技术资料

Gastric cancer (GC) is one of the most common human cancers. Spheroid colony formation is an effective model for characterization of cancer stem cells. However,gene expression profiles of spheroid colonies obtained from GC cells have not been examined. We performed microarray analyses by Human Genome U133 Plus 2.0 Array in spheroid body-forming and parental cells from MKN-45 and MKN-74 GC cell lines. Kinesin family member C1 (KIFC1) was expressed textgreater2-fold higher in spheroid body-forming cells than in parental cells in both GC lines. Both the number and size of spheres from MKN-45 cells were significantly reduced upon KIFC1 siRNA-transfection compared with negative control siRNA-transfection. Immunohistochemical analysis of 114 GC tissue samples revealed that 42 (37%) of GC cases were positive for KIFC1 expression. GC cases positive for KIFC1 were found more frequently in stage III/IV cases than in stage I/II cases. GC cases positive for KIFC1 were found more frequently in intestinal type GC cases than in diffuse type GC cases. Furthermore,KIFC1-positive GC cases showed high Ki-67 labeling index. Kaplan-Meier analysis demonstrated that KIFC1 expression was not associated with survival. We found positive expression of KIFC1 in CD44‑positive GC and aldehyde dehydrogenase 1 (ALDH1)-positive GC cells. Our results showed that KIFC1 is overexpressed in GC. Since knockdown of KIFC1 inhibited sphere formation,KIFC1 likely plays an important role in cancer stem cells. View Publication

Autoimmune diseases (AIDs),a heterogeneous group of immune-mediated disorders,are a major and growing health problem. Although AIDs are currently treated primarily with anti-inflammatory and immunosuppressive drugs,the use of stem cell transplantation in patients with AIDs is becoming increasingly common. However,stem cell transplantation therapy has limitations,including a shortage of available stem cells and immune rejection of cells from nonautologous sources. Induced pluripotent stem cell (iPSC) technology,which allows the generation of patient-specific pluripotent stem cells,could offer an alternative source for clinical applications of stem cell therapies in AID patients. We used nonintegrating oriP/EBNA-1-based episomal vectors to reprogram dermal fibroblasts from patients with AIDs such as ankylosing spondylitis (AS),Sjogren's syndrome (SS) and systemic lupus erythematosus (SLE). The pluripotency and multilineage differentiation capacity of each patient-specific iPSC line was validated. The safety of these iPSCs for use in stem cell transplantation is indicated by the fact that all AID-specific iPSCs are integrated transgene free. Finally,all AID-specific iPSCs derived in this study could be differentiated into cells of hematopoietic and mesenchymal lineages in vitro as shown by flow cytometric analysis and induction of terminal differentiation potential. Our results demonstrate the successful generation of integration-free iPSCs from patients with AS,SS and SLE. These findings support the possibility of using iPSC technology in autologous and allogeneic cell replacement therapy for various AIDs,including AS,SS and SLE. View Publication

Many pathogens evade cytotoxic T lymphocytes (CTLs) by downregulating HLA molecules on infected cells,but the loss of HLA can trigger NK cell-mediated lysis. HIV-1 is thought to subvert CTLs while preserving NK cell inhibition by Nef-mediated downregulation of HLA-A and -B but not HLA-C molecules. We find that HLA-C is downregulated by most primary HIV-1 clones,including transmitted founder viruses,in contrast to the laboratory-adapted NL4-3 virus. HLA-C reduction is mediated by viral Vpu and reduces the ability of HLA-C restricted CTLs to suppress viral replication in CD4+ cells in vitro. HLA-A/B are unaffected by Vpu,and primary HIV-1 clones vary in their ability to downregulate HLA-C,possibly in response to whether CTLs or NK cells dominate immune pressure through HLA-C. HIV-2 also suppresses HLA-C expression through distinct mechanisms,underscoring the immune pressure HLA-C exerts on HIV. This viral immune evasion casts new light on the roles of CTLs and NK cells in immune responses against HIV. View Publication

Small Kinetochore-Associated Protein (SKAP)/Kinastrin is a multifunctional protein with proposed roles in mitosis,apoptosis and cell migration. Exact mechanisms underlying its activities in these cellular processes are not completely understood. SKAP is predicted to have different isoforms,however,previous studies did not differentiate between them. Since distinct molecular architectures of protein isoforms often influence their localization and functions,this study aimed to examine the expression profile and functional differences between SKAP isoforms in human and mouse. Analyses of various human tissues and cells of different origin by RT-PCR,and by Western blotting and immunocytochemistry applying newly generated anti-SKAP monoclonal antibodies revealed that human SKAP exists in two protein isoforms: ubiquitously expressed SKAP16 and testis/sperm-specific SKAP1. In mouse,SKAP1 expression is detectable in testis at 4 weeks postnatally,when the first wave of spermatogenesis in mice is complete and the elongated spermatids are present in the testes. Furthermore,we identified Pontin as a new SKAP1 interaction partner. SKAP1 and Pontin co-localized in the flagellar region of human sperm suggesting a functional relevance for SKAP1-Pontin interaction in sperm motility. Since most previous studies on SKAP were performed with the testis-specific isoform SKAP1,our findings provide a new basis for future studies on the role of SKAP in both human somatic cells and male germ cells,including studies on male fertility. View Publication

Not provided. View Publication

HCMV is a highly sophisticated virus that has developed various mechanisms for immune evasion and viral dissemination throughout the body (partially mediated by neutrophils). NK cells play an important role in elimination of HCMV-infected cells. Both neutrophils and NK cells utilize similar sets of chemokine receptors to traffic,to and from,various organs. However,the mechanisms by which HCMV attracts neutrophils and not NK cells are largely unknown. Here,we show a unique viral protein,vCXCL1,which targets three chemokine receptors: CXCR1 and CXCR2 expressed on neutrophils and CXCR1 and CX3CR1 expressed on NK cells. Although vCXCL1 attracted both cell types,neutrophils migrated faster and more efficiently than NK cells through the binding of CXCR2. Therefore,we propose that HCMV has developed vCXCL1 to orchestrate its rapid systemic dissemination through preferential attraction of neutrophils and uses alternative mechanisms to counteract the later attraction of NK cells. View Publication

BACKGROUND Alcohol abuse produces an enormous impact on health,society,and the economy. Currently,there are very limited therapies available,largely due to the poor understanding of mechanisms underlying alcohol use disorders (AUDs) in humans. Oxidative damage of mitochondria and cellular proteins aggravates the progression of neuroinflammation and neurological disorders initiated by alcohol abuse. RESULTS Here we show that ethanol exposure causes neuroinflammation in both human induced pluripotent stem (iPS) cells and human neural progenitor cells (NPCs). Ethanol exposure for 24 hours or 7 days does not affect the proliferation of iPS cells and NPCs,but primes an innate immune-like response by activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. This leads to an increase of microtubule-associated protein 1A/1B-light chain 3(+) (LC3B(+)) autophagic puncta and impairment of the mitochondrial and lysosomal distribution. In addition,a decrease of mature neurons derived from differentiating NPCs is evident in ethanol pre-exposed compared to control NPCs. Moreover,a second insult of a pro-inflammatory factor in addition to ethanol preexposure enhances innate cellular inflammation in human iPS cells. CONCLUSIONS This study provides strong evidence that neuronal inflammation contributes to the pathophysiology of AUDs through the activation of the inflammasome pathway in human cellular models. View Publication

As multiple sclerosis research progresses,it is pertinent to continue to develop suitable paradigms to allow for ever more sophisticated investigations. Animal models of multiple sclerosis,despite their continuing contributions to the field,may not be the most prudent for every experiment. Indeed,such may be either insufficient to reflect the functional impact of human genetic variations or unsuitable for drug screenings. Thus,we have established a cell- and patient-specific paradigm to provide an in vitro model within which to perform future genetic investigations. Renal proximal tubule epithelial cells were isolated from multiple sclerosis patients' urine and transfected with pluripotency-inducing episomal factors. Subsequent induced pluripotent stem cells were formed into embryoid bodies selective for ectodermal lineage,resulting in neural tube-like rosettes and eventually neural progenitor cells. Differentiation of these precursors into primary neurons was achieved through a regimen of neurotrophic and other factors. These patient-specific primary neurons displayed typical morphology and functionality,also staining positive for mature neuronal markers. The development of such a non-invasive procedure devoid of permanent genetic manipulation during the course of differentiation,in the context of multiple sclerosis,provides an avenue for studies with a greater cell- and human-specific focus,specifically in the context of genetic contributions to neurodegeneration and drug discovery. View Publication

Genome-wide association studies (GWASs) have reported many single nucleotide polymorphisms (SNPs) associated with psychiatric disorders,but knowledge is lacking regarding molecular mechanisms. Here we show that risk alleles spanning multiple genes across the 10q24.32 schizophrenia-related locus are associated in the human brain selectively with an increase in the expression of both BLOC-1 related complex subunit 7 (BORCS7) and a previously uncharacterized,human-specific arsenite methyltransferase (AS3MT) isoform (AS3MT(d2d3)),which lacks arsenite methyltransferase activity and is more abundant in individuals with schizophrenia than in controls. Conditional-expression analysis suggests that BORCS7 and AS3MT(d2d3) signals are largely independent. GWAS risk SNPs across this region are linked with a variable number tandem repeat (VNTR) polymorphism in the first exon of AS3MT that is associated with the expression of AS3MT(d2d3) in samples from both Caucasians and African Americans. The VNTR genotype predicts promoter activity in luciferase assays,as well as DNA methylation within the AS3MT gene. Both AS3MT(d2d3) and BORCS7 are expressed in adult human neurons and astrocytes,and they are upregulated during human stem cell differentiation toward neuronal fates. Our results provide a molecular explanation for the prominent 10q24.32 locus association,including a novel and evolutionarily recent protein that is involved in early brain development and confers risk for psychiatric illness. View Publication

Pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) are an attractive alternative to primary human hepatocytes (PHHs) used in applications ranging from therapeutics to drug safety testing studies. It would be critical to improve and maintain mature hepatocyte functions of the hPSC-HLCs,especially for long-term studies. If 3D culture systems were to be used for such purposes,it would be important that the system can support formation and maintenance of optimal-sized spheroids for long periods of time,and can also be directly deployed in liver drug testing assays. We report the use of 3-dimensional (3D) cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold has a macroporous network which helps to control the formation and maintenance of the spheroids for weeks. Our results show that culturing hPSC-HLCs in 3D cellulosic scaffolds increases functionality,as demonstrated by improved urea production and hepatic marker expression. In addition,hPSC-HLCs in the scaffolds exhibit a more mature phenotype,as shown by enhanced cytochrome P450 activity and induction. This enables the system to show a higher sensitivity to hepatotoxicants and a higher degree of similarity to PHHs when compared to conventional 2D systems. These results suggest that 3D cellulosic scaffolds are ideal for the long-term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs with improved cellular function can be continually maintained in the scaffolds and directly used for hepatotoxicity assays,making this system highly attractive for drug testing applications. View Publication

IL-18 is a member of the IL-1 family involved in innate immunity and inflammation. Deregulated levels of IL-18 are involved in the pathogenesis of multiple disorders including inflammatory and metabolic diseases,yet relatively little is known regarding its regulation. Liver X receptors or LXRs are key modulators of macrophage cholesterol homeostasis and immune responses. Here we show that LXR ligands negatively regulate LPS-induced mRNA and protein expression of IL-18 in bone marrow-derived macrophages. Consistent with this being an LXR-mediated process,inhibition is abolished in the presence of a specific LXR antagonist and in LXR-deficient macrophages. Additionally,IL-18 processing of its precursor inactive form to its bioactive state is inhibited by LXR through negative regulation of both pro-caspase 1 expression and activation. Finally,LXR ligands further modulate IL-18 levels by inducing the expression of IL-18BP,a potent endogenous inhibitor of IL-18. This regulation occurs via the transcription factor IRF8,thus identifying IL-18BP as a novel LXR and IRF8 target gene. In conclusion,LXR activation inhibits IL-18 production through regulation of its transcription and maturation into an active pro-inflammatory cytokine. This novel regulation of IL-18 by LXR could be applied to modulate the severity of IL-18 driven metabolic and inflammatory disorders. View Publication

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that increases the expression of detoxifying enzymes upon ligand stimulation. Recent studies now suggest that novel endogenous roles of the AHR exist throughout development. In an effort to create an optimized model system for the study of AHR signaling in several cellular lineages,we have employed a CRISPR/CAS9 genome editing strategy in induced pluripotent stem cells (iPSCs) to incorporate a reporter cassette at the transcription start site of one of its canonical targets,cytochrome P450 1A1 (CYP1A1). This cell line faithfully reports on CYP1A1 expression,with luciferase levels as its functional readout,when treated with an endogenous AHR ligand (FICZ) at escalating doses. iPSC-derived fibroblast-like cells respond to acute exposure to environmental and endogenous AHR ligands,and iPSC-derived hepatocytes increase CYP1A1 in a similar manner to primary hepatocytes. This cell line is an important innovation that can be used to map AHR activity in discrete cellular subsets throughout developmental ontogeny. As further endogenous ligands are proposed,this line can be used to screen for safety and efficacy and can report on the ability of small molecules to regulate critical cellular processes by modulating the activity of the AHR. View Publication