技术资料

Human embryonic stem cells (hESCs) hold great value for future clinical applications. However,standard culture conditions maintain hESCs in a primed state,which bears heterogeneity in pluripotency and a tendency for spontaneous differentiation. To counter these drawbacks,primed hESCs have been converted to a naive state,but this has restricted the efficiency of existing directed differentiation protocols. In mouse,WNT inhibition by inhibitor of WNT production-2,together with a higher dose of fibroblast growth factor 2 (12 ng/mL) in DMEM/F12 basal medium (DhiFI),markedly improved derivation and maintenance of primed mouse epiblast stem cells. In this study,we show that DhiFI conditions similarly improved primed hESC traits,such as conferring a primed transcriptional signature with high levels of pluripotency markers and reduced levels of differentiation markers. When triggered to differentiate to neuronal and cardiac lineages,DhiFI hESCs and isogenic primed hESCs progressed similarly. Moreover,DhiFI conditions supported the derivation of hESC lines from a post-inner cell mass intermediate (PICMI). DhiFI-derived hESCs showed less spontaneous differentiation and expressed significantly lower levels of lineage-specific markers,compared to primed-derived lines from the same PICMI. Overall,DhiFI hESCs retained advantages of both primed and naive pluripotency and may ultimately represent a more favorable starting point for differentiation toward clinically desired cell types. View Publication

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here,we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1{\&}2),a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS,for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1{\&}2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1{\&}2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore,PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway,and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1{\&}2 reversed arthritis in mice,providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies. View Publication

NF-$\kappa$B is a key regulator of inflammation and cancer progression,with an important role in leukemogenesis. Despite its therapeutic potential,targeting NF-$\kappa$B using pharmacologic inhibitors has proven challenging. Here,we describe a myeloid cell-selective NF-$\kappa$B inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a,C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6),thereby blocking activation of NF-$\kappa$B in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-$\kappa$B activation in myeloid cells,and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly,C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions,miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set,we found an inverse correlation of miR-146a levels with NF-$\kappa$B-related genes and with patient survival. Correspondingly,C-miR146a induced cytotoxic effects in human MDSL,HL-60,and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-$\kappa$B target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders. View Publication

R-2-hydroxyglutarate (R-2HG),produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes,was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically,R-2HG inhibits fat mass and obesity-associated protein (FTO) activity,thereby increasing global N6-methyladenosine (m6A) RNA modification in R-2HG-sensitive leukemia cells,which in turn decreases the stability of MYC/CEBPA transcripts,leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG,whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively,while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation,our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m6A/MYC/CEBPA signaling. View Publication

Leukocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in host defense and immune regulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD),characterized by recurrent bacterial and fungal infections and aberrant inflammation. Key drivers of hyper-inflammation induced by fungal cell walls in CGD are still incompletely defined. Here,we found that CGD (CYBB-null) neutrophils produced higher amounts of leukotriene B4 (LTB4) in vitro following activation with zymosan or Immune complexes,as compared to wild type (WT) neutrophils. This correlated with increased calcium influx in CGD neutrophils,which is restrained in WT neutrophils by the electrogenic activity of the NADPH oxidase. Increased LTB4 generation by CGD neutrophils was also augmented by paracrine cross-talk with the LTB4 receptor BLT1. CGD neutrophils formed more numerous and larger clusters in the presence of zymosan in vitro compared to WT,which was also LTB4- and BLT1-dependent. In zymosan-induced lung inflammation,focal neutrophil infiltrates were increased in CGD compared to WT mice and associated with higher LTB4 levels. Inhibiting LTB4 synthesis or antagonizing the BLT1 receptor following zymosan challenge reduced lung neutrophil recruitment in CGD to WT levels. Thus,LTB4 was the major driver of excessive neutrophilic lung inflammation in CGD mice in the early response to fungal cell walls,likely by a dysregulated feed-forward loop involving amplified neutrophil production of LTB4. This study identifies neutrophil LTB4 generation as a target of NADPH oxidase regulation,which could potentially be exploited therapeutically to reduce excessive inflammation in CGD. View Publication

Immune surveillance against pathogens and tumours in the central nervous system is thought to be limited owing to the lack of lymphatic drainage. However,the characterization of the meningeal lymphatic network has shed light on previously unappreciated ways that an immune response can be elicited to antigens that are expressed in the brain1-3. Despite progress in our understanding of the development and structure of the meningeal lymphatic system,the contribution of this network in evoking a protective antigen-specific immune response in the brain remains unclear. Here,using a mouse model of glioblastoma,we show that the meningeal lymphatic vasculature can be manipulated to mount better immune responses against brain tumours. The immunity that is mediated by CD8 T cells to the glioblastoma antigen is very limited when the tumour is confined to the central nervous system,resulting in uncontrolled tumour growth. However,ectopic expression of vascular endothelial growth factor C (VEGF-C) promotes enhanced priming of CD8 T cells in the draining deep cervical lymph nodes,migration of CD8 T cells into the tumour,rapid clearance of the glioblastoma and a long-lasting antitumour memory response. Furthermore,transfection of an mRNA construct that expresses VEGF-C works synergistically with checkpoint blockade therapy to eradicate existing glioblastoma. These results reveal the capacity of VEGF-C to promote immune surveillance of tumours,and suggest a new therapeutic approach to treat brain tumours. 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

PURPOSE Glioblastoma (GBM) is a fatal primary malignant brain tumor. GBM stem cells (GSC) contribute to resistance to the DNA-damaging chemotherapy,temozolomide. The epidermal growth factor receptor (EGFR) displays genomic alterations enabling DNA repair mechanisms in half of GBMs. We aimed to investigate EGFR/DNA combi-targeting in GBM. EXPERIMENTAL DESIGN ZR2002 is a combi-molecule" designed to inflict DNA damage through its chlorethyl moiety and induce irreversible EGFR tyrosine kinase inhibition. We assessed its in vitro efficacy in temozolomide-resistant patient-derived GSCs mesenchymal temozolomide-sensitive and resistant in vivo-derived GSC sublines and U87/EGFR isogenic cell lines stably expressing EGFR/wild-type or variant III (EGFRvIII). We evaluated its antitumor activity in mice harboring orthotopic EGFRvIII or mesenchymal TMZ-resistant GSC tumors. RESULTS ZR2002 induced submicromolar antiproliferative effects and inhibited neurosphere formation of all GSCs with marginal effects on normal human astrocytes. ZR2002 inhibited EGF-induced autophosphorylation of EGFR downstream Erk1/2 phosphorylation increased DNA strand breaks and induced activation of wild-type p53; the latter was required for its cytotoxicity through p53-dependent mechanism. ZR2002 induced similar effects on U87/EGFR cell lines and its oral administration significantly increased survival in an orthotopic EGFRvIII mouse model. ZR2002 improved survival of mice harboring intracranial mesenchymal temozolomide-resistant GSC line decreased EGFR Erk1/2 and AKT phosphorylation and was detected in tumor brain tissue by MALDI imaging mass spectrometry. CONCLUSIONS These findings provide the molecular basis of binary EGFR/DNA targeting and uncover the oral bioavailability blood-brain barrier permeability and antitumor activity of ZR2002 supporting potential evaluation of this first-in-class drug in recurrent GBM." View Publication

Short-chain fatty acids (SCFAs) butyrate and propionate are metabolites from dietary fiber's fermentation by gut microbiota that can affect differentiation or functions of T cells,macrophages and dendritic cells. We show here that at low doses these SCFAs directly impact B cell intrinsic functions to moderately enhance class-switch DNA recombination (CSR),while decreasing at higher doses over a broad physiological range,AID and Blimp1 expression,CSR,somatic hypermutation and plasma cell differentiation. In human and mouse B cells,butyrate and propionate decrease B cell Aicda and Prdm1 by upregulating select miRNAs that target Aicda and Prdm1 mRNA-3'UTRs through inhibition of histone deacetylation (HDAC) of those miRNA host genes. By acting as HDAC inhibitors,not as energy substrates or through GPR-engagement signaling in these B cell-intrinsic processes,these SCFAs impair intestinal and systemic T-dependent and T-independent antibody responses. Their epigenetic impact on B cells extends to inhibition of autoantibody production and autoimmunity in mouse lupus models. View Publication

Microelectrode material and cell culture medium have significant roles in the signal-to-noise ratio and cell well-being in in vitro electrophysiological studies. Here,we report an ion beam assisted e-beam deposition (IBAD) based process as an alternative titanium nitride (TiN) deposition method for sputtering in the fabrication of state-of-the-art TiN microelectrode arrays (MEAs). The effects of evaporation and nitrogen flow rates were evaluated while developing the IBAD TiN deposition process. Moreover,the produced IBAD TiN microelectrodes were characterized by impedance,charge transfer capacity (CTC) and noise measurements for electrical properties,AFM and SEM for topological imaging,and EDS for material composition. The impedance (at 1 kHz) of brand new 30 $\mu$m IBAD TiN microelectrodes was found to be double but still below 100 k$\Omega$ compared with commercial reference MEAs with sputtered TiN microelectrodes of the same size. On the contrary,the noise level of IBAD TiN MEAs was lower compared with that of commercial sputtered TiN MEAs in equal conditions. In CTC IBAD TiN electrodes (3.3 mC/cm2) also outperformed the sputtered counterparts (2.0 mC/cm2). To verify the suitability of IBAD TiN microelectrodes for cell measurements,human pluripotent stem cell (hPSC)-derived neuronal networks were cultured on IBAD TiN MEAs and commercial sputtered TiN MEAs in two different media: neural differentiation medium (NDM) and BrainPhys (BPH). The effect of cell culture media to hPSC derived neuronal networks was evaluated to gain more stable and more active networks. Higher spontaneous activity levels were measured from the neuronal networks cultured in BPH compared with those in NDM in both MEA types. However,BPH caused more problems in cell survival in long-term cultures by inducing neuronal network retraction and clump formation after 1-2 weeks. In addition,BPH was found to corrode the Si3N4 insulator layer more than NDM medium. The developed IBAD TiN process gives MEA manufacturers more choices to choose which method to use to deposit TiN electrodes and the medium evaluation results remind that not only electrode material but also insulator layer and cell culturing medium have crucial role in successful long term MEA measurements. View Publication

The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts1. Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types2,3. However,whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis,we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E2 (PGE2). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic,autochthonous tumorigenesis. Mechanistically,single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE2 drives the expansion οf a population of Sca-1+ reserve-like stem cells. These express a strong regenerative/tumorigenic program,driven by the Hippo pathway effector Yap. In vivo,Yap is indispensable for Sca-1+ cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments,we identified a molecular mechanism whereby PGE2 promotes Yap dephosphorylation,nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1+ cell expansion and sporadic tumour initiation in mutant mice,thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE2-Ptger4. Analyses of patient-derived organoids established that PGE2-PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE2-Ptger4-Yap signalling axis. View Publication

CRISPR genome engineering has become a powerful tool to functionally investigate the complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity,the utility of current approaches is restricted by poor knockout efficiencies or limited specificity for mature cell lineages in vivo. Here,we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of mature primary mouse innate lymphocyte cells (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore,we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naive natural killer (NK) cells and bone-marrow-derived conventional dendritic cell precursors (cDCPs). This resource will enhance target gene discovery and offer a specific and simplified approach to gene editing in the mouse innate immune system. View Publication