Scientific Resources

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

Platelet (PLT) transfusions are potentially life saving for individuals with low PLT numbers; however,previous work revealed that PLT transfusions are associated with increased infection risk. During storage,PLT intended for transfusion continuously shed ectosomes (Ecto) from their surface,which express immunomodulatory molecules like phosphatidylserine or TGF-β1. Recently,PLT-Ecto were shown to reduce proinflammatory cytokine release by macrophages and to favor the differentiation of naive T cells toward regulatory T cells. Whether PLT-Ecto modify NK cells remains unclear. We exposed purified NK cells and full PBMCs from healthy donors to PLT-Ecto. We found a reduced expression of several activating surface receptors (NKG2D,NKp30,and DNAM-1) and decreased NK cell function,as measured by CD107a expression and IFN-γ production. Pretreatment of PLT-Ecto with anti-TGF-β1 neutralizing Ab restored surface receptor expression and NK cell function. We further observed a TGF-β1-mediated upregulation of miR-183,which,in turn,reduced DAP12,an important protein for stabilization and downstream signaling of several activating NK cell receptors. Again,these effects could antagonized,in part,when PLT-Ecto were preincubated with anti-TGF-β1 Ab. Erythrocyte Ecto did not affect NK cells. Polymorphonuclear cell Ecto expressed MHC class I and inhibited NK cell function. In addition,they induced the secretion of TGF-β1 by NK cells,which participated in an auto/paracrine manner in the suppressive activity of polymorphonuclear cell-derived Ecto. In sum,our study showed that PLT-Ecto could inhibit NK cell effector function in a TGF-β1-dependent manner,suggesting that recipients of PLT transfusions may experience reduced NK cell function. View Publication

Binding of ICAM-1 (intercellular adhesion molecule-1) to the β2-integrin LFA-1 (leukocyte function associated antigen-1) is known to induce crosstalk to the α4β1 integrin. Using different LFA-1 monoclonal antibodies we have been able to study the requirement and mechanism of action for the crosstalk in considerable detail. LFA-1 activating antibodies and those inhibitory antibodies that signal to α4β1 induce phosphorylation of Thr-758 on the β2-chain,which is followed by binding of 14-3-3 proteins and signaling through the G protein exchange factor Tiam1. This results in dephosphorylation of Thr-788/789 on the β1-chain of α4β1 and loss of binding to its ligand VCAM-1 (vascular cell adhesion molecule-1). The results show that with LFA-1 antibodies,we can either 1) activate LFA-1 and inhibit α4β1,2) inhibit both LFA-1 and α4β1,3) inhibit LFA-1 but not α4β1 or 4) not affect LFA-1 or α4β1 These findings are important for the understanding of integrin regulation and for the interpretation of the effect of integrin antibodies and their use in clinical applications. View Publication

Functional variability among human clones of induced pluripotent stem cells (hiPSCs) remains a limitation in assembling high-quality biorepositories. Beyond inter-person variability,the root cause of intra-person variability remains unknown. Mitochondria guide the required transition from oxidative to glycolytic metabolism in nuclear reprogramming. Moreover,mitochondria have their own genome (mitochondrial DNA [mtDNA]). Herein,we performed mtDNA next-generation sequencing (NGS) on 84 hiPSC clones derived from a cohort of 19 individuals,including mitochondrial and non-mitochondrial patients. The analysis of mtDNA variants showed that low levels of potentially pathogenic mutations in the original fibroblasts are revealed through nuclear reprogramming,generating mutant hiPSCs with a detrimental effect in their differentiated progeny. Specifically,hiPSC-derived cardiomyocytes with expanded mtDNA mutations non-related with any described human disease,showed impaired mitochondrial respiration,being a potential cause of intra-person hiPSC variability. We propose mtDNA NGS as a new selection criterion to ensure hiPSC quality for drug discovery and regenerative medicine. View Publication

Aberrant antioxidant activity and excessive deposition of extracellular matrix (ECM) are hallmarks of interstitial lung diseases (ILD). It is known that oxidative stress alters the ECM,but extracellular antioxidant defence mechanisms in ILD are incompletely understood. Here,we extracted abundance and detergent solubility of extracellular antioxidant enzymes from a proteomic dataset of bleomycin-induced lung fibrosis in mice and assessed regulation and distribution of glutathione peroxidase 3 (GPX3) in murine and human lung fibrosis. Superoxide dismutase 3 (Sod3),Gpx3,and Gpx activity were increased in mouse BALF during bleomycin-induced lung fibrosis. In lung tissue homogenates,Gpx3,but not Sod3,was upregulated and detergent solubility profiling indicated that Gpx3 associated with ECM proteins. Immunofluorescence analysis showed that Gpx3 was expressed by bronchial epithelial cells and interstitial fibroblasts and localized to the basement membrane and interstitial ECM in lung tissue. As to human ILD samples,BALF of some patients contained high levels of GPX3,and GPX3 was upregulated in lung homogenates from IPF patients. GPX3 expression in primary human bronchial epithelial cells and lung fibroblasts was downregulated by TNF-$$,but more variably regulated by TGF-$$1 and menadione. In conclusion,the antioxidant enzyme GPX3 localizes to lung ECM and is variably upregulated in ILD. View Publication

The vertebrate retina is a highly multilayered nervous tissue with a large diversity of cellular components. With the development of stem cell technologies,human retinas can be generated in three-dimensional (3-D) culture in vitro. However,understanding the factors modulating key productive processes and the way that they influence development are far from clear. Oxygen,as the most essential element participating in metabolism,is a critical factor regulating organic development. In this study,using 3-D culture of human stem cells,we examined the effect of intermittent high oxygen treatment (40% O2) on the formation and cellular behavior of neural retinas (NR) in the embryonic body (EB). The volume of EB and number of proliferating cells increased significantly under 40% O2 on day 38,50,and 62. Additionally,the ratio of PAX6+ cells within NR was significantly increased. The neural rosettes could only develop with correct apical-basal polarity under 40% O2. In addition,the generation,migration and maturation of retinal ganglion cells were enhanced under 40% O2. All of these results illustrated that 40% O2 strengthened the formation of NR in EB with characteristics similar to the in vivo state,suggesting that the hyperoxic state facilitated the retinal development in vitro. View Publication

While distinct stages of natural killer (NK) cell development have been defined,the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which,through contact-dependent mechanisms,promote the generation of mature,functional human NK cells from CD34(+) precursors. We show that developing NK cells undergo unique,developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells,and that the relative motility of NK cells increases as they move through development in vitro and ex vivo. These interactions include the formation of a synapse between developing NK cells and stromal cells,which we term the developmental synapse. Finally,we identify a role for CD56 in developmental synapse structure,NK cell motility and NK cell development. Thus,we define the developmental synapse leading to human NK cell functional maturation. View Publication

B cell aggregates in the central nervous system (CNS) have been associated with rapid disease progression in patients with multiple sclerosis (MS). Here we demonstrate a key role of carcinoembryogenic antigen-related cell adhesion molecule1 (CEACAM1) in B cell aggregate formation in MS patients and a B cell-dependent mouse model of MS. CEACAM1 expression was increased on peripheral blood B cells and CEACAM1(+) B cells were present in brain infiltrates of MS patients. Administration of the anti-CEACAM1 antibody T84.1 was efficient in blocking aggregation of B cells derived from MS patients. Along these lines,application of the monoclonal anti-CEACAM1 antibody mCC1 was able to inhibit CNS B cell aggregate formation and significantly attenuated established MS-like disease in mice in the absence of any adverse effects. CEACAM1 was co-expressed with the regulator molecule T cell immunoglobulin and mucin domain -3 (TIM-3) on B cells,a novel molecule that has recently been described to induce anergy in T cells. Interestingly,elevated coexpression on B cells coincided with an autoreactive T helper cell phenotype in MS patients. Overall,these data identify CEACAM1 as a clinically highly interesting target in MS pathogenesis and open new therapeutic avenues for the treatment of the disease. View Publication

Insights into the expression of pacemaker-speci�?c markers in human induced pluripotent stemcell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentia-tion and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date,no study hasdirectly assessed gene expression in each pacemaker-,atria-,and ventricular-like cardiomyocytesubtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytescan only be identi�?ed by action potential pro�?les. Traditional acquisition of action potentialsusing patch-clamp recordings renders the cells unviable for subsequent analysis. We circum-vented these issues by acquiring the action potential pro�?le of a single cell optically followedby assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the �?rst time revealed expression of proposed pacemaker-speci�?cmarkers—hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet(Isl)1—at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expressionwas found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- andventricular-like subtypes but its downregulation over time in all subtypes diminished the differ-ences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statisti-cally different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentiallyexpressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes,thesemarkers alone are insuf�?cient in identifying hiPSC-derived pacemaker-like cardiomyocytes. View Publication

In order to develop a novel method of visualizing possible Ca(2+) signaling during the early differentiation of hESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters,we expressed the bioluminescent Ca(2+) reporter,apo-aequorin,in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca(2+) transients (generated by release from intracellular stores) were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or CaCl2,indicating that holo-f-aequorin was functional in these cells. Furthermore,following the addition of exogenous ATP,an inositol trisphosphate receptor (IP3R) agonist,small Ca(2+) transients were generated from day 1 onward. That ATP was inducing Ca(2+) release from functional IP3Rs was demonstrated by treatment with 2-APB,a known IP3R antagonist. In contrast,following treatment with caffeine,a ryanodine receptor (RyR) agonist,a minimal Ca(2+) response was observed at day 8 of differentiation only. Thus,our data indicate that unlike RyRs,IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation. View Publication