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Inflammation is characterized by the recruitment of leukocytes from the bloodstream. The rapid arrival of neutrophils is followed by a wave of inflammatory lymphocyte antigen 6 complex (Ly6C)-positive monocytes. In contrast Ly6C(low) monocytes survey the endothelium in the steady state,but their role in inflammation is still unclear. Here,using confocal intravital microscopy,we show that upon Toll-like receptor 7/8 (TLR7/8)-mediated inflammation of mesenteric veins,platelet activation drives the rapid mobilization of Ly6C(low) monocytes to the luminal side of the endothelium. After repeatedly interacting with platelets,Ly6C(low) monocytes commit to a meticulous patrolling of the endothelial wall and orchestrate the subsequent arrival and extravasation of neutrophils through the production of proinflammatory cytokines and chemokines. At a molecular level,we show that cysteine-rich protein 61 (CYR61)/CYR61 connective tissue growth factor nephroblastoma overexpressed 1 (CCN1) protein is released by activated platelets and enables the recruitment of Ly6C(low) monocytes upon vascular inflammation. In addition endothelium-bound CCN1 sustains the adequate patrolling of Ly6C(low) monocytes both in the steady state and under inflammatory conditions. Blocking CCN1 or platelets with specific antibodies impaired the early arrival of Ly6C(low) monocytes and abolished the recruitment of neutrophils. These results refine the leukocyte recruitment cascade model by introducing endothelium-bound CCN1 as an inflammation mediator and by demonstrating a role for platelets and patrolling Ly6C(low) monocytes in acute vascular inflammation. View Publication

Enterotoxigenic Escherichia coli (ETEC) causes 20% of the acute infectious diarrhea (AID) episodes worldwide,often by producing heat-stable enterotoxins (STs),which are peptides structurally homologous to paracrine hormones of the intestinal guanylate cyclase C (GUCY2C) receptor. While molecular mechanisms mediating ST-induced intestinal secretion have been defined,advancements in therapeutics have been hampered for decades by the paucity of disease models that integrate molecular and functional endpoints amenable to high-throughput screening. Here,we reveal that mouse and human intestinal enteroids in three-dimensional ex vivo cultures express the components of the GUCY2C secretory signaling axis. ST and its structural analog,linaclotide,an FDA-approved oral secretagog,induced fluid accumulation quantified simultaneously in scores of enteroid lumens,recapitulating ETEC-induced intestinal secretion. Enteroid secretion depended on canonical molecular signaling events responsible for ETEC-induced diarrhea,including cyclic GMP (cGMP) produced by GUCY2C,activation of cGMP-dependent protein kinase (PKG),and opening of the cystic fibrosis transmembrane conductance regulator (CFTR). Importantly,pharmacological inhibition of CFTR abrogated enteroid fluid secretion,providing proof of concept for the utility of this model to screen antidiarrheal agents. Intestinal enteroids offer a unique model,integrating the GUCY2C signaling axis and luminal fluid secretion,to explore the pathophysiology of,and develop platforms for,high-throughput drug screening to identify novel compounds to prevent and treat ETEC diarrheal disease. View Publication

When SUCNR1/GPR91-expressing macrophages are activated by inflammatory signals,they change their metabolism and accumulate succinate. In this study,we show that during this activation,macrophages release succinate into the extracellular milieu. They simultaneously up-regulate GPR91,which functions as an autocrine and paracrine sensor for extracellular succinate to enhance IL-1β production. GPR91-deficient mice lack this metabolic sensor and show reduced macrophage activation and production of IL-1β during antigen-induced arthritis. Succinate is abundant in synovial fluids from rheumatoid arthritis (RA) patients,and these fluids elicit IL-1β release from macrophages in a GPR91-dependent manner. Together,we reveal a GPR91/succinate-dependent feed-forward loop of macrophage activation and propose GPR91 antagonists as novel therapeutic principles to treat RA. View Publication

Recent studies have suggested that human pluripotent stem cells (hPSCs) depend primarily on glycolysis and only increase oxidative metabolism during differentiation. Here,we demonstrate that both glycolytic and oxidative metabolism can support hPSC growth and that the metabolic phenotype of hPSCs is largely driven by nutrient availability. We comprehensively characterized hPSC metabolism by using 13C/2H stable isotope tracing and flux analysis to define the metabolic pathways supporting hPSC bioenergetics and biosynthesis. Although glycolytic flux consistently supported hPSC growth,chemically defined media strongly influenced the state of mitochondrial respiration and fatty acid metabolism. Lipid deficiency dramatically reprogramed pathways associated with fatty acid biosynthesis and NADPH regeneration,altering the mitochondrial function of cells and driving flux through the oxidative pentose phosphate pathway. Lipid supplementation mitigates this metabolic reprogramming and increases oxidative metabolism. These results demonstrate that self-renewing hPSCs can present distinct metabolic states and highlight the importance of medium nutrients on mitochondrial function and development. Zhang et al. apply metabolic flux analysis to comprehensively characterize the metabolism of human pluripotent stem cells cultured in different media. Cells maintained in chemically defined media significantly upregulate lipid biosynthesis and redox pathways to compensate for medium lipid deficiency while downregulating oxidative mitochondrial metabolism. View Publication

Pluripotent stem cells (PSCs) can self-renew or differentiate from naive or more differentiated,primed,pluripotent states established by specific culture conditions. Increased intracellular α-ketoglutarate (αKG) was shown to favor self-renewal in naive mouse embryonic stem cells (mESCs). The effect of αKG or αKG/succinate levels on differentiation from primed human PSCs (hPSCs) or mouse epiblast stem cells (EpiSCs) remains unknown. We examined primed hPSCs and EpiSCs and show that increased αKG or αKG-to-succinate ratios accelerate,and elevated succinate levels delay,primed PSC differentiation. αKG has been shown to inhibit the mitochondrial ATP synthase and to regulate epigenome-modifying dioxygenase enzymes. Mitochondrial uncoupling did not impede αKG-accelerated primed PSC differentiation. Instead,αKG induced,and succinate impaired,global histone and DNA demethylation in primed PSCs. The data support αKG promotion of self-renewal or differentiation depending on the pluripotent state. View Publication

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by lack of CD55 and CD59 on blood cell membrane leading to increased sensitivity of blood cells to complement. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for PNH,however,lack of HLA-matched donors and post-transplant complications are major concerns. Induced pluripotent stem cells (iPSCs) derived from patients are an attractive source for generating autologous HSCs to avoid adverse effects resulting from allogeneic HSCT. The disease involves only HSCs and their progeny; therefore,other tissues are not affected by the mutation and may be used to produce disease-free autologous HSCs. This study aimed to derive PNH patient-specific iPSCs from human dermal fibroblasts (HDFs),characterize and differentiate to hematopoietic cells using a feeder-free protocol. Analysis of CD55 and CD59 expression was performed before and after reprogramming,and hematopoietic differentiation. Patients' dermal fibroblasts expressed CD55 and CD59 at normal levels and the normal expression remained after reprogramming. The iPSCs derived from PNH patients had typical pluripotent properties and differentiation capacities with normal karyotype. After hematopoietic differentiation,the differentiated cells expressed early hematopoietic markers (CD34 and CD43) with normal CD59 expression. The iPSCs derived from HDFs of PNH patients have normal levels of CD55 and CD59 expression and hold promise as a potential source of HSCs for autologous transplantation to cure PNH patients. View Publication

Human monoclonal antibodies against RSV have high potential for use as prophylaxis or therapeutic molecules,and they also can be used to define the structure of protective epitopes for rational vaccine design. In the past,however,isolation of human monoclonal antibodies was difficult and inefficient. Here,we describe contemporary methods for activation and proliferation of primary human memory B cells followed by cytofusion to non-secreting myeloma cells by dielectrophoresis to generate human hybridomas secreting RSV-specific monoclonal antibodies. We also provide experimental methods for screening human B cell lines to obtain RSV-specific lines,especially lines secreting neutralizing antibodies. View Publication

CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical,iPSC generation must be rapid and efficient. Therefore,we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector,rCLAE-R6,that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently,the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% ($$(A)/[$$(S)+$$(A)]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected,patient-specific iPSCs for therapeutic applications. View Publication

The epigenetic background of pluripotent stem cells can influence transcriptional and functional behavior. Most of these data have been obtained in standard monolayer cell culture systems. In this study,we used exome sequencing,array comparative genomic hybridization (CGH),miRNA array,DNA methylation array,three-dimensional (3D) tissue engineering,and immunostaining to conduct a comparative analysis of two induced pluripotent stem cell (iPSC) lines used in engineering of 3D human epidermal equivalent (HEE),which more closely approximates epidermis. Exome sequencing and array CGH suggested that their genome was stable following 3 months of feeder-free culture. While the miRNAome was also not affected,≈7% of CpG sites were differently methylated between the two lines. Analysis of the epidermal differentiation complex,a region on chromosome 1 that contains multiple genes involved in skin barrier maturation (including trichohyalin,TCHH),found that in one of the iPSC clones (iKCL004),TCHH retained a DNA methylation signature characteristic of the original somatic cells,whereas in other iPSC line (iKCL011),the TCHH methylation signature matched that of the human embryonic stem cell line KCL034. The difference between the two iPSC clones in TCHH methylation did not have an obvious effect on its expression in 3D HEE,suggesting that differentiation and tissue formation may mitigate variations in the iPSC methylome. View Publication

OBJECTIVE Gastric cancer (GC) is one of the most common human cancers. A useful method of gastric cancer stem cell (CSC) characterization is spheroid colony formation. Previously,we reported that KIF11 expression is textgreater2-fold in spheroid-body-forming GC cells compared with parental cells. Here,we analyzed the expression and distribution of KIF11 in human GC by immunohistochemistry. METHODS Expression of KIF11 in 165 GC cases was determined using immunohistochemistry. For mucin phenotypic expression analysis of GC,immunostaining of MUC5AC,MUC6,MUC2 and CD10 was evaluated. RNA interference was used to inhibit KIF11 expression in GC cell lines. RESULTS In total,119 of 165 GC cases (72%) were positive for KIF11. Expression of KIF11 was not associated with any clinicopathologic characteristics; however,it was observed frequently in GC exhibiting an intestinal phenotype. Both the number and size of spheres formed by MKN-74 cells were significantly reduced following transfection of KIF11-targeting siRNA compared with negative-control siRNA. Furthermore,levels of phosphorylated Erk1/2 were lower in KIF11 siRNA-transfected cells than with negative-control siRNA-transfected cells. CONCLUSION These results indicate that KIF11 is involved in intestinal mucin phenotype GC. View Publication

Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A,the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing,which were rescued by a Nav1.1 transgene,whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons. View Publication

SHIP is an important regulator of immune cell signaling that functions to dephosphorylate the phosphoinositide phosphatidylinositol 3,4,5-trisphosphate at the plasma membrane and mediate protein-protein interactions. One established paradigm for SHIP activation involves its recruitment to the phospho-ITIM motif of the inhibitory receptor FcγRIIB. Although SHIP is essential for the inhibitory function of FcγRIIB,it also has critical modulating functions in signaling initiated from activating immunoreceptors such as B cell Ag receptor. In this study,we found that SHIP is indistinguishably recruited to the plasma membrane after BCR stimulation with or without FcγRIIB coligation in human cell lines and primary cells. Interestingly,fluorescence recovery after photobleaching analysis reveals differential mobility of SHIP-enhanced GFP depending on the mode of stimulation,suggesting that although BCR and FcγRIIB can both recruit SHIP,this occurs via distinct molecular complexes. Mutagenesis of a SHIP-enhanced GFP fusion protein reveals that the SHIP-Src homology 2 domain is essential in both cases whereas the C terminus is required for recruitment via BCR stimulation,but is less important with FcγRIIB coligation. Experiments with pharmacological inhibitors reveal that Syk activity is required for optimal stimulation-induced membrane localization of SHIP,whereas neither PI3K or Src kinase activity is essential. BCR-induced association of SHIP with binding partner Shc1 is dependent on Syk,as is tyrosine phosphorylation of both partners. Our results indicate that FcγRIIB is not uniquely able to promote membrane recruitment of SHIP,but rather modulates its function via formation of distinct signaling complexes. Membrane recruitment of SHIP via Syk-dependent mechanisms may be an important factor modulating immunoreceptor signaling. View Publication