentration-dependently (0.3 microM-3 microM) reversed the inhibition of LTC4 synthesis by the PDE inhibitors,indicating that theophylline and rolipram suppress the mobilization of arachidonic acid. The beta 2-adrenoceptor agonist salbutamol inhibited eosinophil LTC4 synthesis (IC50 = 0.08 microM). The combination of salbutamol with theophylline (10 microM) or rolipram (3 nM) appeared to be additive. 5. Theophylline (IC50 approximately 40 microM),rolipram (IC50 approximately 0.02 microM [C5a],approximately 0.6 microM [PAF]) and PGE2 (IC50 approximately 3 nM) inhibited C5a- and PAF-stimulated eosinophil chemotaxis. The combination of PGE2 with theophylline resulted in an additive effect. 6. Both C5a- and PAF-stimulated eosinophil chemotaxis and LTC4 generation were significantly elevated in eosinophils from atopic individuals compared to normal subjects. However,eosinophils from normal and atopic individuals were not different with respect to their total cyclic AMP-PDE and PDE4 isoenzyme activities as well as the potencies of theophylline and rolipram to suppress LTC4 generation and chemotaxis. View Publication

Cancer can be treated by adoptive cell transfer (ACT) of T lymphocytes. However,how to optimally raise human T cells to a differentiation state allowing the best persistence in ACT is a challenge. It is possible to differentiate mouse CD8(+) T cells towards stem cell-like memory (T(SCM)) phenotype upon TCR stimulation with Wnt/ß-catenin pathway activation. Here,we evaluated if T(SCM) can be obtained from human mature CD8(+) T cells following TCR and Wnt/ß-catenin activation through treatment with the chemical agent 4,6-disubstituted pyrrolopyrimidine (TWS119),which inhibits the glycogen synthase kinase-3β (GSK-3β),key inhibitor of the Wnt pathway. Human CD8(+) T cells isolated from peripheral blood or tumor-infiltrating lymphocytes (TIL),and treated with TWS119 gave rise to CD62L(+)CD45RA(+) cells,indicative of early differentiated stage,also expressing CD127 which is normally found on memory cells,and CD133,an hematopoietic stem cell marker. T(SCM) cells raised from either TIL or blood secreted numerous inflammatory mediators,but in lower amounts than those measured without TWS119. Finally,generated T(SCM) CD8(+) T cells expressed elevated Bcl-2 and no detectable caspase-3 activity,suggesting increased persistence. Our data support a role for Wnt/ß-catenin pathway in promoting the T(SCM) subset in human CD8(+) T cells from TIL and the periphery,which are relevant for ACT. View Publication

BackgroundAdaptor proteins associated with the T cell receptor (TCR) play critical roles in regulating immune responses by Translating receptor engagement into intracellular signals. T cell Receptor Associated Transmembrane Adaptor 1 (TRAT1) has been implicated in modulating TCR complex stability,but its functional role in human effector and regulatory CD4⁺ T cell subsets remains poorly understood. This study aimed to elucidate the role of TRAT1 in regulating T cell activation and differentiation,particularly in helper T cells function and regulatory T cells.MethodsPrimary human CD4⁺ T cells,including thymus-derived and induced regulatory T cells (Treg),were genetically modified by CRISPR/Cas9-mediated gene deletion or retro-/lentiviral overexpression of TRAT1. Functional assays,flow cytometry,cytokine quantification,and RNA sequencing were performed to evaluate modulation of T cell functions. Mechanistic studies included pathway inhibition using small molecules and phospho-protein analysis. The influence of TRAT1 on Treg function was further assessed in a CAR Treg context in an immune organoid model of allo-rejection.ResultsThymus-derived,TGFb-induced and FOXP3-transgenic Treg displayed reduced expression of TRAT1 compared to effector T cells,which showed pronounced up-regulation of TRAT1 following activation. In effector T cells,deletion of TRAT1 led to increased signaling through the phosphoinositide 3-kinase pathway resulting in enhanced proliferation and increased expression of activation markers. However,this was accompanied by reduced production of interleukin-17,which was linked to elevated activity of STAT6 as shown by inhibition experiments using small molecule inhibitors. Overexpression and CRISPR/Cas9-mediated knockout of TRAT1 in Treg enhanced suppression of CD4⁺ target cells via up-regulation of LAP/GARP but reduced suppression of CD8⁺ target cells,an effect confirmed in HLA-A2-specific CAR Treg in a human organoid model of allo-rejection.ConclusionsTRAT1 acts as a dual regulator of human CD4⁺ T cell function,limiting effector activation through modulation of intracellular signaling and supporting regulatory T cell-mediated suppression. These findings reveal a novel mechanism of immune regulation with potential implications for the development of cell-based immunotherapies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-025-02429-z. View Publication

Imatinib mesylate (IM) induces clinical remissions in chronic-phase chronic myeloid leukemia (CML) patients but IM resistance remains a problem. We recently identified several features of CML CD34(+) stem/progenitor cells expected to confer resistance to BCR-ABL-targeted therapeutics. From a study of 25 initially chronic-phase patients,we now demonstrate that some,but not all,of these parameters correlate with subsequent clinical response to IM therapy. CD34(+) cells from the 14 IM nonresponders demonstrated greater resistance to IM than the 11 IM responders in colony-forming cell assays in vitro (P textless .001) and direct sequencing of cloned transcripts from CD34(+) cells further revealed a higher incidence of BCR-ABL kinase domain mutations in the IM nonresponders (10%-40% vs 0%-20% in IM responders,P textless .003). In contrast,CD34(+) cells from IM nonresponders and IM responders were not distinguished by differences in BCR-ABL or transporter gene expression. Interestingly,one BCR-ABL mutation (V304D),predicted to destabilize the interaction between p210(BCR-ABL) and IM,was detectable in 14 of 20 patients. T315I mutant CD34(+) cells found before IM treatment in 2 of 20 patients examined were preferentially amplified after IM treatment. Thus,2 properties of pretreatment CML stem/progenitor cells correlate with subsequent response to IM therapy. Prospective assessment of these properties may allow improved patient management. View Publication

BACKGROUND: Although methods for generating cardiomyocytes from pluripotent stem cells have been reported,current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here,we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs.backslashnbackslashnMETHOD AND RESULTS: Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs,an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes,a mouse cardiomyocyte cell line,but textless3% of 4 noncardiomyocyte cell types in flow cytometry analysis,which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes,which supports the specificity of MBs. Finally,MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures,and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics,which was verified by spontaneous beating,electrophysiological studies,and expression of cardiac proteins. When transplanted in a myocardial infarction model,MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors.backslashnbackslashnCONCLUSIONS: We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery. View Publication