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Sample records for human cells suggest

  1. Human BCAS3 expression in embryonic stem cells and vascular precursors suggests a role in human embryogenesis and tumor angiogenesis.

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    Kavitha Siva

    Full Text Available Cancer is often associated with multiple and progressive genetic alterations in genes that are important for normal development. BCAS3 (Breast Cancer Amplified Sequence 3 is a gene of unknown function on human chromosome 17q23, a region associated with breakpoints of several neoplasms. The normal expression pattern of BCAS3 has not been studied, though it is implicated in breast cancer progression. Rudhira, a murine WD40 domain protein that is 98% identical to BCAS3 is expressed in embryonic stem (ES cells, erythropoiesis and angiogenesis. This suggests that BCAS3 expression also may not be restricted to mammary tissue and may have important roles in other normal as well as malignant tissues. We show that BCAS3 is also expressed in human ES cells and during their differentiation into blood vascular precursors. We find that BCAS3 is aberrantly expressed in malignant human brain lesions. In glioblastoma, hemangiopericytoma and brain abscess we note high levels of BCAS3 expression in tumor cells and some blood vessels. BCAS3 may be associated with multiple cancerous and rapidly proliferating cells and hence the expression, function and regulation of this gene merits further investigation. We suggest that BCAS3 is mis-expressed in brain tumors and could serve as a human ES cell and tumor marker.

  2. Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation.

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    David M Truong

    2015-08-01

    Full Text Available Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a "ribozyme" and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed "retrohoming". Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have

  3. The presence of centrioles and centrosomes in ovarian mature cystic teratoma cells suggests human parthenotes developed in vitro can differentiate into mature cells without a sperm centriole

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    Lee, Bo Yon, E-mail: boyonlee@gmail.com [Department of Obstetrics and Gynecology, Kyung Hee University Hospital, Kyung Hee University, School of Medicine, Seoul (Korea, Republic of); Shim, Sang Woo; Kim, Young Sun; Kim, Seung Bo [Department of Obstetrics and Gynecology, Kyung Hee University Hospital, Kyung Hee University, School of Medicine, Seoul (Korea, Republic of)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer The sperm centriole is the progenitor of centrosomes in all somatic cells. Black-Right-Pointing-Pointer Centrioles and centrosomes exist in parthenogenetic ovarian teratoma cells. Black-Right-Pointing-Pointer Without a sperm centriole, parthenogenetic oocytes produce centrioles and centrosomes. Black-Right-Pointing-Pointer Parthenogenetic human oocytes can develop and differentiate into mature cells. -- Abstract: In most animals, somatic cell centrosomes are inherited from the centriole of the fertilizing spermatozoa. The oocyte centriole degenerates during oogenesis, and completely disappears in metaphase II. Therefore, the embryos generated by in vitro parthenogenesis are supposed to develop without any centrioles. Exceptional acentriolar and/or acentrosomal developments are possible in mice and in some experimental cells; however, in most animals, the full developmental potential of parthenogenetic cells in vitro and the fate of their centrioles/centrosomes are not clearly understood. To predict the future of in vitro human parthenogenesis, we explored the centrioles/centrosomes in ovarian mature cystic teratoma cells by immunofluorescent staining and transmission electron microscopy. We confirmed the presence of centrioles and centrosomes in these well-known parthenogenetic ovarian tumor cells. Our findings clearly demonstrate that, even without a sperm centriole, parthenotes that develop from activated oocytes can produce their own centrioles/centrosomes, and can even develop into the well-differentiated mature tissue.

  4. Gene expression profiling suggests a pathological role of human bone marrow-derived mesenchymal stem cells in aging-related skeletal diseases.

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    Jiang, Shih Sheng; Chen, Chung-Hsing; Tseng, Kuo-Yun; Tsai, Fang-Yu; Wang, Ming Jen; Chang, I-Shou; Lin, Jiunn-Liang; Lin, Shankung

    2011-07-01

    Aging is associated with bone loss and degenerative joint diseases, in which the aging of bone marrow-derived mesenchymal stem cell (bmMSC)[1] may play an important role. In this study, we analyzed the gene expression profiles of bmMSC from 14 donors between 36 and 74 years old, and obtained age-associated genes (in the background of osteoarthritis) and osteoarthritis-associated genes (in the background of old age). Pathway analysis of these genes suggests that alterations in glycobiology might play an important role in the aging of human bmMSC. On the other hand, antigen presentation and signaling of immune cells were the top pathways enriched by osteoarthritis-associated genes, suggesting that alteration in immunology of bmMSC might be involved in the pathogenesis of osteoarthritis. Most intriguingly, we found significant age-associated differential expression of HEXA, HEXB, CTSK, SULF1, ADAMTS5, SPP1, COL8A2, GPNMB, TNFAIP6, and RPL29; those genes have been implicated in the bone loss and the pathology of osteoporosis and osteoarthritis in aging. Collectively, our results suggest a pathological role of bmMSC in aging-related skeletal diseases, and suggest the possibility that alteration in the immunology of bmMSC might also play an important role in the etiology of adult-onset osteoarthritis.

  5. Expression of PROKR1 and PROKR2 in human enteric neural precursor cells and identification of sequence variants suggest a role in HSCR.

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    Macarena Ruiz-Ferrer

    Full Text Available BACKGROUND: The enteric nervous system (ENS is entirely derived from neural crest and its normal development is regulated by specific molecular pathways. Failure in complete ENS formation results in aganglionic gut conditions such as Hirschsprung's disease (HSCR. Recently, PROKR1 expression has been demonstrated in mouse enteric neural crest derived cells and Prok-1 was shown to work coordinately with GDNF in the development of the ENS. PRINCIPAL FINDINGS: In the present report, ENS progenitors were isolated and characterized from the ganglionic gut from children diagnosed with and without HSCR, and the expression of prokineticin receptors was examined. Immunocytochemical analysis of neurosphere-forming cells demonstrated that both PROKR1 and PROKR2 were present in human enteric neural crest cells. In addition, we also performed a mutational analysis of PROKR1, PROKR2, PROK1 and PROK2 genes in a cohort of HSCR patients, evaluating them for the first time as susceptibility genes for the disease. Several missense variants were detected, most of them affecting highly conserved amino acid residues of the protein and located in functional domains of both receptors, which suggests a possible deleterious effect in their biological function. CONCLUSIONS: Our results suggest that not only PROKR1, but also PROKR2 might mediate a complementary signalling to the RET/GFRα1/GDNF pathway supporting proliferation/survival and differentiation of precursor cells during ENS development. These findings, together with the detection of sequence variants in PROKR1, PROK1 and PROKR2 genes associated to HSCR and, in some cases in combination with RET or GDNF mutations, provide the first evidence to consider them as susceptibility genes for HSCR.

  6. Functional characterization of a competitive peptide antagonist of p65 in human macrophage-like cells suggests therapeutic potential for chronic inflammation

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    Srinivasan M

    2014-12-01

    Full Text Available Mythily Srinivasan,1 Corinne Blackburn,1 Debomoy K Lahiri2,3 1Department of Oral Pathology, Medicine and Radiology, Indiana University School of Dentistry, 2Institute of Psychiatry Research, Department of Psychiatry, 3Department of Medical and Molecular Genetics, School of Medicine, Indiana University-Purdue University, Indianapolis, IN, USA Abstract: Glucocorticoid-induced leucine zipper (GILZ is a glucocorticoid responsive protein that links the nuclear factor-kappa B (NFκB and the glucocorticoid signaling pathways. Functional and binding studies suggest that the proline-rich region at the carboxy terminus of GILZ binds the p65 subunit of NFκB and suppresses the immunoinflammatory response. A widely-used strategy in the discovery of peptide drugs involves exploitation of the complementary surfaces of naturally occurring binding partners. Previously, we observed that a synthetic peptide (GILZ-P derived from the proline-rich region of GILZ bound activated p65 and ameliorated experimental encephalomyelitis. Here we characterize the secondary structure of GILZ-P by circular dichroic analysis. GILZ-P adopts an extended polyproline type II helical conformation consistent with the structural conformation commonly observed in interfaces of transient intermolecular interactions. To determine the potential application of GILZ-P in humans, we evaluated the toxicity and efficacy of the peptide drug in mature human macrophage-like THP-1 cells. Treatment with GILZ-P at a wide range of concentrations commonly used for peptide drugs was nontoxic as determined by cell viability and apoptosis assays. Functionally, GILZ-P suppressed proliferation and glutamate secretion by activated macrophages by inhibiting nuclear translocation of p65. Collectively, our data suggest that the GILZ-P has therapeutic potential in chronic CNS diseases where persistent inflammation leads to neurodegeneration such as multiple sclerosis and Alzheimer’s disease. Keywords

  7. Exosomes secreted by human placenta carry functional Fas ligand and TRAIL molecules and convey apoptosis in activated immune cells, suggesting exosome-mediated immune privilege of the fetus.

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    Stenqvist, Ann-Christin; Nagaeva, Olga; Baranov, Vladimir; Mincheva-Nilsson, Lucia

    2013-12-01

    Apoptosis is crucially important in mediating immune privilege of the fetus during pregnancy. We investigated the expression and in vitro apoptotic activity of two physiologically relevant death messengers, the TNF family members Fas ligand (FasL) and TRAIL in human early and term placentas. Both molecules were intracellularly expressed, confined to the late endosomal compartment of the syncytiotrophoblast, and tightly associated to the generation and secretion of placental exosomes. Using immunoelectron microscopy, we show that FasL and TRAIL are expressed on the limiting membrane of multivesicular bodies where, by membrane invagination, intraluminal microvesicles carrying membranal bioactive FasL and TRAIL are formed and released in the extracellular space as exosomes. Analyzing exosomes secreted from placental explant cultures, to our knowledge, we demonstrate for the first time that FasL and TRAIL are clustered on the exosomal membrane as oligomerized aggregates ready to form death-inducing signaling complex. Consistently, placental FasL- and TRAIL-carrying exosomes triggered apoptosis in Jurkat T cells and activated PBMC in a dose-dependent manner. Limiting the expression of functional FasL and TRAIL to exosomes comprise a dual benefit: 1) storage of exosomal FasL and TRAIL in multivesicular bodies is protected from proteolytic cleavage and 2) upon secretion, delivery of preformed membranal death molecules by exosomes rapidly triggers apoptosis. Our results suggest that bioactive FasL- and TRAIL-carrying exosomes, able to convey apoptosis, are secreted by the placenta and tie up the immunomodulatory and protective role of human placenta to its exosome-secreting ability.

  8. Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus

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    Röttger, S; White, J; Wandall, H H

    1998-01-01

    O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation......, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation...... of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we...

  9. Analysis of cytotoxicity and genotoxicity on E. coli, human blood cells and Allium cepa suggests a greater toxic potential of hair dye.

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    Maiti, Swati; Sasmal, Kankaayan; Sinha, Sudarson Sekhar; Singh, Mukesh

    2016-02-01

    Pharmaceuticals and personal care products (PPCPs) are among the most important emerging environmental contaminants in recent time. PPCPs include wide range of cosmetics, among which hair dyes, are immensely popular in modern society. However, impact of hair dye and its residual discharged to the environment in relation to human health and ecological imbalance have not been widely studied. Based on the result of initial survey among the group of populations of eastern India, three most popular and commonly used permanent hair dyes are selected. Working sample of dye is prepared as recommended on the instructions booklet of the hair dye. The effect of three dyes is studied on Escherichia coli, human red blood cells (RBC), white blood cells (WBC) and Allium cepa bulbs by growth inhibition, hemolysis, 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay and A. cepa micronuclei assays respectively. The Lethal dose (LD) demonstrated significant differences among three dyes and the model systems. In vitro hemolytic assays performed on RBC, and MTT assays on WBC show the cytotoxic effects of hair dye. Significant growth inhibition of E. coli has also been noted. In addition, the root tips of A. cepa treated with the dye have shown major chromosomal abnormalities coupled with cell division retardation. Here low mitotic index confirm cell division retardation. Finally, results of in vitro studies of dye-DNA interactions demonstrate electrostatic interaction. Combing all these results it confirms that hair dyes are cytotoxic and may cause mutagenic effect on living cells irrespective of microbes, plant and animal system.

  10. Suggestion: Human Factor Based User Interface Design Tool

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    S.Q. Abbas,; Rizwan Beg; Shahnaz Fatima

    2011-01-01

    In this paper, we introduce HFBUIT, Human Factor based user interface tool that enables designers and engineers to create human factor based user interface. This tool will help the designer to utilize the knowledge about the user to configure the interface for different users, i.e. each user may have different skills, level of experience, or cognitive and physical disabilities. The tool makes it easy to knowhuman factors & to reduce the number of usability problems. HFBUIT can be used in real...

  11. Repression of mammosphere formation of human breast cancer cells by soy isoflavone genistein and blueberry polyphenolic acids suggests diet-mediated targeting of cancer stem-like/progenitor cells

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    Mammary stem cells are undifferentiated epithelial cells which initiate mammary tumors and render them resistant to anticancer therapies, when deregulated. Diets rich in fruits and vegetables are implicated in breast cancer risk reduction, yet underlying mechanisms are poorly understood. Here, we ad...

  12. Evidences Suggesting Involvement of Viruses in Oral Squamous Cell Carcinoma

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    Kanupriya Gupta

    2013-01-01

    Full Text Available Oral cancer is one of the most common cancers and it constitutes a major health problem particularly in developing countries. Oral squamous cell carcinoma (OSCC represents the most frequent of all oral neoplasms. Several risk factors have been well characterized to be associated with OSCC with substantial evidences. The etiology of OSCC is complex and involves many factors. The most clearly defined potential factors are smoking and alcohol, which substantially increase the risk of OSCC. However, despite this clear association, a substantial proportion of patients develop OSCC without exposure to them, emphasizing the role of other risk factors such as genetic susceptibility and oncogenic viruses. Some viruses are strongly associated with OSCC while the association of others is less frequent and may depend on cofactors for their carcinogenic effects. Therefore, the exact role of viruses must be evaluated with care in order to improve the diagnosis and treatment of OSCC. Although a viral association within a subset of OSCC has been shown, the molecular and histopathological characteristics of these tumors have yet to be clearly defined.

  13. Human PTCHD3 nulls: rare copy number and sequence variants suggest a non-essential gene

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    Lionel Anath C

    2011-03-01

    Full Text Available Abstract Background Copy number variations (CNVs can contribute to variable degrees of fitness and/or disease predisposition. Recent studies show that at least 1% of any given genome is copy number variable when compared to the human reference sequence assembly. Homozygous deletions (or CNV nulls that are found in the normal population are of particular interest because they may serve to define non-essential genes in human biology. Results In a genomic screen investigating CNV in Autism Spectrum Disorders (ASDs we detected a heterozygous deletion on chromosome 10p12.1, spanning the Patched-domain containing 3 (PTCHD3 gene, at a frequency of ~1.4% (6/427. This finding seemed interesting, given recent discoveries on the role of another Patched-domain containing gene (PTCHD1 in ASD. Screening of another 177 ASD probands yielded two additional heterozygous deletions bringing the frequency to 1.3% (8/604. The deletion was found at a frequency of ~0.73% (27/3,695 in combined control population from North America and Northern Europe predominately of European ancestry. Screening of the human genome diversity panel (HGDP-CEPH covering worldwide populations yielded deletions in 7/1,043 unrelated individuals and those detected were confined to individuals of European/Mediterranean/Middle Eastern ancestry. Breakpoint mapping yielded an identical 102,624 bp deletion in all cases and controls tested, suggesting a common ancestral event. Interestingly, this CNV occurs at a break of synteny between humans and mouse. Considering all data, however, no significant association of these rare PTCHD3 deletions with ASD was observed. Notwithstanding, our RNA expression studies detected PTCHD3 in several tissues, and a novel shorter isoform for PTCHD3 was characterized. Expression in transfected COS-7 cells showed PTCHD3 isoforms colocalize with calnexin in the endoplasmic reticulum. The presence of a patched (Ptc domain suggested a role for PTCHD3 in various biological

  14. Similarity in gene-regulatory networks suggests that cancer cells share characteristics of embryonic neural cells.

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    Zhang, Zan; Lei, Anhua; Xu, Liyang; Chen, Lu; Chen, Yonglong; Zhang, Xuena; Gao, Yan; Yang, Xiaoli; Zhang, Min; Cao, Ying

    2017-08-04

    Cancer cells are immature cells resulting from cellular reprogramming by gene misregulation, and redifferentiation is expected to reduce malignancy. It is unclear, however, whether cancer cells can undergo terminal differentiation. Here, we show that inhibition of the epigenetic modification enzyme enhancer of zeste homolog 2 (EZH2), histone deacetylases 1 and 3 (HDAC1 and -3), lysine demethylase 1A (LSD1), or DNA methyltransferase 1 (DNMT1), which all promote cancer development and progression, leads to postmitotic neuron-like differentiation with loss of malignant features in distinct solid cancer cell lines. The regulatory effect of these enzymes in neuronal differentiation resided in their intrinsic activity in embryonic neural precursor/progenitor cells. We further found that a major part of pan-cancer-promoting genes and the signal transducers of the pan-cancer-promoting signaling pathways, including the epithelial-to-mesenchymal transition (EMT) mesenchymal marker genes, display neural specific expression during embryonic neurulation. In contrast, many tumor suppressor genes, including the EMT epithelial marker gene that encodes cadherin 1 (CDH1), exhibited non-neural or no expression. This correlation indicated that cancer cells and embryonic neural cells share a regulatory network, mediating both tumorigenesis and neural development. This observed similarity in regulatory mechanisms suggests that cancer cells might share characteristics of embryonic neural cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Suggested alternative starch utilization system from the human gut bacterium Bacteroides thetaiotaomicron.

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    Chaudet, Marcia M; Rose, David R

    2016-06-01

    The human digestive system is host to a highly populated ecosystem of bacterial species that significantly contributes to our assimilation of dietary carbohydrates. Bacteroides thetaiotaomicron is a member of this ecosystem, and participates largely in the role of the gut microbiome by breaking down dietary complex carbohydrates. This process of acquiring glycans from the colon lumen is predicted to rely on the mechanisms of proteins that are part of a classified system known as polysaccharide utilization loci (PUL). These loci are responsible for binding substrates at the cell outer membrane, internalizing them, and then hydrolyzing them within the periplasm into simple sugars. Here we report our investigation into specific components of a PUL, and suggest an alternative starch utilization system in B. thetaiotaomicron. Our analysis of an outer membrane binding protein, a SusD homolog, highlights its contribution to this PUL by acquiring starch-based sugars from the colon lumen. Through our structural characterization of two Family GH31 α-glucosidases, we reveal the flexibility of this bacterium with respect to utilizing a range of starch-derived glycans with an emphasis on branched substrates. With these results we demonstrate the predicted function of a gene locus that is capable of contributing to starch hydrolysis in the human colon.

  16. Widespread divergence of the CEACAM/PSG genes in vertebrates and humans suggests sensitivity to selection.

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    Chia Lin Chang

    Full Text Available In mammals, carcinoembryonic antigen cell adhesion molecules (CEACAMs and pregnancy-specific glycoproteins (PSGs play important roles in the regulation of pathogen transmission, tumorigenesis, insulin signaling turnover, and fetal-maternal interactions. However, how these genes evolved and to what extent they diverged in humans remain to be investigated specifically. Based on syntenic mapping of chordate genomes, we reveal that diverging homologs with a prototypic CEACAM architecture-including an extracellular domain with immunoglobulin variable and constant domain-like regions, and an intracellular domain containing ITAM motif-are present from cartilaginous fish to humans, but are absent in sea lamprey, cephalochordate or urochordate. Interestingly, the CEACAM/PSG gene inventory underwent radical divergence in various vertebrate lineages: from zero in avian species to dozens in therian mammals. In addition, analyses of genetic variations in human populations showed the presence of various types of copy number variations (CNVs at the CEACAM/PSG locus. These copy number polymorphisms have 3-80% frequency in select populations, and encompass single to more than six PSG genes. Furthermore, we found that CEACAM/PSG genes contain a significantly higher density of nonsynonymous single nucleotide polymorphism (SNP compared to the chromosome average, and many CEACAM/PSG SNPs exhibit high population differentiation. Taken together, our study suggested that CEACAM/PSG genes have had a more dynamic evolutionary history in vertebrates than previously thought. Given that CEACAM/PSGs play important roles in maternal-fetal interaction and pathogen recognition, these data have laid the groundwork for future analysis of adaptive CEACAM/PSG genotype-phenotypic relationships in normal and complicated pregnancies as well as other etiologies.

  17. Human mesenchymal stem cells

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    Abdallah, Basem; Kassem, Moustapha

    2008-01-01

    Mesenchymal stem cells (MSC) are a group of clonogenic cells present among the bone marrow stroma and capable of multilineage differentiation into mesoderm-type cells such as osteoblasts, adipocytes and chondrocytes. Due to their ease of isolation and their differentiation potential, MSC are being...... introduced into clinical medicine in variety of applications and through different ways of administration. Here, we discuss approaches for isolation, characterization and directing differentiation of human mesenchymal stem cells (hMSC). An update of the current clinical use of the cells is also provided....

  18. Mitochondrial Genome Analyses Suggest Multiple Trichuris Species in Humans, Baboons, and Pigs from Different Geographical Regions.

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    Mohamed B F Hawash

    Full Text Available The whipworms Trichuris trichiura and Trichuris suis are two parasitic nematodes of humans and pigs, respectively. Although whipworms in human and non-human primates historically have been referred to as T. trichiura, recent reports suggest that several Trichuris spp. are found in primates.We sequenced and annotated complete mitochondrial genomes of Trichuris recovered from a human in Uganda, an olive baboon in the US, a hamadryas baboon in Denmark, and two pigs from Denmark and Uganda. Comparative analyses using other published mitochondrial genomes of Trichuris recovered from a human and a porcine host in China and from a françois' leaf-monkey (China were performed, including phylogenetic analyses and pairwise genetic and amino acid distances. Genetic and protein distances between human Trichuris in Uganda and China were high (~19% and 15%, respectively suggesting that they represented different species. Trichuris from the olive baboon in US was genetically related to human Trichuris in China, while the other from the hamadryas baboon in Denmark was nearly identical to human Trichuris from Uganda. Baboon-derived Trichuris was genetically distinct from Trichuris from françois' leaf monkey, suggesting multiple whipworm species circulating among non-human primates. The genetic and protein distances between pig Trichuris from Denmark and other regions were roughly 9% and 6%, respectively, while Chinese and Ugandan whipworms were more closely related.Our results indicate that Trichuris species infecting humans and pigs are phylogenetically distinct across geographical regions, which might have important implications for the implementation of suitable and effective control strategies in different regions. Moreover, we provide support for the hypothesis that Trichuris infecting primates represents a complex of cryptic species with some species being able to infect both humans and non-human primates.

  19. Mitochondrial genome analyses suggest multiple Trichuris species in humans, baboons, and pigs from different geographical regions

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    Hawash, Mohamed B. F.; Andersen, Lee O.; Gasser, Robin B.;

    2015-01-01

    BACKGROUND: The whipworms Trichuris trichiura and Trichuris suis are two parasitic nematodes of humans and pigs, respectively. Although whipworms in human and non-human primates historically have been referred to as T. trichiura, recent reports suggest that several Trichuris spp. are found...... in primates. METHODS AND FINDINGS: We sequenced and annotated complete mitochondrial genomes of Trichuris recovered from a human in Uganda, an olive baboon in the US, a hamadryas baboon in Denmark, and two pigs from Denmark and Uganda. Comparative analyses using other published mitochondrial genomes...... of Trichuris recovered from a human and a porcine host in China and from a françois' leaf-monkey (China) were performed, including phylogenetic analyses and pairwise genetic and amino acid distances. Genetic and protein distances between human Trichuris in Uganda and China were high (~19% and 15%, respectively...

  20. Early modern human diversity suggests subdivided population structure and a complex out-of-Africa scenario.

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    Gunz, Philipp; Bookstein, Fred L; Mitteroecker, Philipp; Stadlmayr, Andrea; Seidler, Horst; Weber, Gerhard W

    2009-04-14

    The interpretation of genetic evidence regarding modern human origins depends, among other things, on assessments of the structure and the variation of ancient populations. Because we lack genetic data from the time when the first anatomically modern humans appeared, between 200,000 and 60,000 years ago, instead we exploit the phenotype of neurocranial geometry to compare the variation in early modern human fossils with that in other groups of fossil Homo and recent modern humans. Variation is assessed as the mean-squared Procrustes distance from the group average shape in a representation based on several hundred neurocranial landmarks and semilandmarks. We find that the early modern group has more shape variation than any other group in our sample, which covers 1.8 million years, and that they are morphologically similar to recent modern humans of diverse geographically dispersed populations but not to archaic groups. Of the currently competing models of modern human origins, some are inconsistent with these findings. Rather than a single out-of-Africa dispersal scenario, we suggest that early modern humans were already divided into different populations in Pleistocene Africa, after which there followed a complex migration pattern. Our conclusions bear implications for the inference of ancient human demography from genetic models and emphasize the importance of focusing research on those early modern humans, in particular, in Africa.

  1. New class of gene-termini-associated human RNAs suggests a novel RNA copying mechanism.

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    Kapranov, Philipp; Ozsolak, Fatih; Kim, Sang Woo; Foissac, Sylvain; Lipson, Doron; Hart, Chris; Roels, Steve; Borel, Christelle; Antonarakis, Stylianos E; Monaghan, A Paula; John, Bino; Milos, Patrice M

    2010-07-29

    Small (<200 nucleotide) RNA (sRNA) profiling of human cells using various technologies demonstrates unexpected complexity of sRNAs with hundreds of thousands of sRNA species present. Genetic and in vitro studies show that these RNAs are not merely degradation products of longer transcripts but could indeed have a function. Furthermore, profiling of RNAs, including the sRNAs, can reveal not only novel transcripts, but also make clear predictions about the existence and properties of novel biochemical pathways operating in a cell. For example, sRNA profiling in human cells indicated the existence of an unknown capping mechanism operating on cleaved RNA, a biochemical component of which was later identified. Here we show that human cells contain a novel type of sRNA that has non-genomically encoded 5' poly(U) tails. The presence of these RNAs at the termini of genes, specifically at the very 3' ends of known mRNAs, strongly argues for the presence of a yet uncharacterized endogenous biochemical pathway in cells that can copy RNA. We show that this pathway can operate on multiple genes, with specific enrichment towards transcript-encoding components of the translational machinery. Finally, we show that genes are also flanked by sense, 3' polyadenylated sRNAs that are likely to be capped.

  2. Satellite cells in human skeletal muscle plasticity.

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    Snijders, Tim; Nederveen, Joshua P; McKay, Bryon R; Joanisse, Sophie; Verdijk, Lex B; van Loon, Luc J C; Parise, Gianni

    2015-01-01

    Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models.

  3. Cell encoding recombinant human erythropoietin

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    Beck, A.K.; Withy, R.M.; Zabrecky, J.R.; Masiello, N.C.

    1990-09-04

    This patent describes a C127 cell transformed with a recombinant DNA vector. It comprises: a DNA sequence encoding human erythropoietin, the transformed cell being capable of producing N-linked and O-linked glycosylated human erythropoietin.

  4. Cutaneous epithelioid cell histiocytoma: immunohistochemical and ultrastructural findings suggesting endothelial origin.

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    Manente, L; Schmitt, I; Onetti, A M; Peris, K; Caracciolo, E; Chimenti, S

    1997-10-01

    A 13-year-old boy presented with a polypoid nodule localized in the groin. Although the clinical and histopathological features corresponded to previously described diagnostic criteria of epithelioid cell histiocytoma, immunohistochemical and ultrastructural findings suggested vascular differentiation of the epithelioid cells. In particular, the immunohistochemical negativity of the epithelioid cell elements for Factor XIIIa failed to substantiate the previously forwarded hypothesis that this lesion represents a dendrocytoma. Instead, the presence of histiocytoid, vacuolated cells occasionally containing sparse red blood cells, positive staining for Factor VIII-related antigen, and ultrastructural evidence of endothelial characteristics in epithelioid neoplastic cells favor the hypothesis that "epithelioid cell histiocytoma" is of endothelial origin. We suggest the descriptive term cutaneous histiocytoid hemangioendothelioma for this lesion.

  5. Human regulatory B cells control the TFH cell response.

    Science.gov (United States)

    Achour, Achouak; Simon, Quentin; Mohr, Audrey; Séité, Jean-François; Youinou, Pierre; Bendaoud, Boutahar; Ghedira, Ibtissem; Pers, Jacques-Olivier; Jamin, Christophe

    2017-07-01

    Follicular helper T (TFH) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of TFH cell-dependent humoral immune responses is unknown. We sought to assess the role of Breg cells on TFH cell development and function. Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate TFH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing TFH cell maturation. In cocultures they differentiated B cells into CD138(+) plasma and IgD(-)CD27(+) memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of B cells prevented TFH cell development. Added to TFH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3(+)CXCR5(+)PD-1(+) follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on TFH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-β. Human Breg cells control TFH cell maturation, expand follicular regulatory T cells, and inhibit the TFH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the TFH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. Current demographics suggest future energy supplies will be inadequate to slow human population growth.

    Directory of Open Access Journals (Sweden)

    John P DeLong

    Full Text Available Influential demographic projections suggest that the global human population will stabilize at about 9-10 billion people by mid-century. These projections rest on two fundamental assumptions. The first is that the energy needed to fuel development and the associated decline in fertility will keep pace with energy demand far into the future. The second is that the demographic transition is irreversible such that once countries start down the path to lower fertility they cannot reverse to higher fertility. Both of these assumptions are problematic and may have an effect on population projections. Here we examine these assumptions explicitly. Specifically, given the theoretical and empirical relation between energy-use and population growth rates, we ask how the availability of energy is likely to affect population growth through 2050. Using a cross-country data set, we show that human population growth rates are negatively related to per-capita energy consumption, with zero growth occurring at ∼13 kW, suggesting that the global human population will stop growing only if individuals have access to this amount of power. Further, we find that current projected future energy supply rates are far below the supply needed to fuel a global demographic transition to zero growth, suggesting that the predicted leveling-off of the global population by mid-century is unlikely to occur, in the absence of a transition to an alternative energy source. Direct consideration of the energetic constraints underlying the demographic transition results in a qualitatively different population projection than produced when the energetic constraints are ignored. We suggest that energetic constraints be incorporated into future population projections.

  7. Expression of FACT in mammalian tissues suggests its role in maintaining of undifferentiated state of cells.

    Science.gov (United States)

    Garcia, Henry; Fleyshman, Daria; Kolesnikova, Katerina; Safina, Alfiya; Commane, Mairead; Paszkiewicz, Geraldine; Omelian, Angela; Morrison, Carl; Gurova, Kateryna

    2011-10-01

    The Facilitates Chromatin Transcription (FACT) chromatin remodeling complex, comprised of two subunits, SSRP1 and SPT16, is involved in transcription, replication and DNA repair. We recently showed that curaxins, small molecules with anti-cancer activity, target FACT and kill tumor cells in a FACT-dependent manner. We also found that FACT is overexpressed in human and mouse tumors and that tumor cells are sensitive to FACT downregulation. To clarify the clinical potential of FACT inhibition, we were interested in physiological role(s) of FACT in multicellular organisms. We analyzed SSRP1 and SPT16 expression in different cells, tissues and conditions using Immunohistochemical (IHC) staining of mouse and human tissues and analysis of publically available high-content gene expression datasets. Both approaches demonstrated coordinated expression of the two FACT subunits, which was primarily associated with the stage of cellular differentiation. Most cells of adult tissues do not have detectable protein level of FACT. High FACT expression was associated with stem or less-differentiated cells, while low FACT levels were seen in more differentiated cells. Experimental manipulation of cell differentiation and proliferation in vitro, as well as tissue staining for the Ki67 proliferation marker, showed that FACT expression is related more to differentiation than to proliferation. Thus, FACT may be part of a stem cell-like gene expression signature and play a role in maintaining cells in an undifferentiated state, which is consistent with its potential role as an anti-cancer target.

  8. Widening Spectrum of Cellular and Subcellular Expression of Human GLUD1 and GLUD2 Glutamate Dehydrogenases Suggests Novel Functions.

    Science.gov (United States)

    Spanaki, Cleanthe; Kotzamani, Dimitra; Plaitakis, Andreas

    2017-01-01

    in the cytoplasm of gray and white matter astrocytes within coarse structures resembling mitochondria. Additionally, hGDH1 localized to the nuclear membrane of a subpopulation of astrocytes and of the vast majority of oligodendrocytes and their precursors. Remarkably, hGDH2-specific staining was detected in human cortical neurons, with different expression patterns having emerged. One pattern, observed in large cortical neurons (some with pyramidal morphology), was a hGDH2-specific labeling of cytoplasmic structures resembling mitochondria. These were distributed either in the cell body-axon or on the cell surface in close proximity to astrocytic end-feet that encircle glutamatergic synapses. Another pattern was observed in small cortical neurons with round dense nuclei in which the hGDH2-specific staining was found in the nuclear membrane. A detailed description of these observations and their functional implications, suggesting that the GDH flux is used by different cells to serve some of their unique functions, is presented below.

  9. An orthotopic xenograft model of intraneural NF1 MPNST suggests a potential association between steroid hormones and tumor cell proliferation.

    Science.gov (United States)

    Perrin, George Q; Li, Hua; Fishbein, Lauren; Thomson, Susanne A; Hwang, Min S; Scarborough, Mark T; Yachnis, Anthony T; Wallace, Margaret R; Mareci, Thomas H; Muir, David

    2007-11-01

    Malignant peripheral nerve sheath tumors (MPNST) are the most aggressive cancers associated with neurofibromatosis type 1 (NF1). Here we report a practical and reproducible model of intraneural NF1 MPNST, by orthotopic xenograft of an immortal human NF1 tumor-derived Schwann cell line into the sciatic nerves of female scid mice. Intraneural injection of the cell line sNF96.2 consistently produced MPNST-like tumors that were highly cellular and showed extensive intraneural growth. These xenografts had a high proliferative index, were angiogenic, had significant mast cell infiltration and rapidly dominated the host nerve. The histopathology of engrafted intraneural tumors was consistent with that of human NF1 MPNST. Xenograft tumors were readily examined by magnetic resonance imaging, which also was used to assess tumor vascularity. In addition, the intraneural proliferation of sNF96.2 cell tumors was decreased in ovariectomized mice, while replacement of estrogen or progesterone restored tumor cell proliferation. This suggests a potential role for steroid hormones in supporting tumor cell growth of this MPNST cell line in vivo. The controlled orthotopic implantation of sNF96.2 cells provides for the precise initiation of intraneural MPNST-like tumors in a model system suitable for therapeutic interventions, including inhibitors of angiogenesis and further study of steroid hormone effects on tumor cell growth.

  10. Search for naive human pluripotent stem cells

    Institute of Scientific and Technical Information of China (English)

    Simone Aparecida Siqueira Fonseca; Roberta Montero Costas; Lygia Veiga Pereira

    2015-01-01

    Normal mouse pluripotent stem cells were originallyderived from the inner cell mass (ICM) of blastocystsand shown to be the in vitro equivalent of those preimplantationembryonic cells, and thus were calledembryonic stem cells (ESCs). More than a decade later,pluripotent cells were isolated from the ICM of humanblastocysts. Despite being called human ESCs, thesecells differ significantly from mouse ESCs, includingdifferent morphology and mechanisms of control ofpluripotency, suggesting distinct embryonic originsof ESCs from the two species. Subsequently, mousepluripotent stem cells were established from the ICMderivedepiblast of post-implantation embryos. Thesemouse epiblast stem cells (EpiSCs) are morphologicaland epigenetically more similar to human ESCs. Thisraised the question of whether cells from the humanICM are in a more advanced differentiation stage thantheir murine counterpart, or whether the availableculture conditions were not adequate to maintain thosehuman cells in their in vivo state, leading to a transitioninto EpiSC-like cells in vitro . More recently, novel cultureconditions allowed the conversion of human ESCs intomouse ESC-like cells called naive (or ground state)human ESCs, and the derivation of naive human ESCsfrom blastocysts. Here we will review the characteristicsof each type of pluripotent stem cells, how (andwhether) these relate to different stages of embryonicdevelopment, and discuss the potential implications ofnaive human ESCs in research and therapy.

  11. Immunocalization of telomerase in cells of lizard tail after amputation suggests cell activation for tail regeneration.

    Science.gov (United States)

    Alibardi, L

    2016-02-01

    Tail amputation (autotomy) in most lizards elicits a remarkable regenerative response leading to a new although simplified tail. No information on the trigger mechanism following wounding is known but cells from the stump initiate to proliferate and form a regenerative blastema. The present study shows that telomerases are mainly activated in the nuclei of various connective and muscle satellite cells of the stump, and in other tissues, probably responding to the wound signals. Western blotting detection also indicates that telomerase positive bands increases in the regenerating blastema in comparison to the normal tail. Light and ultrastructural immunocytochemistry localization of telomerase shows that 4-14 days post-amputation in lizards immunopositive nuclei of sparse cells located among the wounded tissues are accumulating into the forming blastema. These cells mainly include fibroblasts and fat cells of the connective tissue and satellite cells of muscles. Also some immature basophilic and polychromatophilic erytroblasts, lymphoblasts and myelocytes present within the Bone Marrow of the vertebrae show telomerase localization in their nuclei, but their contribution to the formation of the regenerative blastema remains undetermined. The study proposes that one of the initial mechanisms triggering cell proliferation for the formation of the blastema in lizards involve gene activation for the production of telomerase that stimulates the following signaling pathways for cell division and migration.

  12. The autosomal dominant spinocerebellar ataxias: emerging mechanistic themes suggest pervasive Purkinje cell vulnerability.

    Science.gov (United States)

    Hekman, Katherine E; Gomez, Christopher M

    2015-05-01

    The spinocerebellar ataxias are a genetically heterogeneous group of disorders with clinically overlapping phenotypes arising from Purkinje cell degeneration, cerebellar atrophy and varying degrees of degeneration of other grey matter regions. For 22 of the 32 subtypes, a genetic cause has been identified. While recurring themes are emerging, there is no clear correlation between the clinical phenotype or penetrance, the type of genetic defect or the category of the disease mechanism, or the neuronal types involved beyond Purkinje cells. These phenomena suggest that cerebellar Purkinje cells may be a uniquely vulnerable neuronal cell type, more susceptible to a wider variety of genetic/cellular insults than most other neuron types.

  13. The suggestion of common cause of disease, characteristics of human body, and medical treatment

    Directory of Open Access Journals (Sweden)

    Byung-Jun Cho

    2011-06-01

    Full Text Available Objectives & Methods: This suggestion was attempted to be elevated the recognition of common characteristics in disease. So, we performed to analyze the correlation of common cause of disease, characteristics of human body, and medical treatment. And the results are as follows. Results: 1. The cause of disease is consist of genetic factor, aging, habit, food of not good in health, weather, environment, deficit of the physical activity, stress and so on. 2. Generally, human has common and individual weakness. Individual weakness is appeared similar to the occurrence of volcano and lapse. 3. The correlation of disease and medical treatments is possible to explain using the quotation of the law of motion made by Isaac Newton, the great physicist. 4. When the process of the medical treatment was not progressed, the prognosis is determined by the correlation of the homeostasis(H' in human body and the homeostasis(H of disease. 5. The prognosis of disease is determined by the relationship between the energy of disease(F and medical treatment(F'. 6. The exact diagnosis is possible to predict the treatment sequence, and the facts that homeostasis in human body and disease, relationship between the energy of disease(F and medical treatment(F', action and reaction are important to determine the prognosis. 7. The careful observation of improving response and worsening action of disease becomes available for exact prognosis. Conclusion: The above described contents may be useful in clinical studies, and the concrete clinical reports about this will be made afterward.

  14. Connectivity analysis of suggestive brain areas involved in middle ear pressure regulation in humans

    DEFF Research Database (Denmark)

    SA, Sami; Gaihede, Michael

    2010-01-01

    , MEP counter-regulation presented as Eustachian tube openings with steep and fast pressure changes toward 0 Pa, whereas in others, gradual and slow pressure changes presented related to the mastoid; these changes sometimes crossed 0 Pa into opposite pressures. In many cases, combinations......HYPOTHESIS:: Middle ear pressure (MEP) is actively regulated by both the Eustachian tube and the mastoid air cell system. BACKGROUND:: MEP is a highly significant factor involved in many clinical conditions related to otitis media. Basic knowledge on its overall regulation remains insufficient...... of these distinct mechanisms were found. CONCLUSION:: The human mastoid as well as the Eustachian tube was capable of active counter-regulation of the MEP in short-term experimental pressure changes in healthy ears. Thus, these 2 systems seemed to function in a complementary way, where the mastoid was related...

  15. Trophoblast lineage cells derived from human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying, E-mail: ying.chen@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Wang, Kai; Chandramouli, Gadisetti V.R. [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Knott, Jason G. [Developmental Epigenetics Laboratory, Department of Animal Science, Michigan State University (United States); Leach, Richard, E-mail: Richard.leach@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group (United States)

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  16. Evidence to suggest that teeth act as human ornament displays signalling mate quality.

    Directory of Open Access Journals (Sweden)

    Colin A Hendrie

    Full Text Available Ornament displays seen in animals convey information about genetic quality, developmental history and current disease state to both prospective sexual partners and potential rivals. In this context, showing of teeth through smiles etc is a characteristic feature of human social interaction. Tooth development is influenced by genetic and environmental factors. Adult teeth record environmental and traumatic events, as well as the effects of disease and ageing. Teeth are therefore a rich source of information about individuals and their histories. This study examined the effects of digital manipulations of tooth colour and spacing. Results showed that deviation away from normal spacing and/or the presence of yellowed colouration had negative effects on ratings of attractiveness and that these effects were markedly stronger in female models. Whitening had no effect beyond that produced by natural colouration. This indicates that these colour induced alterations in ratings of attractiveness are mediated by increased/decreased yellowing rather than whitening per se. Teeth become yellower and darker with age. Therefore it is suggested that whilst the teeth of both sexes act as human ornament displays, the female display is more complex because it additionally signals residual reproductive value.

  17. Human embryonic stem cells derived by somatic cell nuclear transfer.

    Science.gov (United States)

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L; Wolf, Don; Mitalipov, Shoukhrat

    2013-06-06

    Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

  18. Widespread Epigenetic Abnormalities Suggest a Broad DNA Methylation Erasure Defect in Abnormal Human Sperm

    Science.gov (United States)

    Siegmund, Kimberly; Yang, Allen; Laird, Peter W.; Sokol, Rebecca Z.

    2007-01-01

    Background Male-factor infertility is a common condition, and etiology is unknown for a high proportion of cases. Abnormal epigenetic programming of the germline is proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. During germ cell maturation and gametogenesis, cells of the germ line undergo extensive epigenetic reprogramming. This process involves widespread erasure of somatic-like patterns of DNA methylation followed by establishment of sex-specific patterns by de novo DNA methylation. Incomplete reprogramming of the male germ line could, in theory, result in both altered sperm DNA methylation and compromised spermatogenesis. Methodology/Principal Finding We determined concentration, motility and morphology of sperm in semen samples collected by male members of couples attending an infertility clinic. Using MethyLight and Illumina assays we measured methylation of DNA isolated from purified sperm from the same samples. Methylation at numerous sequences was elevated in DNA from poor quality sperm. Conclusions This is the first report of a broad epigenetic defect associated with abnormal semen parameters. Our results suggest that the underlying mechanism for these epigenetic changes may be improper erasure of DNA methylation during epigenetic reprogramming of the male germ line. PMID:18074014

  19. Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm.

    Directory of Open Access Journals (Sweden)

    Sahar Houshdaran

    Full Text Available BACKGROUND: Male-factor infertility is a common condition, and etiology is unknown for a high proportion of cases. Abnormal epigenetic programming of the germline is proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. During germ cell maturation and gametogenesis, cells of the germ line undergo extensive epigenetic reprogramming. This process involves widespread erasure of somatic-like patterns of DNA methylation followed by establishment of sex-specific patterns by de novo DNA methylation. Incomplete reprogramming of the male germ line could, in theory, result in both altered sperm DNA methylation and compromised spermatogenesis. METHODOLOGY/PRINCIPAL FINDING: We determined concentration, motility and morphology of sperm in semen samples collected by male members of couples attending an infertility clinic. Using MethyLight and Illumina assays we measured methylation of DNA isolated from purified sperm from the same samples. Methylation at numerous sequences was elevated in DNA from poor quality sperm. CONCLUSIONS: This is the first report of a broad epigenetic defect associated with abnormal semen parameters. Our results suggest that the underlying mechanism for these epigenetic changes may be improper erasure of DNA methylation during epigenetic reprogramming of the male germ line.

  20. Stem cells in the human breast

    DEFF Research Database (Denmark)

    Petersen, Ole William; Polyak, Kornelia

    2010-01-01

    The origins of the epithelial cells participating in the development, tissue homeostasis, and cancer of the human breast are poorly understood. However, emerging evidence suggests a role for adult tissue-specific stem cells in these processes. In a hierarchical manner, these generate the two main...

  1. Genome engineering in human cells.

    Science.gov (United States)

    Song, Minjung; Kim, Young-Hoon; Kim, Jin-Soo; Kim, Hyongbum

    2014-01-01

    Genome editing in human cells is of great value in research, medicine, and biotechnology. Programmable nucleases including zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases recognize a specific target sequence and make a double-strand break at that site, which can result in gene disruption, gene insertion, gene correction, or chromosomal rearrangements. The target sequence complexities of these programmable nucleases are higher than 3.2 mega base pairs, the size of the haploid human genome. Here, we briefly introduce the structure of the human genome and the characteristics of each programmable nuclease, and review their applications in human cells including pluripotent stem cells. In addition, we discuss various delivery methods for nucleases, programmable nickases, and enrichment of gene-edited human cells, all of which facilitate efficient and precise genome editing in human cells.

  2. Genomic Analysis of the Human Gut Microbiome Suggests Novel Enzymes Involved in Quinone Biosynthesis

    Science.gov (United States)

    Ravcheev, Dmitry A.; Thiele, Ines

    2016-01-01

    Ubiquinone and menaquinone are membrane lipid-soluble carriers of electrons that are essential for cellular respiration. Eukaryotic cells can synthesize ubiquinone but not menaquinone, whereas prokaryotes can synthesize both quinones. So far, most of the human gut microbiome (HGM) studies have been based on metagenomic analysis. Here, we applied an analysis of individual HGM genomes to the identification of ubiquinone and menaquinone biosynthetic pathways. In our opinion, the shift from metagenomics to analysis of individual genomes is a pivotal milestone in investigation of bacterial communities, including the HGM. The key results of this study are as follows. (i) The distribution of the canonical pathways in the HGM genomes was consistent with previous reports and with the distribution of the quinone-dependent reductases for electron acceptors. (ii) The comparative genomics analysis identified four alternative forms of the previously known enzymes for quinone biosynthesis. (iii) Genes for the previously unknown part of the futalosine pathway were identified, and the corresponding biochemical reactions were proposed. We discuss the remaining gaps in the menaquinone and ubiquinone pathways in some of the microbes, which indicate the existence of further alternate genes or routes. Together, these findings provide further insight into the biosynthesis of quinones in bacteria and the physiology of the HGM. PMID:26904004

  3. Topography of Gng2- and NetrinG2-Expression Suggests an Insular Origin of the Human Claustrum

    Science.gov (United States)

    Pirone, Andrea; Cozzi, Bruno; Edelstein, Larry; Peruffo, Antonella; Lenzi, Carla; Quilici, Francesca; Antonini, Rita; Castagna, Maura

    2012-01-01

    The claustrum has been described in the forebrain of all mammals studied so far. It has been suggested that the claustrum plays a role in the integration of multisensory information: however, its detailed structure and function remain enigmatic. The human claustrum is a thin, irregular, sheet of grey matter located between the inner surface of the insular cortex and the outer surface of the putamen. Recently, the G-protein gamma2 subunit (Gng2) was proposed as a specific claustrum marker in the rat, and used to better delineate its anatomical boundaries and connections. Additional claustral markers proposed in mammals include Netrin-G2 in the monkey and latexin in the cat. Here we report the expression and distribution of Gng2 and Netrin-G2 in human post-mortem samples of the claustrum and adjacent structures. Gng2 immunoreactivity was detected in the neuropil of the claustrum and of the insular cortex but not in the putamen. A faint labelling was present also in the external and extreme capsules. Double-labelling experiments indicate that Gng2 is also expressed in glial cells. Netrin-G2 labelling was seen in neuronal cell bodies throughout the claustrum and the insular cortex but not in the medially adjacent putamen. No latexin immunoreactive element was detected in the claustrum or adjacent structures. Our results confirm that both the Gng2 and the Netrin-G2 proteins show an affinity to the claustrum and related formations also in the human brain. The presence of Gng2 and Netrin-G2 immunoreactive elements in the insular cortex, but not in the putamen, suggests a possible common ontogeny of the claustrum and insula. PMID:22957104

  4. International Human Resource Management Education: A Survey of HR Professionals, Suggestions for Skill Dissemination.

    Science.gov (United States)

    Barclay, Lizabeth A.; Wagner-Marsh, Fraya; Loewe, G. Michael

    2002-01-01

    Surveyed a human resource professional association about training and interest in international human resources management. Based on results, offers recommendations for expanding coverage of this topic in credit and non-credit courses. (EV)

  5. Genome-Wide Analyses Suggest Mechanisms Involving Early B-Cell Development in Canine IgA Deficiency

    Science.gov (United States)

    Frankowiack, Marcel; Kierczak, Marcin; Bergvall, Kerstin; Axelsson, Erik; Tintle, Linda; Marti, Eliane; Roosje, Petra; Leeb, Tosso; Hedhammar, Åke; Hammarström, Lennart; Lindblad-Toh, Kerstin

    2015-01-01

    Immunoglobulin A deficiency (IgAD) is the most common primary immune deficiency disorder in both humans and dogs, characterized by recurrent mucosal tract infections and a predisposition for allergic and other immune mediated diseases. In several dog breeds, low IgA levels have been observed at a high frequency and with a clinical resemblance to human IgAD. In this study, we used genome-wide association studies (GWAS) to identify genomic regions associated with low IgA levels in dogs as a comparative model for human IgAD. We used a novel percentile groups-approach to establish breed-specific cut-offs and to perform analyses in a close to continuous manner. GWAS performed in four breeds prone to low IgA levels (German shepherd, Golden retriever, Labrador retriever and Shar-Pei) identified 35 genomic loci suggestively associated (p IgA levels. In German shepherd, three genomic regions (candidate genes include KIRREL3 and SERPINA9) were genome-wide significantly associated (p IgA levels. A ~20kb long haplotype on CFA28, significantly associated (p = 0.0005) to IgA levels in Shar-Pei, was positioned within the first intron of the gene SLIT1. Both KIRREL3 and SLIT1 are highly expressed in the central nervous system and in bone marrow and are potentially important during B-cell development. SERPINA9 expression is restricted to B-cells and peaks at the time-point when B-cells proliferate into antibody-producing plasma cells. The suggestively associated regions were enriched for genes in Gene Ontology gene sets involving inflammation and early immune cell development. PMID:26225558

  6. Incomplete lineage sorting patterns among human, chimpanzee and orangutan suggest recent orangutan speciation and widespread selection

    DEFF Research Database (Denmark)

    Hobolth, Asger; Dutheil, Julien; Hawks, John

    2011-01-01

    We search the complete orangutan genome for regions where humans are more closely related to orangutans than to chimpanzees due to incomplete lineage sorting (ILS) in the ancestor of human and chimpanzees. The search uses our recently developed coalescent HMM framework. We find ILS present in ~1%...

  7. Endocannabinoids and Human Sperm Cells

    Directory of Open Access Journals (Sweden)

    Giovanna Zolese

    2010-10-01

    Full Text Available N-acylethanolamides (NAEs are naturally occurring signaling lipids consisting of amides and esters of long-chain polyunsaturated fatty acids. Usually they are present in a very small amounts in many mammalian tissues and cells, including human reproductive tracts and fluids. Recently, the presence of N-arachidonoylethanolamide (anandamide, AEA, the most characterised member of endocannabinoids, and its congeners palmitoylethanolamide (PEA and oleylethanolamide (OEA in seminal plasma, oviductal fluid, and follicular fluids was demonstrated. AEA has been shown to bind not only type-1 (CB1 and type-2 (CB2 cannabinoid receptors, but also type-1 vanilloid receptor (TRPV1, while PEA and OEA are inactive with respect to classical cannabinoid CB1 and CB2 but activate TRPV1 or peroxisome proliferator activate receptors (PPARs. This review concerns the most recent experimental data on PEA and OEA, endocannabinoid-like molecules which appear to exert their action exclusively on sperm cells with altered features, such as membrane characteristics and kinematic parameters. Their beneficial effects on these cells could suggest a possible pharmacological use of PEA and OEA on patients affected by some forms of idiopathic infertility.

  8. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Varga, Nora [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Vereb, Zoltan; Rajnavoelgyi, Eva [Department of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen (Hungary); Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Apati, Agota, E-mail: apati@kkk.org.hu [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  9. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    Science.gov (United States)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  10. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    Science.gov (United States)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  11. Cellular and Molecular Changes Associated with Onion Skin Formation Suggest Involvement of Programmed Cell Death

    Science.gov (United States)

    Galsurker, Ortal; Doron-Faigenboim, Adi; Teper-Bamnolker, Paula; Daus, Avinoam; Fridman, Yael; Lers, Amnon; Eshel, Dani

    2017-01-01

    Skin formation of onion (Allium cepa L.) bulb involves scale desiccation accompanied by scale senescence, resulting in cell death and tissue browning. Understanding the mechanism of skin formation is essential to improving onion skin and bulb qualities. Although onion skin plays a crucial role in postharvest onion storage and shelf life, its formation is poorly understood. We investigated the mode of cell death in the outermost scales that are destined to form the onion skin. Surprisingly, fluorescein diacetate staining and scanning electron microscopy indicated that the outer scale desiccates from the inside out. This striking observation suggests that cell death in the outer scales, during skin formation, is an internal and organized process that does not derive only from air desiccation. DNA fragmentation, a known hallmark of programmed cell death (PCD), was revealed in the outer scales and gradually decreased toward the inner scales of the bulb. Transmission electron microscopy further revealed PCD-related structural alterations in the outer scales which were absent from the inner scales. De novo transcriptome assembly for three different scales: 1st (outer), 5th (intermediate) and 8th (inner) fleshy scales identified 2,542 differentially expressed genes among them. GO enrichment for cluster analysis revealed increasing metabolic processes in the outer senescent scale related to defense response, PCD processes, carbohydrate metabolism and flavonoid biosynthesis, whereas increased metabolism and developmental growth processes were identified in the inner scales. High expression levels of PCD-related genes were identified in the outer scale compared to the inner ones, highlighting the involvement of PCD in outer-skin development. These findings suggest that a program to form the dry protective skin exists and functions only in the outer scales of onion. PMID:28119713

  12. Diffusion inside living human cells

    DEFF Research Database (Denmark)

    Leijnse, N.; Jeon, J. -H.; Loft, Steffen

    2012-01-01

    Naturally occurring lipid granules diffuse in the cytoplasm and can be used as tracers to map out the viscoelastic landscape inside living cells. Using optical trapping and single particle tracking we found that lipid granules exhibit anomalous diffusion inside human umbilical vein endothelial...... cells. For these cells the exact diffusional pattern of a particular granule depends on the physiological state of the cell and on the localization of the granule within the cytoplasm. Granules located close to the actin rich periphery of the cell move less than those located towards to the center...... of the cell or within the nucleus. Also, granules in cells which are stressed by intense laser illumination or which have attached to a surface for a long period of time move in a more restricted fashion than those within healthy cells. For granules diffusing in healthy cells, in regions away from the cell...

  13. Fractal 1/f Dynamics Suggest Entanglement of Measurement and Human Performance

    Science.gov (United States)

    Holden, John G.; Choi, Inhyun; Amazeen, Polemnia G.; Van Orden, Guy

    2011-01-01

    Variability of repeated measurements in human performances exhibits fractal 1/f noise. Yet the relative strength of this fractal pattern varies widely across conditions, tasks, and individuals. Four experiments illustrate how subtle details of the conditions of measurement change the fractal patterns observed across task conditions. The results…

  14. Transcriptional interactions suggest niche segregation among microorganisms in the human gut

    DEFF Research Database (Denmark)

    Plichta, Damian Rafal; Juncker, Agnieszka; dos Santos, Marcelo Bertalan Quintanilha

    2016-01-01

    The human gastrointestinal (GI) tract is the habitat for hundreds of microbial species, of which many cannot be cultivated readily, presumably because of the dependencies between species 1. Studies of microbial co-occurrence in the gut have indicated community substructures that may reflect funct...

  15. Social Networking Web Sites and Human Resource Personnel: Suggestions for Job Searches

    Science.gov (United States)

    Roberts, Sherry J.; Roach, Terry

    2009-01-01

    Social Networking Web sites (SNWs) are now being used as reference checks by human resource personnel. For this reason, SNW users, particularly university students and other soon-to-be job applicants, should ask the following questions: Am I loading information that I want the world to see? Is this really a picture that shows me in the best light?…

  16. Social Networking Web Sites and Human Resource Personnel: Suggestions for Job Searches

    Science.gov (United States)

    Roberts, Sherry J.; Roach, Terry

    2009-01-01

    Social Networking Web sites (SNWs) are now being used as reference checks by human resource personnel. For this reason, SNW users, particularly university students and other soon-to-be job applicants, should ask the following questions: Am I loading information that I want the world to see? Is this really a picture that shows me in the best light?…

  17. Human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Zaher, Walid; Al-Nbaheen, May

    2012-01-01

    Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self-renewal and......Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self...... of clinical applications, e.g., non-healing bone fractures and defects and also non-skeletal degenerative diseases like heart failure. Currently, the numbers of clinical trials that employ MSC are increasing. However, several biological and biotechnological challenges need to be overcome to benefit from...

  18. Human hair genealogies and stem cell latency

    Directory of Open Access Journals (Sweden)

    Tavaré Simon

    2006-02-01

    Full Text Available Abstract Background Stem cells divide to reproduce themselves and produce differentiated progeny. A fundamental problem in human biology has been the inability to measure how often stem cells divide. Although it is impossible to observe every division directly, one method for counting divisions is to count replication errors; the greater the number of divisions, the greater the numbers of errors. Stem cells with more divisions should produce progeny with more replication errors. Methods To test this approach, epigenetic errors (methylation in CpG-rich molecular clocks were measured from human hairs. Hairs exhibit growth and replacement cycles and "new" hairs physically reappear even on "old" heads. Errors may accumulate in long-lived stem cells, or in their differentiated progeny that are eventually shed. Results Average hair errors increased until two years of age, and then were constant despite decades of replacement, consistent with new hairs arising from infrequently dividing bulge stem cells. Errors were significantly more frequent in longer hairs, consistent with long-lived but eventually shed mitotic follicle cells. Conclusion Constant average hair methylation regardless of age contrasts with the age-related methylation observed in human intestine, suggesting that error accumulation and therefore stem cell latency differs among tissues. Epigenetic molecular clocks imply similar mitotic ages for hairs on young and old human heads, consistent with a restart with each new hair, and with genealogies surreptitiously written within somatic cell genomes.

  19. Liver cell adenoma showing sequential alteration of radiological findings suggestive of well-differentiated hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Takayuki Kogure; Yoshiyuki Ueno; Satoshi Sekiguchi; Kazuyuki Ishida; Takehiko Igarashi; Yuta Wakui; Takao Iwasaki; Tooru Shimosegawa

    2009-01-01

    A liver tumor 35 mm in diameter was found incidentally in a 40-year-old woman who had no history of liver diseases or the use of oral contraceptives. Radiological diagnostics showed the typical findings of liver cell adenoma (LCA). Dynamic computed tomography revealed that the tumor showed a homogenous enhancement in the arterial phase and almost the same enhancement as the surrounding liver parenchyma in the delayed phase. The tumor was found to contain fat on magnetic resonance imaging. A benign fat containing liver tumor was suggested. However, radiological findings altered, which caused us to suspect that a welldifferentiated hepatocellular carcinoma (HCC) containing fat was becoming dedifferentiated. Partial hepatectomy was performed and the pathological findings showed the typical findings of LCA. This case was an extremely rare LCA, which had no background of risk for LCA and developed the sequential alteration of the radiological findings to suspect well-differentiated HCC.

  20. Human neutrophils facilitate tumor cell transendothelial migration.

    LENUS (Irish Health Repository)

    Wu, Q D

    2012-02-03

    Tumor cell extravasation plays a key role in tumor metastasis. However, the precise mechanisms by which tumor cells migrate through normal vascular endothelium remain unclear. In this study, using an in vitro transendothelial migration model, we show that human polymorphonuclear neutrophils (PMN) assist the human breast tumor cell line MDA-MB-231 to cross the endothelial barrier. We found that tumor-conditioned medium (TCM) downregulated PMN cytocidal function, delayed PMN apoptosis, and concomitantly upregulated PMN adhesion molecule expression. These PMN treated with TCM attached to tumor cells and facilitated tumor cell migration through different endothelial monolayers. In contrast, MDA-MB-231 cells alone did not transmigrate. FACScan analysis revealed that these tumor cells expressed high levels of intercellular adhesion molecule-1 (ICAM-1) but did not express CD11a, CD11b, or CD18. Blockage of CD11b and CD18 on PMN and of ICAM-1 on MDA-MB-231 cells significantly attenuated TCM-treated, PMN-mediated tumor cell migration. These tumor cells still possessed the ability to proliferate after PMN-assisted transmigration. These results indicate that TCM-treated PMN may serve as a carrier to assist tumor cell transendothelial migration and suggest that tumor cells can exploit PMN and alter their function to facilitate their extravasation.

  1. Nuclear envelope dynamics during plant cell division suggest common mechanisms between kingdoms.

    Science.gov (United States)

    Graumann, Katja; Evans, David E

    2011-05-01

    Behaviour of the NE (nuclear envelope) during open mitosis has been explored extensively in metazoans, but lack of native markers has limited similar investigations in plants. In the present study, carried out using living synchronized tobacco BY-2 suspension cultures, the non-functional NE marker LBR (lamin B receptor)-GFP (green fluorescent protein) and two native, functional NE proteins, AtSUN1 [Arapidopsis thaliana SUN (Sad1/UNC84) 1] and AtSUN2, we provide evidence that the ER (endoplasmic reticulum)-retention theory for NE membranes is applicable in plants. We also observe two apparently unique plant features: location of the NE-membrane components in close proximity to chromatin throughout division, and spatially distinct reformation of the NE commencing at the chromatin surface facing the spindle poles and concluding at the surface facing the cell plate. Mobility of the proteins was investigated in the interphase NE, during NE breakdown and reformation, in the spindle membranes and the cell plate. A role for AtSUN2 in nuclear envelope breakdown is suggested.

  2. Discovery of intramolecular trans-sialidases in human gut microbiota suggests novel mechanisms of mucosal adaptation

    Science.gov (United States)

    Tailford, Louise E.; Owen, C. David; Walshaw, John; Crost, Emmanuelle H.; Hardy-Goddard, Jemma; Le Gall, Gwenaelle; de Vos, Willem M.; Taylor, Garry L.; Juge, Nathalie

    2015-07-01

    The gastrointestinal mucus layer is colonized by a dense community of microbes catabolizing dietary and host carbohydrates during their expansion in the gut. Alterations in mucosal carbohydrate availability impact on the composition of microbial species. Ruminococcus gnavus is a commensal anaerobe present in the gastrointestinal tract of >90% of humans and overrepresented in inflammatory bowel diseases (IBD). Using a combination of genomics, enzymology and crystallography, we show that the mucin-degrader R. gnavus ATCC 29149 strain produces an intramolecular trans-sialidase (IT-sialidase) that cleaves off terminal α2-3-linked sialic acid from glycoproteins, releasing 2,7-anhydro-Neu5Ac instead of sialic acid. Evidence of IT-sialidases in human metagenomes indicates that this enzyme occurs in healthy subjects but is more prevalent in IBD metagenomes. Our results uncover a previously unrecognized enzymatic activity in the gut microbiota, which may contribute to the adaptation of intestinal bacteria to the mucosal environment in health and disease.

  3. Regulating private human suborbital flight at the international and European level: Tendencies and suggestions

    Science.gov (United States)

    Masson-Zwaan, Tanja; Moro-Aguilar, Rafael

    2013-12-01

    In the context of the FAST20XX project (Future High-Altitude High-Speed Transport) that started in 2009 under the 7th Framework Programme of the European Union (EU), the authors reexamined the legal status of private human suborbital flight, and researched whether it might be regulated as aviation or as spaceflight. International space law is ambiguous as to accommodating suborbital activities. While some provisions of the UN outer space treaties would seem to exclude them, generally there is not any explicit condition in terms of reaching orbit as a requirement for application. International air law presents equal difficulties in dealing with this activity. The classic definition of "aircraft" as contained in the Annexes to the Chicago Convention does not really encompass the kind of rocket-powered vehicles that are envisaged here. As a result, it is unclear whether the United Nations Committee on the Peaceful Uses of Outer Space (COPUOS), the International Civil Aviation Organization (ICAO), or both could be involved in an eventual international regulation of suborbital flight. In the absence of a uniform international regime, each state has the sovereign right to regulate human suborbital flights operating within its airspace. So far, two practical solutions have been realised or proposed, and will be analyzed. On the one hand, the USA granted power for regulation and licensing over private human suborbital flight to the Office of Commercial Space Transportation of the Federal Aviation Administration (FAA/AST). Subsequent regulations by the FAA have set out a series of requirements for companies that want to operate these flights, enabling a market to develop. On the other side of the Atlantic, both the European Space Agency (ESA) and a group of representatives of the European Aviation Safety Agency (EASA) of the European Union (EU) seem to rather regard this activity as aviation, potentially subject to the regulation and certification competences of EASA

  4. T-cell response in human leishmaniasis

    DEFF Research Database (Denmark)

    Kharazmi, A; Kemp, K; Ismail, A

    1999-01-01

    In the present communication we provide evidence for the existence of a Th1/Th2 dichotomy in the T-cell response to Leishmania antigens in human leishmaniasis. Our data suggest that the pattern of IL-4 and IFN-gamma response is polarised in these patients. Lymphocytes from individuals recovered......+. Furthermore, IL-10 plays an important role in the development of post kala azar dermal leishmaniasis (PKDL) from VL. The balance between the parasitic-specific T-cell response plays an important regulatory role in determining the outcome of Leishmania infections in humans....

  5. Ancient human genomes suggest three ancestral populations for present-day Europeans

    Science.gov (United States)

    Lazaridis, Iosif; Patterson, Nick; Mittnik, Alissa; Renaud, Gabriel; Mallick, Swapan; Kirsanow, Karola; Sudmant, Peter H.; Schraiber, Joshua G.; Castellano, Sergi; Lipson, Mark; Berger, Bonnie; Economou, Christos; Bollongino, Ruth; Fu, Qiaomei; Bos, Kirsten I.; Nordenfelt, Susanne; Li, Heng; de Filippo, Cesare; Prüfer, Kay; Sawyer, Susanna; Posth, Cosimo; Haak, Wolfgang; Hallgren, Fredrik; Fornander, Elin; Rohland, Nadin; Delsate, Dominique; Francken, Michael; Guinet, Jean-Michel; Wahl, Joachim; Ayodo, George; Babiker, Hamza A.; Bailliet, Graciela; Balanovska, Elena; Balanovsky, Oleg; Barrantes, Ramiro; Bedoya, Gabriel; Ben-Ami, Haim; Bene, Judit; Berrada, Fouad; Bravi, Claudio M.; Brisighelli, Francesca; Busby, George B. J.; Cali, Francesco; Churnosov, Mikhail; Cole, David E. C.; Corach, Daniel; Damba, Larissa; van Driem, George; Dryomov, Stanislav; Dugoujon, Jean-Michel; Fedorova, Sardana A.; Romero, Irene Gallego; Gubina, Marina; Hammer, Michael; Henn, Brenna M.; Hervig, Tor; Hodoglugil, Ugur; Jha, Aashish R.; Karachanak-Yankova, Sena; Khusainova, Rita; Khusnutdinova, Elza; Kittles, Rick; Kivisild, Toomas; Klitz, William; Kučinskas, Vaidutis; Kushniarevich, Alena; Laredj, Leila; Litvinov, Sergey; Loukidis, Theologos; Mahley, Robert W.; Melegh, Béla; Metspalu, Ene; Molina, Julio; Mountain, Joanna; Näkkäläjärvi, Klemetti; Nesheva, Desislava; Nyambo, Thomas; Osipova, Ludmila; Parik, Jüri; Platonov, Fedor; Posukh, Olga; Romano, Valentino; Rothhammer, Francisco; Rudan, Igor; Ruizbakiev, Ruslan; Sahakyan, Hovhannes; Sajantila, Antti; Salas, Antonio; Starikovskaya, Elena B.; Tarekegn, Ayele; Toncheva, Draga; Turdikulova, Shahlo; Uktveryte, Ingrida; Utevska, Olga; Vasquez, René; Villena, Mercedes; Voevoda, Mikhail; Winkler, Cheryl; Yepiskoposyan, Levon; Zalloua, Pierre; Zemunik, Tatijana; Cooper, Alan; Capelli, Cristian; Thomas, Mark G.; Ruiz-Linares, Andres; Tishkoff, Sarah A.; Singh, Lalji; Thangaraj, Kumarasamy; Villems, Richard; Comas, David; Sukernik, Rem; Metspalu, Mait; Meyer, Matthias; Eichler, Evan E.; Burger, Joachim; Slatkin, Montgomery; Pääbo, Svante; Kelso, Janet; Reich, David; Krause, Johannes

    2014-01-01

    We sequenced the genomes of a ~7,000 year old farmer from Germany and eight ~8,000 year old hunter-gatherers from Luxembourg and Sweden. We analyzed these and other ancient genomes1–4 with 2,345 contemporary humans to show that most present Europeans derive from at least three highly differentiated populations: West European Hunter-Gatherers (WHG), who contributed ancestry to all Europeans but not to Near Easterners; Ancient North Eurasians (ANE) related to Upper Paleolithic Siberians3, who contributed to both Europeans and Near Easterners; and Early European Farmers (EEF), who were mainly of Near Eastern origin but also harbored WHG-related ancestry. We model these populations’ deep relationships and show that EEF had ~44% ancestry from a “Basal Eurasian” population that split prior to the diversification of other non-African lineages. PMID:25230663

  6. Appearance of Human Plasma Cells Following Differentiation of Human B Cells in NOD/SCID Mouse Spleen

    Directory of Open Access Journals (Sweden)

    Kentaro Kikuchi

    2003-01-01

    Full Text Available Relatively little is known for the differentiation and maturation process of human B cells to plasma cells. This is particularly important in reconstitution work involving transfer of autoantibodies. To address this issue, we transplanted human peripheral blood mononuclear cells (PBMC directly into the spleen of irradiated NOD/SCID mice depleted of natural killer cell activity. Within 6 weeks, naïve B cells differentiated into memory B cells and, importantly, the numbers of human CD138+ plasma cells in spleen increased by 100 fold after transplantation. Plasma cell numbers correlated with the detection of human IgM and IgG in serum, indicating that human B cells had differentiated into mature plasma cells in the murine spleen. In addition to CD19+ plasma cells, a distinct CD19- plasma cell population was detected, suggesting that downregulation of CD19 associated with maturation of plasma cells occurred. When purified human B cells were transplanted, those findings were not observed. Our results indicate that differentiation and maturation of human B cells and plasma cells can be investigated by transplantation of human PBMC into the spleen of NOD/SCID mice. The model will be useful for studying the differentiation of human B cells and generation of plasma cells.

  7. Dietary options and behavior suggested by plant biomarker evidence in an early human habitat

    Science.gov (United States)

    Magill, Clayton R.; Ashley, Gail M.; Domínguez-Rodrigo, Manuel; Freeman, Katherine H.

    2016-03-01

    The availability of plants and freshwater shapes the diets and social behavior of chimpanzees, our closest living relative. However, limited evidence about the spatial relationships shared between ancestral human (hominin) remains, edible resources, refuge, and freshwater leaves the influence of local resources on our species' evolution open to debate. Exceptionally well-preserved organic geochemical fossils-biomarkers-preserved in a soil horizon resolve different plant communities at meter scales across a contiguous 25,000 m2 archaeological land surface at Olduvai Gorge from about 2 Ma. Biomarkers reveal hominins had access to aquatic plants and protective woods in a patchwork landscape, which included a spring-fed wetland near a woodland that both were surrounded by open grassland. Numerous cut-marked animal bones are located within the wooded area, and within meters of wetland vegetation delineated by biomarkers for ferns and sedges. Taken together, plant biomarkers, clustered bone debris, and hominin remains define a clear spatial pattern that places animal butchery amid the refuge of an isolated forest patch and near freshwater with diverse edible resources.

  8. Modeling of the human rhinovirus C capsid suggests possible causes for antiviral drug resistance.

    Science.gov (United States)

    Basta, Holly A; Ashraf, Shamaila; Sgro, Jean-Yves; Bochkov, Yury A; Gern, James E; Palmenberg, Ann C

    2014-01-05

    Human rhinoviruses of the RV-C species are recently discovered pathogens with greater clinical significance than isolates in the RV-A+B species. The RV-C cannot be propagated in typical culture systems; so much of the virology is necessarily derivative, relying on comparative genomics, relative to the better studied RV-A+B. We developed a bioinformatics-based structural model for a C15 isolate. The model showed the VP1-3 capsid proteins retain their fundamental cores relative to the RV-A+B, but conserved, internal RV-C residues affect the shape and charge of the VP1 hydrophobic pocket that confers antiviral drug susceptibility. When predictions of the model were tested in organ cultures or ALI systems with recombinant C15 virus, there was a resistance to capsid-binding drugs, including pleconaril, BTA-188, WIN56291, WIN52035 and WIN52084. Unique to all RV-C, the model predicts conserved amino acids within the pocket and capsid surface pore leading to the pocket may correlate with this activity.

  9. Early events following experimental infection with Peste-Des-Petits ruminants virus suggest immune cell targeting.

    Directory of Open Access Journals (Sweden)

    Robert A Pope

    Full Text Available Peste-des-petits ruminants virus (PPRV is a viral pathogen that causes a devastating plague of small ruminants. PPRV is an economically significant disease that continues to be a major obstacle to the development of sustainable agriculture across the developing world. The current understanding of PPRV pathogenesis has been heavily assumed from the closely related rinderpest virus (RPV and other morbillivirus infections alongside data derived from field outbreaks. There have been few studies reported that have focused on the pathogenesis of PPRV and very little is known about the processes underlying the early stages of infection. In the present study, 15 goats were challenged by the intranasal route with a virulent PPRV isolate, Côte d'Ivoire '89 (CI/89 and sacrificed at strategically defined time-points post infection to enable pre- and post-mortem sampling. This approach enabled precise monitoring of the progress and distribution of virus throughout the infection from the time of challenge, through peak viraemia and into a period of convalescence. Observations were then related to findings of previous field studies and experimental models of PPRV to develop a clinical scoring system for PPRV. Importantly, histopathological investigations demonstrated that the initial site for virus replication is not within the epithelial cells of the respiratory mucosa, as has been previously reported, but is within the tonsillar tissue and lymph nodes draining the site of inoculation. We propose that virus is taken up by immune cells within the respiratory mucosa which then transport virus to lymphoid tissues where primary virus replication occurs, and from where virus enters circulation. Based on these findings we propose a novel clinical scoring methodology for PPRV pathogenesis and suggest a fundamental shift away from the conventional model of PPRV pathogenesis.

  10. Early events following experimental infection with Peste-Des-Petits ruminants virus suggest immune cell targeting.

    Science.gov (United States)

    Pope, Robert A; Parida, Satya; Bailey, Dalan; Brownlie, Joe; Barrett, Thomas; Banyard, Ashley C

    2013-01-01

    Peste-des-petits ruminants virus (PPRV) is a viral pathogen that causes a devastating plague of small ruminants. PPRV is an economically significant disease that continues to be a major obstacle to the development of sustainable agriculture across the developing world. The current understanding of PPRV pathogenesis has been heavily assumed from the closely related rinderpest virus (RPV) and other morbillivirus infections alongside data derived from field outbreaks. There have been few studies reported that have focused on the pathogenesis of PPRV and very little is known about the processes underlying the early stages of infection. In the present study, 15 goats were challenged by the intranasal route with a virulent PPRV isolate, Côte d'Ivoire '89 (CI/89) and sacrificed at strategically defined time-points post infection to enable pre- and post-mortem sampling. This approach enabled precise monitoring of the progress and distribution of virus throughout the infection from the time of challenge, through peak viraemia and into a period of convalescence. Observations were then related to findings of previous field studies and experimental models of PPRV to develop a clinical scoring system for PPRV. Importantly, histopathological investigations demonstrated that the initial site for virus replication is not within the epithelial cells of the respiratory mucosa, as has been previously reported, but is within the tonsillar tissue and lymph nodes draining the site of inoculation. We propose that virus is taken up by immune cells within the respiratory mucosa which then transport virus to lymphoid tissues where primary virus replication occurs, and from where virus enters circulation. Based on these findings we propose a novel clinical scoring methodology for PPRV pathogenesis and suggest a fundamental shift away from the conventional model of PPRV pathogenesis.

  11. Early Events following Experimental Infection with Peste-Des-Petits Ruminants Virus Suggest Immune Cell Targeting

    Science.gov (United States)

    Pope, Robert A.; Parida, Satya; Bailey, Dalan; Brownlie, Joe; Barrett, Thomas; Banyard, Ashley C.

    2013-01-01

    Peste-des-petits ruminants virus (PPRV) is a viral pathogen that causes a devastating plague of small ruminants. PPRV is an economically significant disease that continues to be a major obstacle to the development of sustainable agriculture across the developing world. The current understanding of PPRV pathogenesis has been heavily assumed from the closely related rinderpest virus (RPV) and other morbillivirus infections alongside data derived from field outbreaks. There have been few studies reported that have focused on the pathogenesis of PPRV and very little is known about the processes underlying the early stages of infection. In the present study, 15 goats were challenged by the intranasal route with a virulent PPRV isolate, Côte d’Ivoire ’89 (CI/89) and sacrificed at strategically defined time-points post infection to enable pre- and post-mortem sampling. This approach enabled precise monitoring of the progress and distribution of virus throughout the infection from the time of challenge, through peak viraemia and into a period of convalescence. Observations were then related to findings of previous field studies and experimental models of PPRV to develop a clinical scoring system for PPRV. Importantly, histopathological investigations demonstrated that the initial site for virus replication is not within the epithelial cells of the respiratory mucosa, as has been previously reported, but is within the tonsillar tissue and lymph nodes draining the site of inoculation. We propose that virus is taken up by immune cells within the respiratory mucosa which then transport virus to lymphoid tissues where primary virus replication occurs, and from where virus enters circulation. Based on these findings we propose a novel clinical scoring methodology for PPRV pathogenesis and suggest a fundamental shift away from the conventional model of PPRV pathogenesis. PMID:23418464

  12. Functional magnetic resonance imaging suggests automatization of the cortical response to inspiratory threshold loading in humans.

    Science.gov (United States)

    Raux, Mathieu; Tyvaert, Louise; Ferreira, Michael; Kindler, Félix; Bardinet, Eric; Karachi, Carine; Morelot-Panzini, Capucine; Gotman, Jean; Pike, G Bruce; Koski, Lisa; Similowski, Thomas

    2013-12-01

    Inspiratory threshold loading (ITL) induces cortical activation. It is sustained over time and is resistant to distraction, suggesting automaticity. We hypothesized that ITL-induced changes in cerebral activation may differ between single-breath ITL and continuous ITL, with differences resembling those observed after cortical automatization of motor tasks. We analyzed the brain blood oxygen level dependent (BOLD) signal of 11 naive healthy volunteers during 5 min of random, single-breath ITL and 5 min of continuous ITL. Single-breath ITL increased BOLD in many areas (premotor cortices, bilateral insula, cerebellum, reticular formation of the lateral mesencephalon) and decreased BOLD in regions co-localizing with the default mode network. Continuous ITL induced signal changes in a limited number of areas (supplementary motor area). These differences are comparable to those observed before and after overlearning of motor tasks. We conclude that the respiratory-related cortical activation observed in response to ITL is likely due to automated, attention-independent mechanisms. Also, ITL activates cortical circuits right from the first breath.

  13. Differences in the microrheology of human embryonic stem cells and human induced pluripotent stem cells.

    Science.gov (United States)

    Daniels, Brian R; Hale, Christopher M; Khatau, Shyam B; Kusuma, Sravanti; Dobrowsky, Terrence M; Gerecht, Sharon; Wirtz, Denis

    2010-12-01

    Embryonic and adult fibroblasts can be returned to pluripotency by the expression of reprogramming genes. Multiple lines of evidence suggest that these human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells are behaviorally, karyotypically, and morphologically similar. Here we sought to determine whether the physical properties of hiPS cells, including their micromechanical properties, are different from those of hES cells. To this end, we use the method of particle tracking microrheology to compare the viscoelastic properties of the cytoplasm of hES cells, hiPS cells, and the terminally differentiated parental human fibroblasts from which our hiPS cells are derived. Our results indicate that although the cytoplasm of parental fibroblasts is both viscous and elastic, the cytoplasm of hiPS cells does not exhibit any measurable elasticity and is purely viscous over a wide range of timescales. The viscous phenotype of hiPS cells is recapitulated in parental cells with disassembled actin filament network. The cytoplasm of hES cells is predominantly viscous but contains subcellular regions that are also elastic. This study supports the hypothesis that intracellular elasticity correlates with the degree of cellular differentiation and reveals significant differences in the mechanical properties of hiPS cells and hES cells. Because mechanical stimuli have been shown to mediate the precise fate of differentiating stem cells, our results support the concept that stem cell "softness" is a key feature of force-mediated differentiation of stem cells and suggest there may be subtle functional differences between force-mediated differentiation of hiPS cells and hES cells.

  14. Human embryonic stem cells handbook

    Directory of Open Access Journals (Sweden)

    Carlo Alberto Redi

    2013-03-01

    Full Text Available After the Nobel prize in physiology or medicine was awarded jointly to Sir John Gurdon and Shinya Yamanaka for the discovery that mature cells can be reprogrammed to become pluripotent it became imperative to write down the review for a book entirely devoted to human embryonic stem cells (hES, those cells that are a urgent need for researchers, those cells that rekindle the ethical debates and finally, last but not least, those cells whose study paved the way to obtain induced pluripotent stem cells by the OSKC’s Yamanaka method (the OSKC acronim refers, for those not familiar with the topic, to the four stemness genes used to transfect somatic fibroblasts: Oct4, Sox2, Klf4 and c-Myc....

  15. LANGERHANS CELL HISTIOCYTOSIS - EXPRESSION OF LEUKOCYTE CELLULAR ADHESION MOLECULES SUGGESTS ABNORMAL HOMING AND DIFFERENTIATION

    NARCIS (Netherlands)

    DEGRAAF, JH; TAMMINGA, RYJ; KAMPS, WA; TIMENS, W

    1994-01-01

    Langerhans' cell histiocytosis (LCH) is characterized by an accumulation of cells with a Langerhans' cell (LC) phenotype. Most patients present with solitary skin or bone lesions, but multi-organ lesions may appear Twenty-two LCH-tissue sections from 13 children and adolescents, with lesions at diff

  16. Comparative secretome analysis suggests low plant cell wall degrading capacity in Frankia symbionts

    Directory of Open Access Journals (Sweden)

    Normand Philippe

    2008-01-01

    genomes, suggesting that plant cell wall polysaccharide degradation may not be crucial to root infection, or that this degradation varies among strains. We hypothesize that the relative lack of secreted polysaccharide-degrading enzymes in Frankia reflects a strategy used by these bacteria to avoid eliciting host defense responses. The esterases, lipases, and proteases found in the core Frankia secretome might facilitate hyphal penetration through the cell wall, release carbon sources, or modify chemical signals. The core secretome also includes extracellular solute-binding proteins and Frankia-specific hypothetical proteins that may enable the actinorhizal symbiosis.

  17. Phenotypic changes of human cells in human-rat liver during partial hepatectomy-induced regeneration

    Institute of Scientific and Technical Information of China (English)

    Yan Sun; Dong Xiao; Hong-An Li; Jin-Fang Jiang; Qing Li; Ruo-Shuang Zhang; Xi-Gu Chen

    2009-01-01

    AIM: To examine the human hepatic parenchymal and stromal components in rat liver and the phenotypic changes of human cells in liver of human-rat chimera (HRC) generated by in utero transplantation of human cells during partial hepatectomy (PHx)-induced liver regeneration. METHODS: Human hepatic parenchymal and stromal components and phenotypic changes of human cells during liver regeneration were examined by flow cytometry, in situ hybridization and immunohistochemistry. RESULTS: ISH analysis demonstrated human Alupositive cells in hepatic parenchyma and stroma of recipient liver. Functional human hepatocytes generated in this model potentially constituted human hepatic functional units with the presence of donor-derived human endothelial and biliary duct cells in host liver. Alpha fetoprotein (AFP)+, CD34+ and CD45+ cells were observed in the chimeric liver on day 10 after PHxinduced liver regeneration and then disappeared in PHx group, but not in non-PHx group, suggesting that dynamic phenotypic changes of human cells expressing AFP, CD34 and CD45 cells may occur during the chimeric liver regeneration. Additionally, immunostaining for human proliferating cell nuclear antigen (PCNA) showed that the number of PCNA-positive cells in the chimeric liver of PHx group was markedly increased, as compared to that of control group, indicating that donor-derived human cells are actively proliferated during PHx-induced regeneration of HRC liver.

  18. 454 Transcriptome sequencing suggests a role for two-component signalling in cellularization and differentiation of barley endosperm transfer cells.

    Directory of Open Access Journals (Sweden)

    Johannes Thiel

    Full Text Available BACKGROUND: Cell specification and differentiation in the endosperm of cereals starts at the maternal-filial boundary and generates the endosperm transfer cells (ETCs. Besides the importance in assimilate transfer, ETCs are proposed to play an essential role in the regulation of endosperm differentiation by affecting development of proximate endosperm tissues. We attempted to identify signalling elements involved in early endosperm differentiation by using a combination of laser-assisted microdissection and 454 transcriptome sequencing. PRINCIPAL FINDINGS: 454 sequencing of the differentiating ETC region from the syncytial state until functionality in transfer processes captured a high proportion of novel transcripts which are not available in existing barley EST databases. Intriguingly, the ETC-transcriptome showed a high abundance of elements of the two-component signalling (TCS system suggesting an outstanding role in ETC differentiation. All components and subfamilies of the TCS, including distinct kinds of membrane-bound receptors, have been identified to be expressed in ETCs. The TCS system represents an ancient signal transduction system firstly discovered in bacteria and has previously been shown to be co-opted by eukaryotes, like fungi and plants, whereas in animals and humans this signalling route does not exist. Transcript profiling of TCS elements by qRT-PCR suggested pivotal roles for specific phosphorelays activated in a coordinated time flow during ETC cellularization and differentiation. ETC-specificity of transcriptionally activated TCS phosphorelays was assessed for early differentiation and cellularization contrasting to an extension of expression to other grain tissues at the beginning of ETC maturation. Features of candidate genes of distinct phosphorelays and transcriptional activation of genes putatively implicated in hormone signalling pathways hint at a crosstalk of hormonal influences, putatively ABA and ethylene, and

  19. EXPRESSION OF Fas LIGAND IN HUMAN COLON CANCER CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁

    2001-01-01

    To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

  20. Enriched retinal ganglion cells derived from human embryonic stem cells

    Science.gov (United States)

    Gill, Katherine P.; Hung, Sandy S. C.; Sharov, Alexei; Lo, Camden Y.; Needham, Karina; Lidgerwood, Grace E.; Jackson, Stacey; Crombie, Duncan E.; Nayagam, Bryony A.; Cook, Anthony L.; Hewitt, Alex W.; Pébay, Alice; Wong, Raymond C. B.

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  1. Sodium Valproate Induces Cell Senescence in Human Hepatocarcinoma Cells

    Directory of Open Access Journals (Sweden)

    Hong-Mei An

    2013-12-01

    Full Text Available Hepatocarcinogenesis is associated with epigenetic changes, including histone deacetylases (HDACs. Epigenetic modulation by HDAC inhibition is a potentially valuable approach for hepatocellular carcinoma treatment. In present study, we evaluated the anticancer effects of sodium valproate (SVP, a known HDAC inhibitor, in human hepatocarcinoma cells. The results showed SVP inhibited the proliferation of Bel-7402 cells in a dose-dependent manner. Low dose SVP treatment caused a large and flat morphology change, positive SA-β-gal staining, and G0/G1 phase cell cycle arrest in human hepatocarcinoma cells. Low dose SVP treatment also increased acetylation of histone H3 and H4 on p21 promoter, accompanied by up-regulation of p21 and down-regulation of RB phosphorylation. These observations suggested that a low dose of SVP could induce cell senescence in hepatocarcinoma cells, which might correlate with hyperacetylation of histone H3 and H4, up-regulation of p21, and inhibition of RB phosphorylation. Since the effective concentration inducing cell senescence in hepatocarcinoma cells is clinically available, whether a clinical dose of SVP could induce cell senescence in clinical hepatocarcinoma is worthy of further study.

  2. Quercetin Inhibits Cell Migration and Invasion in Human Osteosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Haifeng Lan

    2017-09-01

    Full Text Available Background/Aims: Osteosarcoma is a malignant tumor associated with high mortality; however, no effective therapies for the disease have been developed. Several studies have focused on elucidating the pathogenesis of osteosarcoma and have aimed to develop novel therapies for the disease. Quercetin is a vital dietary flavonoid that has been shown to have a variety of anticancer effects, as it induces cell cycle arrest, apoptosis, and differentiation and is involved in cell adhesion, metastasis and angiogenesis. Herein, we aimed to investigate the effects of quercetin on osteosarcoma migration and invasion in vitro and in vivo and to explore the molecular mechanisms underlying its effects on osteosarcoma migration and invasion. Methods: Cell viability, cell cycle activity and cell apoptosis were measured using CCK-8 assay and flow cytometry, and cell migration and invasion were evaluated by wound healing and transwell assays, respectively. The mRNA and protein expression levels of several proteins of interest were assessed by real-time quantitative PCR and western blotting, respectively. Moreover, a nude mouse model of human osteosarcoma lung metastasis was established to assess the anti-metastatic effects of quercetin in vivo. Results: We noted no significant differences in cell cycle activity and apoptosis between HOS and MG63 cells and control cells. Treatment with quercetin significantly attenuated cell migration and invasion in HOS and MG63 cells compared with treatment with control medium. Moreover HIF-1α, VEGF, MMP2, and MMP9 mRNA and protein expression levels were significantly downregulated in HOS cells treated with quercetin compared with HOS cells treated with controls. Additionally, treatment with quercetin attenuated metastatic lung tumor formation and growth in the nude mouse model of osteosarcoma compared with treatment with controls. Conclusion: Our findings regarding the inhibitory effects of quercetin on cell migration and

  3. Drug screen in patient cells suggests quinacrine to be repositioned for treatment of acute myeloid leukemia

    OpenAIRE

    Eriksson, Anna; Österroos, Albin; Hassan, Sadia Bashir; Gullbo, Joachim; Rickardson, Linda; Jarvius, Malin; Nygren, Peter; Fryknäs, Mårten; Höglund, Martin; Larsson, Rolf

    2015-01-01

    To find drugs suitable for repositioning for use against leukemia, samples from patients with chronic lymphocytic, acute myeloid and lymphocytic leukemias as well as peripheral blood mononuclear cells (PBMC) were tested in response to 1266 compounds from the LOPAC1280 library (Sigma). Twenty-five compounds were defined as hits with activity in all leukemia subgroups (<50% cell survival compared with control) at 10 mu M drug concentration. Only one of these compounds, quinacrine, showed low...

  4. Human fetal mesenchymal stem cells.

    Science.gov (United States)

    O'Donoghue, Keelin; Chan, Jerry

    2006-09-01

    Stem cells have been isolated at all stages of development from the early developing embryo to the post-reproductive adult organism. However, the fetal environment is unique as it is the only time in ontogeny that there is migration of stem cells in large numbers into different organ compartments. While fetal neural and haemopoietic stem cells (HSC) have been well characterised, only recently have mesenchymal stem cells from the human fetus been isolated and evaluated. Our group have characterised in human fetal blood, liver and bone marrow a population of non-haemopoietic, non-endothelial cells with an immunophenotype similar to adult bone marrow-derived mesenchymal stem cells (MSC). These cells, human fetal mesenchymal stem cells (hfMSC), are true multipotent stem cells with greater self-renewal and differentiation capacity than their adult counterparts. They circulate in first trimester fetal blood and have been found to traffic into the maternal circulation, engrafting in bone marrow, where they remain microchimeric for decades after pregnancy. Though fetal microchimerism has been implicated in the pathogenesis of autoimmune disease, the biological role of hfMSC microchimerism is unknown. Potential downstream applications of hfMSC include their use as a target cell for non-invasive pre-natal diagnosis from maternal blood, and for fetal cellular and gene therapy. Using hfMSC in fetal therapy offers the theoretical advantages of avoidance of immune rejection, increased engraftment, and treatment before disease pathology sets in. Aside from allogeneic hfMSC in utero transplantation, the use of autologous hfMSC has been brought a step forward with the development of early blood sampling techniques, efficient viral transduction and clonal expansion. Work is ongoing to determine hfMSC fate post-transplantation in murine models of genetic disease. In this review we will examine what is known about hfMSC biology, as well as discussing areas for future research. The

  5. Human induced pluripotent stem cell-derived models to investigate human cytomegalovirus infection in neural cells.

    Directory of Open Access Journals (Sweden)

    Leonardo D'Aiuto

    Full Text Available Human cytomegalovirus (HCMV infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs, neural progenitor cells (NPCs and neurons suggests that (i iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii Neural stem cells have impaired differentiation when infected by HCMV; (iii NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv most iPS-derived neurons are not permissive to HCMV infection; and (v infected neurons have impaired calcium influx in response to glutamate.

  6. Derivation of a Homozygous Human Androgenetic Embryonic Stem Cell Line.

    Science.gov (United States)

    Ding, Chenhui; Huang, Sunxing; Qi, Quan; Fu, Rui; Zhu, Wanwan; Cai, Bing; Hong, Pingping; Liu, Zhengxin; Gu, Tiantian; Zeng, Yanhong; Wang, Jing; Xu, Yanwen; Zhao, Xiaoyang; Zhou, Qi; Zhou, Canquan

    2015-10-01

    Human embryonic stem cells (hESCs) have long been considered as a promising source for cell replacement therapy. However, one major obstacle for the use of these cells is immune compatibility. Histocompatible human parthenogenetic ESCs have been reported as a new method for generating human leukocyte antigen (HLA)-matched hESCs. To further investigate the possibility of obtaining histocompatible stem cells from uniparental embryos, we tried to produce androgenetic haploid human embryos by injecting a single spermatozoon into enucleated human oocyte, and establish human androgenetic embryonic stem (hAGES) cell lines from androgenetic embryos. In the present study, a diploid hAGES cell line has been established, which exhibits typical features of human ESCs, including the expression of pluripotency markers, having differentiation potential in vitro and in vivo, and stable propagation in an undifferentiated state (>P40). Bisulfite sequencing of the H19, Snrpn, Meg3, and Kv imprinting control regions suggested that hAGES cells maintained to a certain extent a sperm methylation pattern. Genome-wide single nucleotide polymorphism, short tandem repeat, and HLA analyses revealed that the hAGES cell genome was highly homozygous. These results suggest that hAGES cells from spermatozoon could serve as a useful tool for studying the mechanisms underlying genomic imprinting in humans. It might also be used as a potential resource for cell replacement therapy as parthenogenetic stem cells.

  7. Protein conservation and variation suggest mechanisms of cell type-specific modulation of signaling pathways.

    Directory of Open Access Journals (Sweden)

    Martin H Schaefer

    2014-06-01

    Full Text Available Many proteins and signaling pathways are present in most cell types and tissues and yet perform specialized functions. To elucidate mechanisms by which these ubiquitous pathways are modulated, we overlaid information about cross-cell line protein abundance and variability, and evolutionary conservation onto functional pathway components and topological layers in the pathway hierarchy. We found that the input (receptors and the output (transcription factors layers evolve more rapidly than proteins in the intermediary transmission layer. In contrast, protein expression variability decreases from the input to the output layer. We observed that the differences in protein variability between the input and transmission layer can be attributed to both the network position and the tendency of variable proteins to physically interact with constitutively expressed proteins. Differences in protein expression variability and conservation are also accompanied by the tendency of conserved and constitutively expressed proteins to acquire somatic mutations, while germline mutations tend to occur in cell type-specific proteins. Thus, conserved core proteins in the transmission layer could perform a fundamental role in most cell types and are therefore less tolerant to germline mutations. In summary, we propose that the core signal transmission machinery is largely modulated by a variable input layer through physical protein interactions. We hypothesize that the bow-tie organization of cellular signaling on the level of protein abundance variability contributes to the specificity of the signal response in different cell types.

  8. Dynamic behaviour of human neuroepithelial cells in the developing forebrain

    Science.gov (United States)

    Subramanian, Lakshmi; Bershteyn, Marina; Paredes, Mercedes F.; Kriegstein, Arnold R.

    2017-01-01

    To understand how diverse progenitor cells contribute to human neocortex development, we examined forebrain progenitor behaviour using timelapse imaging. Here we find that cell cycle dynamics of human neuroepithelial (NE) cells differ from radial glial (RG) cells in both primary tissue and in stem cell-derived organoids. NE cells undergoing proliferative, symmetric divisions retract their basal processes, and both daughter cells regrow a new process following cytokinesis. The mitotic retraction of the basal process is recapitulated by NE cells in cerebral organoids generated from human-induced pluripotent stem cells. In contrast, RG cells undergoing vertical cleavage retain their basal fibres throughout mitosis, both in primary tissue and in older organoids. Our findings highlight developmentally regulated changes in mitotic behaviour that may relate to the role of RG cells to provide a stable scaffold for neuronal migration, and suggest that the transition in mitotic dynamics can be studied in organoid models. PMID:28139695

  9. Differentiated human stem cells resemble fetal, not adult, β cells.

    Science.gov (United States)

    Hrvatin, Sinisa; O'Donnell, Charles W; Deng, Francis; Millman, Jeffrey R; Pagliuca, Felicia Walton; DiIorio, Philip; Rezania, Alireza; Gifford, David K; Melton, Douglas A

    2014-02-25

    Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic β cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal β cells than adult β cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true β cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional β cells.

  10. Differential phospholipid-labeling suggests two subtypes of phospholipase D in rat Leydig cells

    DEFF Research Database (Denmark)

    Lauritzen, L.; Hansen, Harald S.

    1995-01-01

    The aim of the present study was to compare the transphosphatidylation activity of phospholipase D (PLD) under different substrate labeling conditions, in order to investigate whether PLD in rat Leydig cells exhibited any substrate preferences for the alkyl- or acyl-form of phosphatidylcholine (P...

  11. Acute progression of BCR-FGFR1 induced murine B-lympho/myeloproliferative disorder suggests involvement of lineages at the pro-B cell stage.

    Directory of Open Access Journals (Sweden)

    Mingqiang Ren

    Full Text Available Constitutive activation of FGFR1, through rearrangement with various dimerization domains, leads to atypical myeloproliferative disorders where, although T cell lymphoma are common, the BCR-FGFR1 chimeric kinase results in CML-like leukemia. As with the human disease, mouse bone marrow transduction/transplantation with BCR-FGFR1 leads to CML-like myeloproliferation as well as B-cell leukemia/lymphoma. The murine disease described in this report is virtually identical to the human disease in that both showed bi-lineage involvement of myeloid and B-cells, splenomegaly, leukocytosis and bone marrow hypercellularity. A CD19(+ IgM(- CD43(+ immunophenotype was seen both in primary tumors and two cell lines derived from these tumors. In all primary tumors, subpopulations of these CD19(+ IgM(- CD43(+ were also either B220(+ or B220(-, suggesting a block in differentiation at the pro-B cell stage. The B220(- phenotype was retained in one of the cell lines while the other was B220(+. When the two cell lines were transplanted into syngeneic mice, all animals developed the same B-lymphoblastic leukemia within 2-weeks. Thus, the murine model described here closely mimics the human disease with bilineage myeloid and B-cell leukemia/lymphoma which provides a representative model to investigate therapeutic intervention and a better understanding of the etiology of the disease.

  12. Matrilysin (MMP-7) Inhibition of BMP-7 Induced Renal Tubular Branching Morphogenesis Suggests a Role in the Pathogenesis of Human Renal Dysplasia

    Science.gov (United States)

    Harju-Baker, Susanna; Rims, Cliff; Sheen, Joong-Hyuk; Liapis, Helen

    2012-01-01

    Congenital renal dysplasia (RD) is a severe form of congenital renal malformation characterized by disruption of normal renal development with cyst formation, reduced or absent nephrons, and impaired renal growth. The authors previously identified that matrilysin (matrix metalloproteinase–7) was overexpressed in a microarray gene expression analysis of human RD compared to normal control kidneys. They now find that active matrilysin gene transcription and protein synthesis occur within dysplastic tubules and epithelial cells lining cysts in human RD by RT-PCR and immunohistochemistry. Similar staining patterns were seen in obstructed kidneys of pouch opossums that show histological features similar to that of human RD. In vitro, matrilysin inhibits formation of branching structures in mIMCD-3 cells stimulated by bone morphogenetic protein–7 (BMP-7) but does not inhibit hepatocyte growth factor–stimulated branching. BMP-7 signaling is essential for normal kidney development, and overexpression of catalytically active matrilysin in human embryonic kidney 293 cells reduces endogenous BMP-7 protein levels and inhibits phosphorylation of BMP-7 SMAD signaling intermediates. These findings suggest that matrilysin expression in RD may be an injury response that disrupts normal nephrogenesis by impairing BMP-7 signaling. PMID:22215634

  13. Role of human mast cells and basophils in bronchial asthma.

    Science.gov (United States)

    Marone, Gianni; Triggiani, Massimo; Genovese, Arturo; De Paulis, Amato

    2005-01-01

    Mast cells and basophils are the only cells expressing the tetrameric (alphabetagamma2) structure of the high affinity receptor for IgE (FcepsilonRI) and synthesizing histamine in humans. Human FcepsilonRI+ cells are conventionally considered primary effector cells of bronchial asthma. There is now compelling evidence that these cells differ immunologically, biochemically, and pharmacologically, which suggests that they might play distinct roles in the appearance and fluctuation of the asthma phenotype. Recent data have revealed the complexity of the involvement of human mast cells and basophils in asthma and have shed light on the control of recruitment and activation of these cells in different lung compartments. Preliminary evidence suggests that these cells might not always be detrimental in asthma but, under some circumstances, they might exert a protective effect by modulating certain aspects of innate and acquired immunity and allergic inflammation.

  14. Nanoscale Mechanical Stimulation of Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    H Nikukar

    2014-05-01

    We observed significant responses after 1 and 2-week stimulations in cell number, cell shapes and phenotypical markers. Microarray was performed for all groups. Cell count showed normal cell growth with stimulation. However, cell surface area, cell perimeter, and arboration after 1-week stimulation showed significant increases. Immunofluorescent studies have showed significant increase in osteocalcin production after stimulation. Conclusions: Nanoscale mechanical vibration showed significant changes in human mesenchymal stem cell behaviours. Cell morphology changed to become more polygonal and increased expression of the osteoblast markers were noted. These findings with gene regulation changes suggesting nanoscale mechanostimulation has stimulated osteoblastogenesis.  Keywords:  Mesenchymal, Nanoscale, Stem Cells.

  15. Modeling Pathogenic Mutations of Human Twinkle in Drosophila Suggests an Apoptosis Role in Response to Mitochondrial Defects

    Science.gov (United States)

    Sanchez-Martinez, Alvaro; Calleja, Manuel; Peralta, Susana; Matsushima, Yuichi; Hernandez-Sierra, Rosana; Whitworth, Alexander J.; Kaguni, Laurie S.; Garesse, Rafael

    2012-01-01

    The human gene C10orf2 encodes the mitochondrial replicative DNA helicase Twinkle, mutations of which are responsible for a significant fraction of cases of autosomal dominant progressive external ophthalmoplegia (adPEO), a human mitochondrial disease caused by defects in intergenomic communication. We report the analysis of orthologous mutations in the Drosophila melanogaster mitochondrial DNA (mtDNA) helicase gene, d-mtDNA helicase. Increased expression of wild type d-mtDNA helicase using the UAS-GAL4 system leads to an increase in mtDNA copy number throughout adult life without any noteworthy phenotype, whereas overexpression of d-mtDNA helicase containing the K388A mutation in the helicase active site results in a severe depletion of mtDNA and a lethal phenotype. Overexpression of two d-mtDNA helicase variants equivalent to two human adPEO mutations shows differential effects. The A442P mutation exhibits a dominant negative effect similar to that of the active site mutant. In contrast, overexpression of d-mtDNA helicase containing the W441C mutation results in a slight decrease in mtDNA copy number during the third instar larval stage, and a moderate decrease in life span in the adult population. Overexpression of d-mtDNA helicase containing either the K388A or A442P mutations causes a mitochondrial oxidative phosphorylation (OXPHOS) defect that significantly reduces cell proliferation. The mitochondrial impairment caused by these mutations promotes apoptosis, arguing that mitochondria regulate programmed cell death in Drosophila. Our study of d-mtDNA helicase overexpression provides a tractable Drosophila model for understanding the cellular and molecular effects of human adPEO mutations. PMID:22952820

  16. DNA repair responses in human skin cells

    Energy Technology Data Exchange (ETDEWEB)

    Hanawalt, P.C.; Liu, S.C.; Parsons, C.S.

    1981-07-01

    Sunlight and some environmental chemical agents produce lesions in the DNA of human skin cells that if unrepaired may interfere with normal functioning of these cells. The most serious outcome of such interactions may be malignancy. It is therefore important to develop an understanding of mechanisms by which the lesions may be repaired or tolerated without deleterious consequences. Our models for the molecular processing of damaged DNA have been derived largely from the study of bacterial systems. Some similarities but significant differences are revealed when human cell responses are tested against these models. It is also of importance to learn DNA repair responses of epidermal keratinocytes for comparison with the more extensive studies that have been carried out with dermal fibroblasts. Our experimental results thus far indicate similarities for the excision-repair of ultraviolet-induced pyrimidine dimers in human keratinocytes and fibroblasts. Both the monoadducts and the interstrand crosslinks produced in DNA by photoactivated 8-methoxypsoralen (PUVA) can be repaired in normal human fibroblasts but not in those from xeroderma pigmentosum patients. The monoadducts, like pyrimidine dimers, are probably the more mutagenic/carcinogenic lesions while the crosslinks are less easily repaired and probably result in more effective blocking of DNA function. It is suggested that a split-dose protocol that maximizes the production of crosslinks while minimizing the yield of monoadducts may be more effective and potentially less carcinogenic than the single ultraviolet exposure regimen in PUVA therapy for psoriasis.

  17. Human remains from the Pleistocene-Holocene transition of southwest China suggest a complex evolutionary history for East Asians.

    Science.gov (United States)

    Curnoe, Darren; Xueping, Ji; Herries, Andy I R; Kanning, Bai; Taçon, Paul S C; Zhende, Bao; Fink, David; Yunsheng, Zhu; Hellstrom, John; Yun, Luo; Cassis, Gerasimos; Bing, Su; Wroe, Stephen; Shi, Hong; Parr, William C H; Shengmin, Huang; Rogers, Natalie

    2012-01-01

    Later Pleistocene human evolution in East Asia remains poorly understood owing to a scarcity of well described, reliably classified and accurately dated fossils. Southwest China has been identified from genetic research as a hotspot of human diversity, containing ancient mtDNA and Y-DNA lineages, and has yielded a number of human remains thought to derive from Pleistocene deposits. We have prepared, reconstructed, described and dated a new partial skull from a consolidated sediment block collected in 1979 from the site of Longlin Cave (Guangxi Province). We also undertook new excavations at Maludong (Yunnan Province) to clarify the stratigraphy and dating of a large sample of mostly undescribed human remains from the site. We undertook a detailed comparison of cranial, including a virtual endocast for the Maludong calotte, mandibular and dental remains from these two localities. Both samples probably derive from the same population, exhibiting an unusual mixture of modern human traits, characters probably plesiomorphic for later Homo, and some unusual features. We dated charcoal with AMS radiocarbon dating and speleothem with the Uranium-series technique and the results show both samples to be from the Pleistocene-Holocene transition: ∼14.3-11.5 ka. Our analysis suggests two plausible explanations for the morphology sampled at Longlin Cave and Maludong. First, it may represent a late-surviving archaic population, perhaps paralleling the situation seen in North Africa as indicated by remains from Dar-es-Soltane and Temara, and maybe also in southern China at Zhirendong. Alternatively, East Asia may have been colonised during multiple waves during the Pleistocene, with the Longlin-Maludong morphology possibly reflecting deep population substructure in Africa prior to modern humans dispersing into Eurasia.

  18. Human Remains from the Pleistocene-Holocene Transition of Southwest China Suggest a Complex Evolutionary History for East Asians

    Science.gov (United States)

    Curnoe, Darren; Xueping, Ji; Herries, Andy I. R.; Kanning, Bai; Taçon, Paul S. C.; Zhende, Bao; Fink, David; Yunsheng, Zhu; Hellstrom, John; Yun, Luo; Cassis, Gerasimos; Bing, Su; Wroe, Stephen; Shi, Hong; Parr, William C. H.; Shengmin, Huang; Rogers, Natalie

    2012-01-01

    Background Later Pleistocene human evolution in East Asia remains poorly understood owing to a scarcity of well described, reliably classified and accurately dated fossils. Southwest China has been identified from genetic research as a hotspot of human diversity, containing ancient mtDNA and Y-DNA lineages, and has yielded a number of human remains thought to derive from Pleistocene deposits. We have prepared, reconstructed, described and dated a new partial skull from a consolidated sediment block collected in 1979 from the site of Longlin Cave (Guangxi Province). We also undertook new excavations at Maludong (Yunnan Province) to clarify the stratigraphy and dating of a large sample of mostly undescribed human remains from the site. Methodology/Principal Findings We undertook a detailed comparison of cranial, including a virtual endocast for the Maludong calotte, mandibular and dental remains from these two localities. Both samples probably derive from the same population, exhibiting an unusual mixture of modern human traits, characters probably plesiomorphic for later Homo, and some unusual features. We dated charcoal with AMS radiocarbon dating and speleothem with the Uranium-series technique and the results show both samples to be from the Pleistocene-Holocene transition: ∼14.3-11.5 ka. Conclusions/Significance Our analysis suggests two plausible explanations for the morphology sampled at Longlin Cave and Maludong. First, it may represent a late-surviving archaic population, perhaps paralleling the situation seen in North Africa as indicated by remains from Dar-es-Soltane and Temara, and maybe also in southern China at Zhirendong. Alternatively, East Asia may have been colonised during multiple waves during the Pleistocene, with the Longlin-Maludong morphology possibly reflecting deep population substructure in Africa prior to modern humans dispersing into Eurasia. PMID:22431968

  19. Human remains from the Pleistocene-Holocene transition of southwest China suggest a complex evolutionary history for East Asians.

    Directory of Open Access Journals (Sweden)

    Darren Curnoe

    Full Text Available BACKGROUND: Later Pleistocene human evolution in East Asia remains poorly understood owing to a scarcity of well described, reliably classified and accurately dated fossils. Southwest China has been identified from genetic research as a hotspot of human diversity, containing ancient mtDNA and Y-DNA lineages, and has yielded a number of human remains thought to derive from Pleistocene deposits. We have prepared, reconstructed, described and dated a new partial skull from a consolidated sediment block collected in 1979 from the site of Longlin Cave (Guangxi Province. We also undertook new excavations at Maludong (Yunnan Province to clarify the stratigraphy and dating of a large sample of mostly undescribed human remains from the site. METHODOLOGY/PRINCIPAL FINDINGS: We undertook a detailed comparison of cranial, including a virtual endocast for the Maludong calotte, mandibular and dental remains from these two localities. Both samples probably derive from the same population, exhibiting an unusual mixture of modern human traits, characters probably plesiomorphic for later Homo, and some unusual features. We dated charcoal with AMS radiocarbon dating and speleothem with the Uranium-series technique and the results show both samples to be from the Pleistocene-Holocene transition: ∼14.3-11.5 ka. CONCLUSIONS/SIGNIFICANCE: Our analysis suggests two plausible explanations for the morphology sampled at Longlin Cave and Maludong. First, it may represent a late-surviving archaic population, perhaps paralleling the situation seen in North Africa as indicated by remains from Dar-es-Soltane and Temara, and maybe also in southern China at Zhirendong. Alternatively, East Asia may have been colonised during multiple waves during the Pleistocene, with the Longlin-Maludong morphology possibly reflecting deep population substructure in Africa prior to modern humans dispersing into Eurasia.

  20. Kinetic properties of mouse pancreatic lipase-related protein-2 suggest the mouse may not model human fat digestion.

    Science.gov (United States)

    Xiao, Xunjun; Ross, Leah E; Miller, Rita A; Lowe, Mark E

    2011-05-01

    Genetically engineered mice have been employed to understand the role of lipases in dietary fat digestion with the expectation that the results can be extrapolated to humans. However, little is known about the properties of mouse pancreatic triglyceride lipase (mPTL) and pancreatic lipase-related protein-2 (mPLRP2). In this study, both lipases were expressed in Pichia Pastoris GS115, purified to near homogeneity, and their properties were characterized. Mouse PTL displayed the kinetics typical of PTL from other species. Like mPTL, mPLRP2 exhibited strong activity against various triglycerides. In contrast to mPTL, mPLRP2 was not inhibited by increasing bile salt concentration. Colipase stimulated mPLRP2 activity 2- to 4-fold. Additionally, mPTL absolutely required colipase for absorption to a lipid interface, whereas mPLRP2 absorbed fully without colipase. mPLRP2 had full activity in the presence of BSA, whereas BSA completely inhibited mPTL unless colipase was present. All of these properties of mPLRP2 differ from the properties of human PLRP2 (hPLRP2). Furthermore, mPLRP2 appears capable of compensating for mPTL deficiency. These findings suggest that the molecular mechanisms of dietary fat digestion may be different in humans and mice. Thus, extrapolation of dietary fat digestion in mice to humans should be done with care.

  1. Coevolution of paired receptors in Xenopus carcinoembryonic antigen-related cell adhesion molecule families suggests appropriation as pathogen receptors.

    Science.gov (United States)

    Zimmermann, Wolfgang; Kammerer, Robert

    2016-11-16

    In mammals, CEACAM1 and closely related members represent paired receptors with similar extracellular ligand-binding regions and cytoplasmic domains with opposing functions. Human CEACAM1 and CEACAM3 which have inhibitory ITIM/ITSM and activating ITAM-like motifs, respectively, in their cytoplasmic regions are such paired receptors. Various bacterial pathogens bind to CEACAM1 on epithelial and immune cells facilitating both entry into the host and down-regulation of the immune response whereas interaction with granulocyte-specific CEACAM3 leads to their uptake and destruction. It is unclear whether paired CEACAM receptors also exist in other vertebrate clades. We identified more than 80 ceacam genes in Xenopus tropicalis and X. laevis. They consist of two subgroups containing one or two putative paired receptor pairs each. Analysis of genomic sequences of paired receptors provide evidence that their highly similar ligand binding domains were adjusted by recent gene conversion events. In contrast, selection for diversification is observed among inhibitory receptor orthologs of the two frogs which split some 60 million years ago. The allotetraploid X. laevis arose later by hybridization of two closely related species. Interestingly, despite the conservation of the genomic landscape surrounding the homeologous ceacam loci only one locus resembles the one found in X. tropicalis. From the second X. laevis locus more than 80 % of the ceacam genes were lost including 5 of the 6 paired receptor genes. This suggests that once the gene for one of the paired receptors is lost the remaining gene cluster degrades rapidly probably due to lack of selection pressure exerted by pathogens. The presence of paired receptors and selection for diversification suggests that also in amphibians CEACAM1-related inhibitory proteins are or were used as pathogen receptors.

  2. The potency of human testicular stem cells

    NARCIS (Netherlands)

    Chikhovskaya, J.V.

    2013-01-01

    In this thesis, we evaluate the stem cell state of cells present in primary human testicular cell cultures as well as their origin and relation to germ or somatic lineages within testicular tissue. We conclude that human testis-derived embryonic stem cell-like (htES-like) colonies arising in primary

  3. Stem cell differentiation and human liver disease

    Institute of Scientific and Technical Information of China (English)

    Wen-Li Zhou; Claire N Medine; Liang Zhu; David C Hay

    2012-01-01

    Human stem cells are scalable cell populations capable of cellular differentiation.This makes them a very attractive in vitro cellular resource and in theory provides unlimited amounts of primary cells.Such an approach has the potential to improve our understanding of human biology and treating disease.In the future it may be possible to deploy novel stem cell-based approaches to treat human liver diseases.In recent years,efficient hepatic differentiation from human stem cells has been achieved by several research groups including our own.In this review we provide an overview of the field and discuss the future potential and limitations of stem cell technology.

  4. The human airway epithelial basal cell transcriptome.

    Directory of Open Access Journals (Sweden)

    Neil R Hackett

    Full Text Available BACKGROUND: The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. METHODOLOGY/PRINCIPAL FINDINGS: Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the "human airway basal cell signature" as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. CONCLUSION/SIGNIFICANCE: The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of

  5. Genetic engineering of human pluripotent cells using TALE nucleases.

    Science.gov (United States)

    Hockemeyer, Dirk; Wang, Haoyi; Kiani, Samira; Lai, Christine S; Gao, Qing; Cassady, John P; Cost, Gregory J; Zhang, Lei; Santiago, Yolanda; Miller, Jeffrey C; Zeitler, Bryan; Cherone, Jennifer M; Meng, Xiangdong; Hinkley, Sarah J; Rebar, Edward J; Gregory, Philip D; Urnov, Fyodor D; Jaenisch, Rudolf

    2011-07-07

    Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).

  6. Characterizing cancer cells with cancer stem cell-like features in 293T human embryonic kidney cells

    OpenAIRE

    Buchholz Thomas A; Lacerda Lara; Xu Wei; Robertson Fredika; Ueno Naoto T; Lucci Anthony; Landis Melissa D; Rodriguez Angel A; Li Li; Cohen Evan; Gao Hui; Krishnamurthy Savitri; Zhang Xiaomei; Debeb Bisrat G; Cristofanilli Massimo

    2010-01-01

    Abstract Background Since the first suggestion of prospectively identifiable cancer stem cells in solid tumors, efforts have been made to characterize reported cancer stem cell surrogates in existing cancer cell lines, and cell lines rich with these surrogates have been used to screen for cancer stem cell targeted agents. Although 293T cells were derived from human embryonic kidney, transplantation of these cells into the mammary fat pad yields aggressive tumors that self-renew as evidenced b...

  7. Low density of membrane particles in auditory hair cells of lizards and birds suggests an absence of somatic motility.

    Science.gov (United States)

    Köppl, Christine; Forge, Andrew; Manley, Geoffrey A

    2004-11-08

    Hair cells are the mechanoreceptive cells of the vertebrate lateral line and inner ear. In addition to their sensory function, hair cells display motility and thus themselves generate mechanical energy, which is thought to enhance sensitivity. Two principal cellular mechanism are known that can mediate hair-cell motility in vitro. One of these is based on voltage-dependent changes of an intramembrane protein and has so far been demonstrated only in outer hair cells of the mammalian cochlea. Correlated with this, the cell membranes of outer hair cells carry an extreme density of embedded particles, as revealed by freeze fracturing. The present study explored the possibility of membrane-based motility in hair cells of nonmammals, by determining their density of intramembrane particles. Replicas of freeze-fractured membrane were prepared from auditory hair cells of a lizard, the Tokay gecko, and a bird, the barn owl. These species were chosen because of independent evidence for active cochlear mechanics, in the form of spontaneous otoacoustic emissions. For quantitative comparison, mammalian inner and outer hair cells, as well as vestibular hair, cells were reevaluated. Lizard and bird hair cells displayed median densities of 2,360 and 1,880 intramembrane particles/microm2, respectively. This was not significantly different from the densities in vestibular and mammalian inner hair cells; however, it was about half the density in of mammalian outer hair cells. This suggests that nonmammalian hair cells do not possess high densities of motor protein in their membranes and are thus unlikely to be capable of somatic motility. 2004 Wiley-Liss, Inc.

  8. Embryonic death and the creation of human embryonic stem cells

    OpenAIRE

    Landry, Donald W.; Zucker, Howard A.

    2004-01-01

    The creation of human embryonic stem cells through the destruction of a human embryo pits the value of a potential therapeutic tool against that of an early human life. This contest of values has resulted in a polarized debate that neglects areas of common interest and perspective. We suggest that a common ground for pursuing research on human embryonic stem cells can be found by reconsidering the death of the human embryo and by applying to this research the ethical norms of essential organ ...

  9. Embryonic death and the creation of human embryonic stem cells.

    Science.gov (United States)

    Landry, Donald W; Zucker, Howard A

    2004-11-01

    The creation of human embryonic stem cells through the destruction of a human embryo pits the value of a potential therapeutic tool against that of an early human life. This contest of values has resulted in a polarized debate that neglects areas of common interest and perspective. We suggest that a common ground for pursuing research on human embryonic stem cells can be found by reconsidering the death of the human embryo and by applying to this research the ethical norms of essential organ donation.

  10. Differential representation of liver proteins in obese human subjects suggests novel biomarkers and promising targets for drug development in obesity.

    Science.gov (United States)

    Caira, Simonetta; Iannelli, Antonio; Sciarrillo, Rosaria; Picariello, Gianluca; Renzone, Giovanni; Scaloni, Andrea; Addeo, Pietro

    2017-12-01

    The proteome of liver biopsies from human obese (O) subjects has been compared to those of nonobese (NO) subjects using two-dimensional gel electrophoresis (2-DE). Differentially represented proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based peptide mass fingerprinting (PMF) and nanoflow-liquid chromatography coupled to electrospray-tandem mass spectrometry (nLC-ESI-MS/MS). Overall, 61 gene products common to all of the liver biopsies were identified within 65 spots, among which 25 ones were differently represented between O and NO subjects. In particular, over-representation of short-chain acyl-CoA dehydrogenase, Δ(3,5)-Δ(2,4)dienoyl-CoA isomerase, acetyl-CoA acetyltransferase, glyoxylate reductase/hydroxypyruvate reductase, fructose-biphosphate aldolase B, peroxiredoxin I, protein DJ-1, catalase, α- and β-hemoglobin subunits, 3-mercaptopyruvate S-transferase, calreticulin, aminoacylase 1, phenazine biosynthesis-like domain-containing protein and a form of fatty acid-binding protein, together with downrepresentation of glutamate dehydrogenase, glutathione S-transferase A1, S-adenosylmethionine synthase 1A and a form of apolipoprotein A-I, was associated with the obesity condition. Some of these metabolic enzymes and antioxidant proteins have already been identified as putative diagnostic markers of liver dysfunction in animal models of steatosis or obesity, suggesting additional investigations on their role in these syndromes. Their differential representation in human liver was suggestive of their consideration as obesity human biomarkers and for the development of novel antiobesity drugs.

  11. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

    Science.gov (United States)

    Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián

    2015-09-01

    Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.

  12. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos

    Directory of Open Access Journals (Sweden)

    Daniela Ávila-González

    2015-09-01

    Full Text Available Data from the literature suggest that human embryonic stem cell (hESC lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1 from poor-quality (PQ embryos derived and maintained on human amniotic epithelial cells (hAEC. This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.

  13. Restriction of human adenovirus replication in Chinese hamster cell lines and their hybrids with human cells.

    Science.gov (United States)

    Radna, R L; Foellmer, B; Feldman, L A; Francke, U; Ozer, H L

    1987-11-01

    We have found that the replication of human adenovirus (Ad2) is restricted in multiple Chinese hamster cell lines including CHO and V79. The major site of restriction involves differential accumulation of late viral proteins as demonstrated by immunofluorescence assay and polyacrylamide gel electrophoresis with and without prior immunoprecipitation. Synthesis of fiber and penton base are markedly reduced, whereas others, such as the 100K polypeptide, are synthesized efficiently. This pattern of restriction is similar to that previously reported for Ad2 infection of several monkey cell lines; however, the restriction is more marked in the Chinese hamster cell lines. The restriction is most likely due to a deficient cellular function since stable cell hybrids between V79 or CHO and human cells are permissive for virus replication. By analysis of a series of hybrids with reduced numbers of human chromosomes, fiber synthesis was correlated with the presence of the short arm of human chromosome 3. More hybrids showed restoration of fiber synthesis than production of progeny virus, suggesting that more than one unlinked function is required for the latter.

  14. The core regulatory network in human cells.

    Science.gov (United States)

    Kim, Man-Sun; Kim, Dongsan; Kang, Nam Sook; Kim, Jeong-Rae

    2017-03-04

    In order to discover the common characteristics of various cell types in the human body, many researches have been conducted to find the set of genes commonly expressed in various cell types and tissues. However, the functional characteristics of a cell is determined by the complex regulatory relationships among the genes rather than by expressed genes themselves. Therefore, it is more important to identify and analyze a core regulatory network where all regulatory relationship between genes are active across all cell types to uncover the common features of various cell types. Here, based on hundreds of tissue-specific gene regulatory networks constructed by recent genome-wide experimental data, we constructed the core regulatory network. Interestingly, we found that the core regulatory network is organized by simple cascade and has few complex regulations such as feedback or feed-forward loops. Moreover, we discovered that the regulatory links from genes in the core regulatory network to genes in the peripheral regulatory network are much more abundant than the reverse direction links. These results suggest that the core regulatory network locates at the top of regulatory network and plays a role as a 'hub' in terms of information flow, and the information that is common to all cells can be modified to achieve the tissue-specific characteristics through various types of feedback and feed-forward loops in the peripheral regulatory networks. We also found that the genes in the core regulatory network are evolutionary conserved, essential and non-disease, non-druggable genes compared to the peripheral genes. Overall, our study provides an insight into how all human cells share a common function and generate tissue-specific functional traits by transmitting and processing information through regulatory network.

  15. Gene expression profiling of CD8(+) T cells induced by ovarian cancer cells suggests a possible mechanism for CD8(+) Treg cell production.

    Science.gov (United States)

    Wu, Meng; Lou, Jianfang; Zhang, Shuping; Chen, Xian; Huang, Lei; Sun, Ruihong; Huang, Peijun; Pan, Shiyang; Wang, Fang

    2016-12-01

    The aim of this study was to investigate a possible mechanism of CD8(+) regulatory T-cell (Treg) production in an ovarian cancer (OC) microenvironment. Agilent microarray was used to detect changes in gene expression between CD8(+) T cells cultured with and without the SKOV3 ovarian adenocarcinoma cell line. QRT-PCR was performed to determine glycolysis gene expression in CD8(+) T cells from a transwell culturing system and OC patients. We also detected protein levels of glycolysis-related genes using Western blot analysis. Comparing gene expression profiles revealed significant differences in expression levels of 1420 genes, of which 246 were up-regulated and 1174 were down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis indicated that biological processes altered in CD8(+) Treg are particularly associated with energy metabolism. CD8(+) Treg cells induced by co-culture with SKOV3 had lower glycolysis gene expression compared to CD8(+) T cells cultured alone. Glycolysis gene expression was also decreased in the CD8(+) T cells of OC patients. These findings provide a comprehensive bioinformatics analysis of DEGs in CD8(+) T cells cultured with and without SKOV3 and suggests that metabolic processes may be a possible mechanism for CD8(+) Treg induction. © 2016 John Wiley & Sons Ltd.

  16. The evolution of signaling complexity suggests a mechanism for reducing the genomic search space in human association studies.

    Science.gov (United States)

    Irizarry, K J L; Merriman, B; Bahamonde, M E; Wong, M-L; Licinio, J

    2005-01-01

    the genome. We suggest that the development and use of theoretical models can provide insight into the nature of biological systems and may lead to significant improvements in computational algorithms designed to reduce the complexity of the human genome.

  17. Convergent evidence from mouse and human studies suggests the involvement of zinc finger protein 326 gene in antidepressant treatment response.

    Directory of Open Access Journals (Sweden)

    Ying-Jay Liou

    Full Text Available OBJECTIVES: The forced swim test (FST is a commonly used model to predict antidepressant efficacy. Uncovering the genetic basis of the model may unravel the mechanism of antidepressant treatment. METHODS: FVB/NJ (FVB and C57BL/6J (B6 were first identified as the response and non-response strains to fluoxetine (a serotonin-specific reuptake inhibitor antidepressant treatment in the mouse FST. Simple-interval (SIM and composite-interval (CIM mappings were applied to map the quantitative trait loci (QTLs of the anti-immobility effect of fluoxetine in FST (FST(FLX in 865 male B6×FVB-F2 mice. The brain mRNA expressions of the gene with the maximum QTL-linkage signal for FST(FLX after the FST were compared between B6 and FVB mice and also compared between fluoxetine and saline treatment. The association of the variants in the human homologue of the mouse FST(FLX-QTL gene with major depressive disorder (MDD and antidepressant response were investigated in 1080 human subjects (MDD/control = 582/498. RESULTS: One linkage signal for FST(FLX-QTL was detected at an intronic SNP (rs6215396 of the mouse Zfp326 gene (maximal CIM-LOD = 9.36. The Zfp326 mRNA expression in the FVB thalamus was significantly down-regulated by fluoxetine in the FST, and the higher FVB-to-B6 Zfp326 mRNA expressions in the frontal cortex, striatum and hypothalamus diminished after fluoxetine treatment. Two coding-synonymous SNPs (rs2816881 and rs10922744 in the human homologue of Zfp326, ZNF326, were significantly associated with the 8-week antidepressant treatment response in the MDD patients (Bonferroni-corrected p = 0.004-0.028. CONCLUSIONS: The findings suggest the involvement of the Zfp326 and ZNF326 genes in antidepressant treatment response.

  18. Expression of the human TSPY gene in the brains of transgenic mice suggests a potential role of this Y chromosome gene in neural functions

    Institute of Scientific and Technical Information of China (English)

    Tatsuo Kido; Stephanie Schubert; J(o)rg Schmidtke; Yun-Fai Chris Lau

    2011-01-01

    The testis specific protein Y-encoded (TSPY) is a member of TSPY/SET/NAPl superfamily, encoded within the gonadoblastoma locus on the Y chromosome. TSPY shares a highly conserved SET/NAP-domain responsible for protein-protein interaction among TSPY/SET/NAPl proteins.Accumulating data, so far, support the role of TSPY as the gonadoblastoma gene, involved in germ cell tumorigenesis. The X-chromosome homolog of TSPY, TSPX is expressed in various tissues at both fetal and adult stages, including the brain, and is capable of interacting with the multi-domain adapter protein CASK, thereby influencing the synaptic and transcriptional functions and developmental regulation of CASK in the brain and other neural tissues. Similar to TSPX, we demonstrated that TSPY could interact with CASK at its SET/NAP-domain in cultured cells. Transgenic mice harboring a human TSPY gene and flanking sequences showed specific expression of the human TSPYtransgene in both testis and brain. The neural expression pattern of the human TSPY gene overlapped with those of the endogenous mouse Cask and Tspx gene. Similarly with TSPX, TSPY was co-localized with CASK in neuronal axon fibers in the brain, suggesting a potential role(s) of TSPY in development and/or physiology of the nervous system.

  19. Dopamine receptor repertoire of human granulosa cells

    Directory of Open Access Journals (Sweden)

    Kunz Lars

    2007-10-01

    in the absence of extracellular calcium and was abolished by a D2 blocker (L-741,626. DA treatment (48 h of human GCs resulted in slightly, but significantly enlarged, viable cells. Conclusion A previous study showed D2 in human GCs, which are linked to cAMP, and the present study reveals the full spectrum of DA receptors present in these endocrine cells, which also includes D2-like receptors, linked to calcium. Ovarian DA can act thus via D1,2,4,5, which are co-expressed by endocrine cells of the follicle and the corpus luteum and are linked to different signaling pathways. This suggests a complex role of DA in the regulation of ovarian processes.

  20. Limited Gene Expression Variation in Human Embryonic Stem Cell and Induced Pluripotent Stem Cell Derived Endothelial Cells

    OpenAIRE

    2013-01-01

    Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes, yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified, homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here, we report a differentiation protocol based ...

  1. Human B cells produce chemokine CXCL10 in the presence of Mycobacterium tuberculosis specific T cells

    DEFF Research Database (Denmark)

    Hoff, Soren T; Salman, Ahmed M; Ruhwald, Morten

    2015-01-01

    BACKGROUND: The role of B cells in human host response to Mycobacterium tuberculosis (Mtb) infection is still controversial, but recent evidence suggest that B cell follicle like structures within the lung may influence host responses through regulation of the local cytokine environment....... A candidate for such regulation could be the chemokine CXCL10. CXCL10 is mainly produced by human monocytes, but a few reports have also found CXCL10 production by human B cells. The objective of this study was to investigate CXCL10 production by human B cells in response to in vitro stimulation with Mtb...... antigens. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed human blood samples from 30 volunteer donors using multiparameter flow cytometry, and identified a subgroup of B cells producing CXCL10 in response to in vitro stimulation with antigens. T cells did not produce CXCL10, but CXCL10 production by B cells...

  2. Overlapping gene structure of the human neuropeptide Y receptor subtypes Y1 and Y5 suggests coordinate transcriptional regulation

    Energy Technology Data Exchange (ETDEWEB)

    Herzog, H.; Darby, K.; Ball, H. [St. Vincent`s Hospital, Sydney (Australia)] [and others

    1997-05-01

    The human y1 and y5 receptor genes are transcribed in opposite directions from a common promoter region on chromosome 4q31-q32. One of the alternately spliced 5{prime} exons of the y1 receptor gene (1C) is also an integral part of the coding region of a novel neuropeptide Y receptor, Y5. Exon 1C of the y1 receptor gene, if translated from the opposite strand, encodes sequences corresponding to the large third intracellular loop of the Y5 receptor. The close proximity of the two neuropeptide Y receptor genes suggests that they have evolved from a gene duplication event with the small intron interrupting the coding sequence of the y1 gene being converted into a functional sequence within the y5 gene, while the reverse complementary sequence was utilized as an alternatively spliced 5{prime} exon for the y1 gene. The transcription of both genes from opposite strands of the same DNA sequence suggests that transcriptional activation of one will have an effect on the regulation of gene expression of the other. As both Y1 and Y5 receptors are thought to play an important role in the regulation of food intake, coordinate expression of their specific genes may be important in the modulation of NPY activity. 23 refs., 2 figs.

  3. IMMUNORESPONSES OF HUMANIZED SCID MICE TO HUMAN LUNG CANCER CELLS

    Institute of Scientific and Technical Information of China (English)

    陈力真; 王树蕙; 张云; 王世真

    1996-01-01

    HuPBL-SCID mice were used to explore how they would response to human ttmoor cells of 801/MLC.Living 801/MLC cells appeared to be fetal to the the mice due to the production of human TNF. The huP-BL-SCID rniee did not generate any noticeable amotmt of specific human immunoglobttlin either by single immunization with living 801/MLC cells or by repeated immunization with irradiated 801/MLC cells. Our preliminary experiments with huPBL-SCID mice showed that such chimeras would he a very useful models for tumor immunological researches.

  4. Hsp10 nuclear localization and changes in lung cells response to cigarette smoke suggest novel roles for this chaperonin.

    Science.gov (United States)

    Corrao, Simona; Anzalone, Rita; Lo Iacono, Melania; Corsello, Tiziana; Di Stefano, Antonino; D'Anna, Silvestro Ennio; Balbi, Bruno; Carone, Mauro; Sala, Anna; Corona, Davide; Timperio, Anna Maria; Zolla, Lello; Farina, Felicia; de Macario, Everly Conway; Macario, Alberto J L; Cappello, Francesco; La Rocca, Giampiero

    2014-10-01

    Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Here, we show that Hsp10 in COPD undergoes changes at the molecular and subcellular levels in bronchial cells from human specimens and derived cell lines, intact or subjected to stress induced by cigarette smoke extract (CSE). Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein.

  5. Hsp10 nuclear localization and changes in lung cells response to cigarette smoke suggest novel roles for this chaperonin

    Science.gov (United States)

    Corrao, Simona; Anzalone, Rita; Lo Iacono, Melania; Corsello, Tiziana; Di Stefano, Antonino; D'Anna, Silvestro Ennio; Balbi, Bruno; Carone, Mauro; Sala, Anna; Corona, Davide; Timperio, Anna Maria; Zolla, Lello; Farina, Felicia; Conway de Macario, Everly; Macario, Alberto J. L.; Cappello, Francesco; La Rocca, Giampiero

    2014-01-01

    Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Here, we show that Hsp10 in COPD undergoes changes at the molecular and subcellular levels in bronchial cells from human specimens and derived cell lines, intact or subjected to stress induced by cigarette smoke extract (CSE). Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein. PMID:25355063

  6. Endothelial cells derived from human embryonic stem cells

    Science.gov (United States)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  7. Exposure to Music Alters Cell Viability and Cell Motility of Human Nonauditory Cells in Culture

    Directory of Open Access Journals (Sweden)

    Nathalia R. Lestard

    2016-01-01

    Full Text Available Although music is part of virtually all cultures in the world, little is known about how it affects us. Since the beginning of this century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. The present study was designed to better understand the direct effects of acoustic vibrations, in the form of music, in human cells in culture. Our results suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and nonauditory cells.

  8. Induced pluripotent stem cell lines derived from human somatic cells.

    Science.gov (United States)

    Yu, Junying; Vodyanik, Maxim A; Smuga-Otto, Kim; Antosiewicz-Bourget, Jessica; Frane, Jennifer L; Tian, Shulan; Nie, Jeff; Jonsdottir, Gudrun A; Ruotti, Victor; Stewart, Ron; Slukvin, Igor I; Thomson, James A

    2007-12-21

    Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

  9. Generation of human melanocytes from induced pluripotent stem cells.

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    Shigeki Ohta

    Full Text Available Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders. Additionally, melanomas are considered to arise from mutations that accumulate in melanocyte stem cells. The mechanisms underlying melanocyte differentiation and the defining characteristics of melanocyte stem cells in humans are, however, largely unknown. In the present study, we set out to generate melanocytes from human iPS cells in vitro, leading to a preliminary investigation of the mechanisms of human melanocyte differentiation. We generated iPS cell lines from human dermal fibroblasts using the Yamanaka factors (SOX2, OCT3/4, and KLF4, with or without c-MYC. These iPS cell lines were subsequently used to form embryoid bodies (EBs and then differentiated into melanocytes via culture supplementation with Wnt3a, SCF, and ET-3. Seven weeks after inducing differentiation, pigmented cells expressing melanocyte markers such as MITF, tyrosinase, SILV, and TYRP1, were detected. Melanosomes were identified in these pigmented cells by electron microscopy, and global gene expression profiling of the pigmented cells showed a high similarity to that of human primary foreskin-derived melanocytes, suggesting the successful generation of melanocytes from iPS cells. This in vitro differentiation system should prove useful for understanding human melanocyte biology and revealing the mechanism of various pigment cell disorders, including melanoma.

  10. Modeling of the human rhinovirus C capsid suggests a novel topography with insights on receptor preference and immunogenicity.

    Science.gov (United States)

    Basta, Holly A; Sgro, Jean-Yves; Palmenberg, Ann C

    2014-01-05

    Features of human rhinovirus (RV)-C virions that allow them to use novel cell receptors and evade immune responses are unknown. Unlike the RV-A+B, these isolates cannot be propagated in typical culture systems or grown for structure studies. Comparative sequencing, I-TASSER, MODELLER, ROBETTA, and refined alignment techniques led to a structural approximation for C15 virions, based on the extensive, resolved RV-A+B datasets. The model predicts that all RV-C VP1 proteins are shorter by 21 residues relative to the RV-A, and 35 residues relative to the RV-B, effectively shaving the RV 5-fold plateau from the particle. There are major alterations in VP1 neutralizing epitopes and the structural determinants for ICAM-1 and LDLR receptors. The VP2 and VP3 elements are similar among all RV, but the loss of sequence "words" contributing Nim1ab has increased the apparent selective pressure among the RV-C to fix mutations elsewhere in the VP1, creating a possible compensatory epitope.

  11. Generating trunk neural crest from human pluripotent stem cells.

    Science.gov (United States)

    Huang, Miller; Miller, Matthew L; McHenry, Lauren K; Zheng, Tina; Zhen, Qiqi; Ilkhanizadeh, Shirin; Conklin, Bruce R; Bronner, Marianne E; Weiss, William A

    2016-01-27

    Neural crest cells (NCC) are stem cells that generate different lineages, including neuroendocrine, melanocytic, cartilage, and bone. The differentiation potential of NCC varies according to the level from which cells emerge along the neural tube. For example, only anterior "cranial" NCC form craniofacial bone, whereas solely posterior "trunk" NCC contribute to sympathoadrenal cells. Importantly, the isolation of human fetal NCC carries ethical and scientific challenges, as NCC induction typically occur before pregnancy is detectable. As a result, current knowledge of NCC biology derives primarily from non-human organisms. Important differences between human and non-human NCC, such as expression of HNK1 in human but not mouse NCC, suggest a need to study human NCC directly. Here, we demonstrate that current protocols to differentiate human pluripotent stem cells (PSC) to NCC are biased toward cranial NCC. Addition of retinoic acid drove trunk-related markers and HOX genes characteristic of a posterior identity. Subsequent treatment with bone morphogenetic proteins (BMPs) enhanced differentiation to sympathoadrenal cells. Our approach provides methodology for detailed studies of human NCC, and clarifies roles for retinoids and BMPs in the differentiation of human PSC to trunk NCC and to sympathoadrenal lineages.

  12. Human embryonic stem cells and microenvironment

    Directory of Open Access Journals (Sweden)

    Banu İskender

    2014-09-01

    Full Text Available Human embryonic stem cells (hESCs possess a great potential in the field of regenerative medicine by their virtue of pluripotent potential with indefinite proliferation capabilities. They can self renew themselves and differentiate into three embryonic germ layers. Although they are conventionally grown on mitotically inactivated mouse feeder cells, there are in vitro culture systems utilizing feeder cells of human origin in order to prevent cross-species contamination. Recently established in vitro culture systems suggested that direct interaction with feeder cells is not necessary but rather attachment to a substrate is required to ensure long-term, efficient hESC culture in vitro. This substrate is usually composed of a mixture of extracellular matrix components representing in vivo natural niche. In hESC biology, the mechanism of interaction of hESCs with extracellular matrix molecules remained insufficiently explored area of research due to their transient nature of interaction with the in vivo niche. However, an in vitro culture system established using extracellular matrix molecules may provide a safer alternative to culture systems with feeder cells while paving the way to Good Manufacturing Practice-GMP production of hESCs for therapeutic purposes. Therefore, it is essential to study the interaction of extracellular matrix molecules with hESCs in order to standardize in vitro culture systems for large-scale production of hESCs in a less labor-intensive way. This would not only provide valuable information regarding the mechanisms that control pluripotency but also serve to dissect the molecular signaling pathways of directed differentiation for prospective therapeutic applications in the future. J Clin Exp Invest 2014; 5 (3: 486-495

  13. Human induced pluripotent stem cells on autologous feeders.

    Directory of Open Access Journals (Sweden)

    Kazutoshi Takahashi

    Full Text Available BACKGROUND: For therapeutic usage of induced Pluripotent Stem (iPS cells, to accomplish xeno-free culture is critical. Previous reports have shown that human embryonic stem (ES cells can be maintained in feeder-free condition. However, absence of feeder cells can be a hostile environment for pluripotent cells and often results in karyotype abnormalities. Instead of animal feeders, human fibroblasts can be used as feeder cells of human ES cells. However, one still has to be concerned about the existence of unidentified pathogens, such as viruses and prions in these non-autologous feeders. METHODOLOGY/PRINCIPAL FINDINGS: This report demonstrates that human induced Pluripotent Stem (iPS cells can be established and maintained on isogenic parental feeder cells. We tested four independent human skin fibroblasts for the potential to maintain self-renewal of iPS cells. All the fibroblasts tested, as well as their conditioned medium, were capable of maintaining the undifferentiated state and normal karyotypes of iPS cells. Furthermore, human iPS cells can be generated on isogenic parental fibroblasts as feeders. These iPS cells carried on proliferation over 19 passages with undifferentiated morphologies. They expressed undifferentiated pluripotent cell markers, and could differentiate into all three germ layers via embryoid body and teratoma formation. CONCLUSIONS/SIGNIFICANCE: These results suggest that autologous fibroblasts can be not only a source for iPS cells but also be feeder layers. Our results provide a possibility to solve the dilemma by using isogenic fibroblasts as feeder layers of iPS cells. This is an important step toward the establishment of clinical grade iPS cells.

  14. HIF induces human embryonic stem cell markers in cancer cells.

    Science.gov (United States)

    Mathieu, Julie; Zhang, Zhan; Zhou, Wenyu; Wang, Amy J; Heddleston, John M; Pinna, Claudia M A; Hubaud, Alexis; Stadler, Bradford; Choi, Michael; Bar, Merav; Tewari, Muneesh; Liu, Alvin; Vessella, Robert; Rostomily, Robert; Born, Donald; Horwitz, Marshall; Ware, Carol; Blau, C Anthony; Cleary, Michele A; Rich, Jeremy N; Ruohola-Baker, Hannele

    2011-07-01

    Low oxygen levels have been shown to promote self-renewal in many stem cells. In tumors, hypoxia is associated with aggressive disease course and poor clinical outcomes. Furthermore, many aggressive tumors have been shown to display gene expression signatures characteristic of human embryonic stem cells (hESC). We now tested whether hypoxia might be responsible for the hESC signature observed in aggressive tumors. We show that hypoxia, through hypoxia-inducible factor (HIF), can induce an hESC-like transcriptional program, including the induced pluripotent stem cell (iPSC) inducers, OCT4, NANOG, SOX2, KLF4, cMYC, and microRNA-302 in 11 cancer cell lines (from prostate, brain, kidney, cervix, lung, colon, liver, and breast tumors). Furthermore, nondegradable forms of HIFα, combined with the traditional iPSC inducers, are highly efficient in generating A549 iPSC-like colonies that have high tumorigenic capacity. To test potential correlation between iPSC inducers and HIF expression in primary tumors, we analyzed primary prostate tumors and found a significant correlation between NANOG-, OCT4-, and HIF1α-positive regions. Furthermore, NANOG and OCT4 expressions positively correlated with increased prostate tumor Gleason score. In primary glioma-derived CD133 negative cells, hypoxia was able to induce neurospheres and hESC markers. Together, these findings suggest that HIF targets may act as key inducers of a dynamic state of stemness in pathologic conditions.

  15. Cell cycle regulation by glucosamine in human pulmonary epithelial cells.

    Science.gov (United States)

    Chuang, Kun-Han; Lu, Chih-Shen; Kou, Yu Ru; Wu, Yuh-Lin

    2013-04-01

    Airway epithelial cells play an important role against intruding pathogens. Glucosamine, a commonly used supplemental compound, has recently begun to be regarded as a potential anti-inflammatory molecule. This study aimed to uncover how glucosamine impacts on cellular proliferation in human alveolar epithelial cells (A549) and bronchial epithelial cells (HBECs). With trypan blue-exclusion assay, we observed that glucosamine (10, 20, 50 mM) caused a decrease in cell number at 24 and 48 h; with a flow cytometric analysis, we also noted an enhanced cell accumulation within the G(0)/G(1) phase at 24 h and induction of late apoptosis at 24 and 48 h by glucosamine (10, 20, 50 mM) in A549 cells and HBECs. Examination of phosphorylation in retinoblastoma (Rb) protein, we found an inhibitory effect by glucosamine at 20 and 50 mM. Glucosamine at 50 mM was demonstrated to elevate both the mRNA and protein expression of p53 and heme oxygenase-1 (HO-1), but also caused a reduction in p21 protein expression. In addition, glucosamine attenuated p21 protein stability via the proteasomal proteolytic pathway, as well as inducing p21 nuclear accumulation. Altogether, our results suggest that a high dose of glucosamine may inhibit cell proliferation through apoptosis and disturb cell cycle progression with a halt at G(0)/G(1) phase, and that this occurs, at least in part, by a reduction in Rb phosphorylation together with modulation of p21, p53 and HO-1 expression, and nuclear p21 accumulation.

  16. Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ramasharma, K.; Li, C.H.

    1987-05-01

    Human granulosa cells cultured with calf serum actively proliferated for 18-20 generations and secreted progesterone into the medium; progesterone levels appeared to decline with increase in generation number. Cells cultured under serum-free conditions secreted significant amounts of progesterone and insulin-like growth factor II (IGF-II). The progesterone secretion was enhanced by the addition of human follitropin, lutropin, and chorionic gonadotropin but not by growth hormone. These cells, when challenged to varying concentrations of human growth hormone, human chorionic somatomammotropin, human prolactin, chorionic gonadotropin, follitropin, and lutropin, secreted IGF-II into the medium as measured by specific IGF-II RIA. Among these human hormones, chorionic gonadotropin, follitropin, and lutropin were most effective in inducing IGF-II secretion from these cells. When synthetic lutropin-releasing hormone and ..cap alpha..-inhibin-92 were tested, only lutropin-releasing hormone was effective in releasing IGF-II. The results described suggest that cultured human granulosa cells can proliferate and actively secrete progesterone and IGF-II into the medium. IGF-II production in human granulosa cells was influenced by a multi-hormonal complex including human growth hormone, human chorionic somatomammotropin, and prolactin.

  17. Human Rights Education in Japan: An Historical Account, Characteristics and Suggestions for a Better-Balanced Approach

    Science.gov (United States)

    Takeda, Sachiko

    2012-01-01

    Although human rights are often expressed as universal tenets, the concept was conceived in a particular socio-political and historical context. Conceptualisations and practice of human rights vary across societies, and face numerous challenges. After providing an historical account of the conceptualisation of human rights in Japanese society,…

  18. Human Rights Education in Japan: An Historical Account, Characteristics and Suggestions for a Better-Balanced Approach

    Science.gov (United States)

    Takeda, Sachiko

    2012-01-01

    Although human rights are often expressed as universal tenets, the concept was conceived in a particular socio-political and historical context. Conceptualisations and practice of human rights vary across societies, and face numerous challenges. After providing an historical account of the conceptualisation of human rights in Japanese society,…

  19. In vitro methods to culture primary human breast epithelial cells.

    Science.gov (United States)

    Raouf, Afshin; Sun, Yu Jia

    2013-01-01

    Current evidence suggests that much like leukemia, breast tumors are maintained by a small subpopulation of tumor cells that have stem cell properties. These cancer stem cells are envisaged to be responsible for tumor formation and relapse. Therefore, knowledge about their nature will provide a platform to develop therapies to eliminate these breast cancer stem cells. This concept highlights the need to understand the mechanisms that regulate the normal functions of the breast stem cells and their immediate progeny as alterations to these same mechanisms can cause these primitive cells to act as cancer stem cells. The study of the primitive cell functions relies on the ability to isolate them from primary sources of breast tissue. This chapter describes processing of discarded tissue from reduction mammoplasty samples as sources of normal primary human breast epithelial cells and describes cell culture systems to grow single-cell suspensions prepared from these reduction samples in vitro.

  20. Human Neural Cell-Based Biosensor

    Science.gov (United States)

    2013-05-28

    including incubation with factors such as SHH ) and proceed to Human Neural Progenitor Cells Dopaminergic Differentiation β-III Tubulin/TH...exposure in human embryonic stem cells. J Recept Signal Transduct Res. 2011 Jun;31(3):206-13. Gerwe BA, Angel PM, West FD, Hasneen K, Young A

  1. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

    Energy Technology Data Exchange (ETDEWEB)

    Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.; Hession, Catherine A.; Miatkowski, Konrad; Wildes, Craig P.; Cullen, Patrick F.; Hong, Victor; Hopkins, Brian T.; Mertsching, Elisabeth; Jenkins, Tracy J.; Romanowski, Michael J.; Baker, Darren P.; Silvian, Laura F. (Sunesis); (Biogen)

    2010-11-15

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

  2. Absence of C-type virus production in human leukemic B cell, T cell and null cell lines.

    Directory of Open Access Journals (Sweden)

    Ogura,Hajime

    1978-06-01

    Full Text Available Electron microscope observation of cultured human leukemic B cell, T cell and null cell lines and reverse transcriptase assay of the culture supernatants were all negative for the presence of C-type virus. Bat cell line, which propagates primate C-type viruses well, was cocultivated with the human leukemic cell lines, in the hope of amplification of virus if present. Three weeks after mixed culture, the culture supernatants were again examined for reverse transcriptase activity and the cells were tested for syncytia formation by cocultivation with rat XC, human KC and RSb cell lines. All these tests, except for the positive control using a simian sarcoma virus, were negative, suggesting that no C-type was produced from these human leukemic cell lines.

  3. Comparative analysis of protocadherin-11 X-linked expression among postnatal rodents, non-human primates, and songbirds suggests its possible involvement in brain evolution.

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    Eiji Matsunaga

    Full Text Available BACKGROUND: Protocadherin-11 is a cell adhesion molecule of the cadherin superfamily. Since, only in humans, its paralog is found on the Y chromosome, it is expected that protocadherin-11X/Y plays some role in human brain evolution or sex differences. Recently, a genetic mutation of protocadherin-11X/Y was reported to be associated with a language development disorder. Here, we compared the expression of protocadherin-11 X-linked in developing postnatal brains of mouse (rodent and common marmoset (non-human primate to explore its possible involvement in mammalian brain evolution. We also investigated its expression in the Bengalese finch (songbird to explore a possible function in animal vocalization and human language faculties. METHODOLOGY/PRINCIPAL FINDINGS: Protocadherin-11 X-linked was strongly expressed in the cerebral cortex, hippocampus, amygdala and brainstem. Comparative analysis between mice and marmosets revealed that in certain areas of marmoset brain, the expression was clearly enriched. In Bengalese finches, protocadherin-11 X-linked was expressed not only in nuclei of regions of the vocal production pathway and the tracheosyringeal hypoglossal nucleus, but also in areas homologous to the mammalian amygdala and hippocampus. In both marmosets and Bengalese finches, its expression in pallial vocal control areas was developmentally regulated, and no clear expression was seen in the dorsal striatum, indicating a similarity between songbirds and non-human primates. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the enriched expression of protocadherin-11 X-linked is involved in primate brain evolution and that some similarity exists between songbirds and primates regarding the neural basis for vocalization.

  4. iTRAQ analysis of colorectal cancer cell lines suggests Drebrin (DBN1) is overexpressed during liver metastasis.

    Science.gov (United States)

    Lin, Qifeng; Tan, Hwee Tong; Lim, Teck Kwang; Khoo, Avery; Lim, Kiat Hon; Chung, Maxey C M

    2014-06-01

    Colorectal cancer is currently the third in cancer incidence worldwide and the fourth most common cause of cancer deaths. Mortality in colorectal cancer is often ascribed to liver metastasis. In an effort to elucidate the proteins involved in colorectal cancer liver metastasis, we compared the proteome profiles of the human colon adenocarcinoma cell line HCT-116 with its metastatic derivative E1, using the iTRAQ labelling technology, coupled to 2D-LC and MALDI-TOF/TOF MS. A total of 547 proteins were identified, of which 31 of them were differentially expressed in the E1 cell line. Among these proteins, the differential expressions of translationally controlled tumour protein 1, A-kinase anchor protein 12 and Drebrin (DBN1) were validated using Western blot. In particular, DBN1, a protein not previously known to be involved in colorectal cancer metastasis, was found to be overexpressed in E1 as compared to HCT-116 cells. The overexpression of DBN1 was further validated using immunohistochemistry on colorectal cancer tissue sections with matched lymph node and liver metastasis tissues. DBN1 is currently believed to be involved in actin cytoskeleton reorganisation and suppresses actin filament cross-linking and bundling. Since actin reorganisation is an important process for tumour cell migration and invasion, DBN1 may have an important role during colorectal cancer metastasis.

  5. Growth-stimulatory effect of resveratrol in human cancer cells.

    Science.gov (United States)

    Fukui, Masayuki; Yamabe, Noriko; Kang, Ki Sung; Zhu, Bao Ting

    2010-08-01

    Earlier studies have shown that resveratrol could induce death in several human cancer cell lines in culture. Here we report our observation that resveratrol can also promote the growth of certain human cancer cells when they are grown either in culture or in athymic nude mice as xenografts. At relatively low concentrations (cells, but this effect was not observed in several other human cell lines tested. Analysis of cell signaling molecules showed that resveratrol induced the activation of JNK, p38, Akt, and NF-kappaB signaling pathways in these cells. Further analysis using pharmacological inhibitors showed that only the NF-kappaB inhibitor (BAY11-7082) abrogated the growth-stimulatory effect of resveratrol in cultured cells. In athymic nude mice, resveratrol at 16.5 mg/kg body weight enhanced the growth of MDA-MB-435s xenografts compared to the control group, while resveratrol at the 33 mg/kg body weight dose did not have a similar effect. Additional analyses confirmed that resveratrol stimulated cancer cell growth in vivo through activation of the NF-kappaB signaling pathway. Taken together, these observations suggest that resveratrol at low concentrations could stimulate the growth of certain types of human cancer cells in vivo. This cell type-specific mitogenic effect of resveratrol may also partly contribute to the procarcinogenic effect of alcohol consumption (rich in resveratrol) in the development of certain human cancers.

  6. Spatial expression of CLAVATA3 in the shoot apical meristem suggests it is not a stem cell marker in soybean.

    Science.gov (United States)

    Wong, Chui E; Singh, Mohan B; Bhalla, Prem L

    2013-12-01

    CLAVATA3 (CLV3), a stem cell marker in Arabidopsis thaliana, encodes a secreted peptide that maintains the stem cell population within the shoot apical meristem. This work investigated the CLV3 orthologue in a major legume crop, soybean (GmCLV3). Instead of being expressed in the three outermost layers of the meristem as in Arabidopsis, GmCLV3 was expressed deeper in the central zone beneath the fourth layer (L4) of the meristem, overlapping with the expression of soybean WUSCHEL. Subsequent investigation using an alternative stem cell marker (GmLOG1) revealed its expression within layers L2-L4, indicating that GmCLV3 is not a stem cell marker. Overexpression studies of GmCLV3 in Arabidopsis and complementation of clv3-2 mutant suggest similar functional capacity to that of Arabidopsis CLV3. The expression of soybean CLV1, which encodes a receptor for CLV3 in Arabidopsis, was not detectable in the central zone of the meristem via reverse-transcription PCR analysis of amplified RNA from laser-microdissected samples or in situ, implicating a diverged pathway in soybean. This study also reports the novel expression of GmLOG1 in initials of axillary meristem in the boundary region between the SAM and developing leaf primordia, before the expression of GmWUS or GmCLV3, indicating cytokinin as one of the earliest signals in initiating and specifying the stem cell population.

  7. Cortical network from human embryonic stem cells

    OpenAIRE

    2010-01-01

    Abstract The connection of embryonic stem cell technology and developmental biology provides valuable tools to decipher the mechanisms underlying human brain development and diseases, especially among neuronal populations, that are not readily available in primary cultures. It is obviously the case of neurons forming the human cerebral cortex. In the images that are presented, the neurons were generated in vitro from human embryonic stem cells via forebrain-like progenitors. Maintained in cul...

  8. Intracellular Immunization of Human Fetal Cord Blood Stem/Progenitor Cells with a Ribozyme Against Human Immunodeficiency Virus Type 1

    Science.gov (United States)

    Yu, Mang; Leavitt, Mark C.; Maruyama, Midori; Yamada, Osamu; Young, Dennis; Ho, Anthony D.; Wong-Staal, Flossie

    1995-01-01

    Successful treatment of human immunodeficiency virus infection may ultimately require targeting of hematopoietic stem cells. Here we used retroviral vectors carrying the ribozyme gene to transduce CD34^+ cells from human fetal cord blood. Transduction and ribozyme expression had no apparent adverse effect on cell differentiation and/or proliferation. The macrophage-like cells, differentiated from the stem/progenitor cells in vitro, expressed the ribozyme gene and resisted infection by a macrophage tropic human immunodeficiency virus type 1. These results suggest the feasibility of stem cell gene therapy for human immunodeficiency virus-infected patients.

  9. Immunohistochemistry of Programmed Cell Death in Archival Human Pathology Specimens

    Directory of Open Access Journals (Sweden)

    Takami Matsuyama

    2012-05-01

    Full Text Available Immunohistochemistry (IHC for detecting key signal molecules involved in programmed cell death (PCD in archival human pathology specimens is fairly well established. Detection of cleaved caspase-3 in lymphocytes in rheumatoid arthritis (RA and gastric surface foveolar glandular epithelia but not in synoviocytes in RA, gastric fundic glandular epithelia, or nasal NK/T-cell lymphoma (NKTCL cells suggests anti-apoptotic mechanisms in cell differentiation and in oncogenesis such as the induction of survivin. Enzymatically pretreated and ultra-super sensitive detection of beclin-1 in synoviocytes in RA and gastric fundic glandular epithelia suggests enhanced autophagy. The deposition of beclin-1 in fibrinoid necrosis in RA and expression of beclin-1 in detached gastric fundic glandular cells suggest that enhanced autophagy undergoes autophagic cell death (ACD. NKTCL exhibited enhanced autophagy through LC3 labeling and showed densely LC3 labeled cell-debris in regions of peculiar necrosis without deposition of beclin-1, indicating massive ACD in NKTCL and the alternative pathway enhancing autophagy following autophagic vesicle nucleation. Autophagy progression was monitored by labeling aggregated mitochondria and cathepsin D. The cell-debris in massive ACD in NKTCL were positive for 8-hydroxydeoxyguanosine, suggesting DNA oxidation occurred in ACD. Immunohistochemical autophagy and PCD analysis in archival human pathology specimens may offer new insights into autophagy in humans.

  10. Physiological and proteomic changes suggest an important role of cell walls in the high tolerance to metals of Elodea nuttallii.

    Science.gov (United States)

    Larras, Floriane; Regier, Nicole; Planchon, Sébastien; Poté, John; Renaut, Jenny; Cosio, Claudia

    2013-12-15

    Macrophytes bioaccumulate metals, the suggestion being made that they be considered for phytoremediation. However, a thorough understanding of the mechanisms of metal tolerance in these plants is necessary to allow full optimization of this approach. The present study was undertaken to gain insight into Hg and Cd accumulation and their effects in a representative macrophyte, Elodea nuttallii. Exposure to methyl-Hg (23 ng dm(-3)) had no significant effect while inorganic Hg (70 ng dm(-3)) and Cd (281 μg dm(-3)) affected root growth but did not affect shoots growth, photosynthesis, or antioxidant enzymes. Phytochelatins were confirmed as having a role in Cd tolerance in this plant while Hg tolerance seems to rely on different mechanisms. Histology and subcellular distribution revealed a localized increase in lignification, and an increased proportion of metal accumulation in cell wall over time. Proteomics further suggested that E. nuttallii was able to efficiently adapt its energy sources and the structure of its cells during Hg and Cd exposure. Storage in cell walls to protect cellular machinery is certainly predominant at environmental concentrations of metals in this plant resulting in a high tolerance highlighted by the absence of toxicity symptoms in shoots despite the significant accumulation of metals.

  11. Dissecting the oncogenic and tumorigenic potential of differentiated human induced pluripotent stem cells and human embryonic stem cells.

    Science.gov (United States)

    Ghosh, Zhumur; Huang, Mei; Hu, Shijun; Wilson, Kitchener D; Dey, Devaveena; Wu, Joseph C

    2011-07-15

    Pluripotent stem cells, both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC), can give rise to multiple cell types and hence have tremendous potential for regenerative therapies. However, the tumorigenic potential of these cells remains a great concern, as reflected in the formation of teratomas by transplanted pluripotent cells. In clinical practice, most pluripotent cells will be differentiated into useful therapeutic cell types such as neuronal, cardiac, or endothelial cells prior to human transplantation, drastically reducing their tumorigenic potential. Our work investigated the extent to which these differentiated stem cell derivatives are truly devoid of oncogenic potential. In this study, we analyzed the gene expression patterns from three sets of hiPSC- and hESC-derivatives and the corresponding primary cells, and compared their transcriptomes with those of five different types of cancer. Our analysis revealed a significant gene expression overlap of the hiPSC- and hESC-derivatives with cancer, whereas the corresponding primary cells showed minimum overlap. Real-time quantitative PCR analysis of a set of cancer-related genes (selected on the basis of rigorous functional and pathway analyses) confirmed our results. Overall, our findings suggested that pluripotent stem cell derivatives may still bear oncogenic properties even after differentiation, and additional stringent functional assays to purify these cells should be done before they can be used for regenerative therapy.

  12. Human hematopoietic cell culture, transduction, and analyses

    DEFF Research Database (Denmark)

    Bonde, Jesper; Wirthlin, Louisa; Kohn, Donald B;

    2008-01-01

    This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long...

  13. Human periodontal ligament stem cells repair mental nerve injury*

    Institute of Scientific and Technical Information of China (English)

    Bohan Li; Hun-Jong Jung; Soung-Min Kim; Myung-Jin Kim; Jeong Won Jahng; Jong-Ho Lee

    2013-01-01

    Human periodontal ligament stem cells are easily accessible and can differentiate into Schwann cells. We hypothesized that human periodontal ligament stem cells can be used as an alternative source for the autologous Schwann cells in promoting the regeneration of injured peripheral nerve. To validate this hypothesis, human periodontal ligament stem cells (1 × 106) were injected into the crush-injured left mental nerve in rats. Simultaneously, autologous Schwann cells (1 × 106) and PBS were also injected as controls. Real-time reverse transcriptase polymerase chain reaction showed that at 5 days after injection, mRNA expression of low affinity nerve growth factor receptor was sig-nificantaly increased in the left trigeminal ganglion of rats with mental nerve injury. Sensory tests, histomorphometric evaluation and retrograde labeling demonstrated that at 2 and 4 weeks after in-jection, sensory function was significantly improved, the numbers of retrograde labeled sensory neurons and myelinated axons were significantly increased, and human periodontal ligament stem cells and autologous Schwann cells exhibited similar therapeutic effects. These findings suggest that transplantation of human periodontal ligament stem cells show a potential value in repair of mental nerve injury.

  14. Subsets of human natural killer cells and their regulatory effects

    Science.gov (United States)

    Fu, Binqing; Tian, Zhigang; Wei, Haiming

    2014-01-01

    Human natural killer (NK) cells have distinct functions as NKtolerant, NKcytotoxic and NKregulatory cells and can be divided into different subsets based on the relative expression of the surface markers CD27 and CD11b. CD27+ NK cells, which are abundant cytokine producers, are numerically in the minority in human peripheral blood but constitute the large population of NK cells in cord blood, spleen, tonsil and decidua tissues. Recent data suggest that these NK cells may have immunoregulatory properties under certain conditions. In this review, we will focus on these new NK cell subsets and discuss how regulatory NK cells may serve as rheostats or sentinels in controlling inflammation and maintaining immune homeostasis in various organs. PMID:24303897

  15. Reconstitution of Torso signaling in cultured cells suggests a role for both Trunk and Torso-like in receptor activation.

    Science.gov (United States)

    Amarnath, Smita; Stevens, Leslie M; Stein, David S

    2017-02-15

    Formation of the Drosophila embryonic termini is controlled by the localized activation of the receptor tyrosine kinase Torso. Both Torso and Torso's presumed ligand, Trunk, are expressed uniformly in the early embryo. Polar activation of Torso requires Torso-like, which is expressed by follicle cells adjacent to the ends of the developing oocyte. We find that Torso expressed at high levels in cultured Drosophila cells is activated by individual application of Trunk, Torso-like or another known Torso ligand, Prothoracicotropic Hormone. In addition to assays of downstream signaling activity, Torso dimerization was detected using bimolecular fluorescence complementation. Trunk and Torso-like were active when co-transfected with Torso and when presented to Torso-expressing cells in conditioned medium. Trunk and Torso-like were also taken up from conditioned medium specifically by cells expressing Torso. At low levels of Torso, similar to those present in the embryo, Trunk and Torso-like alone were ineffective but acted synergistically to stimulate Torso signaling. Our results suggest that Torso interacts with both Trunk and Torso-like, which cooperate to mediate dimerization and activation of Torso at the ends of the Drosophila embryo. © 2017. Published by The Company of Biologists Ltd.

  16. Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation.

    Science.gov (United States)

    Düssmann, H; Rehm, M; Concannon, C G; Anguissola, S; Würstle, M; Kacmar, S; Völler, P; Huber, H J; Prehn, J H M

    2010-02-01

    Mitochondrial outer membrane permeabilisation (MOMP) during apoptosis is triggered by the activation and oligomerisation of Bax and Bak, but a quantification of these processes in individual cells has not yet been performed. Single-cell imaging of Bax translocation and oligomerisation in Bax-deficient DU-145 cells expressing CFP-Bax and YFP-Bax revealed that both processes started only minutes before or concomitantly with MOMP, with the majority of Bax translocation and oligomerisation occurring downstream of MOMP. Quantification of YFP-Bax concentrations at mitochondria revealed an increase of only 1.8 + or - 1.5% at MOMP onset. This was increased to 11.2 + or - 3.6% in bak-silenced cells. These data suggested that Bax activation exceeded by far the quantities required for MOMP induction, and that minimal Bax or Bak activation may be sufficient to trigger rapid pore formation. In a cellular automaton modelling approach that incorporated the quantities and movement probabilities of Bax and its inhibitors, activators and enablers in the mitochondrial membrane, we could re-model rapid pore formation kinetics at submaximal Bax activation.

  17. Human Neuroepithelial Cells Express NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Cappell B

    2003-11-01

    Full Text Available Abstract L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1 cerebral endothelial barrier and 2 cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR expression via immunohistochemistry and murine neuroepithelial cell line (V1 were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.

  18. Protein dynamics in individual human cells: experiment and theory.

    Directory of Open Access Journals (Sweden)

    Ariel Aharon Cohen

    Full Text Available A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle-dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell-cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell-cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells.

  19. The phenotype and activation status of T and NK cells in porcine colostrum suggest these are central/effector memory cells.

    Science.gov (United States)

    Hlavova, Karolina; Stepanova, Hana; Faldyna, Martin

    2014-12-01

    In pigs, the epitheliochorial placenta does not allow transfer of maternally derived antibodies or immune cells to the fetus. Thus, piglets are dependent on intake of colostrum for acquisition of passive immunity during the neonatal period. As well as immunoglobulin G (IgG), cellular components of colostrum, mainly lymphocytes, can enter the systemic circulation and secondary lymphoid organs of the neonate. In order to understand the function and immunological role of these cells, a flow cytometric study was undertaken to characterise the cellular profile and phenotype of T cells and NK cells present in porcine colostrum. The results indicated that the greatest numbers of lymphocytes were found on the first day of lactation. The predominant cell types in colostrum were CD8(+) single positive T cells (53.6%), followed by CD4(+)CD8(+) double positive T cells (21.1%), CD2(+)CD8(+) γδ T cells (15.0%) and NK cells (13.5%). CD4(+) single positive T cells (4.4%) and other γδ T cell subpopulations (1.8% CD2(-)CD8(-) and 0.4% CD2(+)CD8(-)) were present in colostrum at low levels. Although the profile of the T cell subpopulations during the first 3 days of lactation remained constant, the absolute numbers of T and NK cells decreased significantly in the first few hours of lactation. Expression of CCR7, CD11b, CD25, CD45RA and MHC class II was used to assess the activation status of T and NK cells in colostrum. T cell subpopulations expressed markers consistent with an effector memory phenotype, indicating that these were antigen-experienced cells. The phenotype of colostral T and NK cells suggests a role in mucosal immunity and potentially in transfer of passive immunity from sow to piglet.

  20. Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

    Directory of Open Access Journals (Sweden)

    José J. Gaforio

    2011-10-01

    Full Text Available Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A or breast cancer cells (MDA-MB-231 and MCF7. We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  1. Hydroxytyrosol protects against oxidative DNA damage in human breast cells.

    Science.gov (United States)

    Warleta, Fernando; Quesada, Cristina Sánchez; Campos, María; Allouche, Yosra; Beltrán, Gabriel; Gaforio, José J

    2011-10-01

    Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol's effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A) or breast cancer cells (MDA-MB-231 and MCF7). We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS) level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  2. A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology.

    Science.gov (United States)

    Shaharuddin, Bakiah; Ahmad, Sajjad; Md Latar, Nani; Ali, Simi; Meeson, Annette

    2016-10-14

    : Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue. Limbal stem cell deficiency is a painful eye condition caused by abnormal maintenance of limbal stem cells. It is treated by transplantation of limbal epithelial cells derived from human tissue. The success of this treatment depends of the quality of the cells transplanted; however, some transplants fail. Understanding more about the immunobiology of these cells within the transplants could

  3. Transcription profiling of Epstein-Barr virus nuclear antigen (EBNA-1 expressing cells suggests targeting of chromatin remodeling complexes.

    Directory of Open Access Journals (Sweden)

    Ramakrishna Sompallae

    Full Text Available The Epstein-Barr virus (EBV encoded nuclear antigen (EBNA-1 regulates virus replication and transcription, and participates in the remodeling of the cellular environment that accompanies EBV induced B-cell immortalization and malignant transformation. The putative cellular targets of these effects of EBNA-1 are largely unknown. To address this issue we have profiled the transcriptional changes induced by short- and long-term expression of EBNA-1 in the EBV negative B-cell lymphoma BJAB. Three hundred and nineteen cellular genes were regulated in a conditional transfectant shortly after EBNA-1 induction while a ten fold higher number of genes was regulated upon continuous EBNA-1 expression. Promoter analysis of the differentially regulated genes demonstrated a significant enrichment of putative EBNA-1 binding sites suggesting that EBNA-1 may directly influence the transcription of a subset of genes. Gene ontology analysis of forty seven genes that were consistently regulated independently on the time of EBNA-1 expression revealed an unexpected enrichment of genes involved in the maintenance of chromatin architecture. The interaction network of the affected gene products suggests that EBNA-1 may promote a broad rearrangement of the cellular transcription landscape by altering the expression of key components of chromatin remodeling complexes.

  4. Human NK cells of mice with reconstituted human immune system components require preactivation to acquire functional competence.

    Science.gov (United States)

    Strowig, Till; Chijioke, Obinna; Carrega, Paolo; Arrey, Frida; Meixlsperger, Sonja; Rämer, Patrick C; Ferlazzo, Guido; Münz, Christian

    2010-11-18

    To investigate human natural killer (NK)-cell reactivity in vivo we have reconstituted human immune system components by transplantation of human hematopoietic progenitor cells into NOD-scid IL2Rγ(null) mice. We demonstrate here that this model allows the development of all NK-cell subsets that are also found in human adult peripheral and cord blood, including NKp46(+)CD56(-) NK cells. Similar to human cord blood, NK cells from these reconstituted mice require preactivation by interleukin-15 to reach the functional competence of human adult NK cells. Mainly the terminally differentiated CD16(+) NK cells demonstrate lower reactivity without this stimulation. After preactivation, both CD16(+) and CD16(-) NK cells efficiently produce interferon-γ and degranulate in response to stimulation with NK cell-susceptible targets, including K562 erythroleukemia cells. NK-cell lines, established from reconstituted mice, demonstrate cytotoxicity against this tumor cell line. Importantly, preactivation can as well be achieved by bystander cell maturation via poly I:C stimulation in vitro and injection of this maturation stimulus in vivo. Preactivation in vivo enhances killing of human leukocyte antigen class I negative tumor cells after their adoptive transfer. These data suggest that a functional, but resting, NK-cell compartment can be established in immune-compromised mice after human hematopoietic progenitor cell transfer.

  5. Derivation of naive human embryonic stem cells.

    Science.gov (United States)

    Ware, Carol B; Nelson, Angelique M; Mecham, Brigham; Hesson, Jennifer; Zhou, Wenyu; Jonlin, Erica C; Jimenez-Caliani, Antonio J; Deng, Xinxian; Cavanaugh, Christopher; Cook, Savannah; Tesar, Paul J; Okada, Jeffrey; Margaretha, Lilyana; Sperber, Henrik; Choi, Michael; Blau, C Anthony; Treuting, Piper M; Hawkins, R David; Cirulli, Vincenzo; Ruohola-Baker, Hannele

    2014-03-25

    The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

  6. Alteration of Cell Cycle Mediated by Zinc in Human Bronchial ...

    Science.gov (United States)

    Zinc (Zn2+), a ubiquitous ambient air contaminant, presents an oxidant challenge to the human lung and is linked to adverse human health effects. To further elucidate the adaptive and apoptotic cellular responses of human airway cells to Zn2+, we performed pilot studies to examine cell cycle perturbation upon exposure using a normal human bronchial epithelial cell culture (BEAS-2B). BEAS-2B cells were treated with low (0, 1, 2 µM) and apoptotic (3 µM) doses of Zn2+ plus 1 µM pyrithione, a Zn2+-specific ionophore facilitating cellular uptake, for up to 24 h. Fixed cells were then stained with propidium iodine (PI) and cell cycle phase was determined by fluorescent image cytometry. Initial results report the percentage of cells in the S phase after 18 h exposure to 1, 2, and 3 µM Zn2+ were similar (8%, 7%, and 12%, respectively) compared with 7% in controls. Cells exposed to 3 µM Zn2+ increased cell populations in G2/M phase (76% versus 68% in controls). Interestingly, exposure to 1 µM Zn2+ resulted in decreased (59%) cells in G2/M. While preliminary, these pilot studies suggest Zn2+ alters cell cycle in BEAS-2B cells, particularly in the G2/M phase. The G2/M checkpoint maintains DNA integrity by enabling initiation of DNA repair or apoptosis. Our findings suggest that the adaptive and apoptotic responses to Zn2+ exposure may be mediated via perturbation of the cell cycle at the G2/M checkpoint. This work was a collaborative summer student project. The st

  7. Anticorrelation between Local Photoluminescence and Photocurrent Suggests Variability in Contact to Active Layer in Perovskite Solar Cells.

    Science.gov (United States)

    Eperon, Giles E; Moerman, David; Ginger, David S

    2016-11-22

    We use high-resolution, spatially resolved, laser beam induced current, confocal photoluminescence, and photoconductive atomic force microscopy (pcAFM) measurements to correlate local solar cell performance with spatially heterogeneous local material properties in methylammonium lead triiodide (CH3NH3PbI3) perovskite solar cells. We find that, for this material and device architecture, the photocurrent heterogeneity measured via pcAFM on devices missing a top selective contact with traditional Au-coated tips is significantly larger than the photocurrent heterogeneity observed in full devices with both electron- and hole-selective extraction layers, indicating that extraction barriers at the Au/perovskite interface are ameliorated by deposition of the organic charge extraction layer. Nevertheless, in completed, efficient device structures (PCE ≈ 16%) with state-of-the-art nickel oxide and [6,6]-phenyl-C61-butyric acid (PCBM) methyl ester contacts, we observe that the local photoluminescence (PL) is weakly anticorrelated with local photocurrent at both short-circuit and open-circuit conditions. We determine that the contact materials are fairly homogeneous; thus the heterogeneity stems from the perovskite itself. We suggest a cause for the anticorrelation as being related to local carrier extraction heterogeneity. However, we find that the contacts are still the dominating source of losses in these devices, which minimizes the impact of the material heterogeneity on device performance at present. These results suggest that further steps to prevent recombination losses at the interfaces are needed to help perovskite-based cells approach theoretical efficiency limits; only at this point will material heterogeneity become crucial.

  8. Memory regulatory T cells reside in human skin.

    Science.gov (United States)

    Sanchez Rodriguez, Robert; Pauli, Mariela L; Neuhaus, Isaac M; Yu, Siegrid S; Arron, Sarah T; Harris, Hobart W; Yang, Sara Hsin-Yi; Anthony, Bryan A; Sverdrup, Francis M; Krow-Lucal, Elisabeth; MacKenzie, Tippi C; Johnson, David S; Meyer, Everett H; Löhr, Andrea; Hsu, Andro; Koo, John; Liao, Wilson; Gupta, Rishu; Debbaneh, Maya G; Butler, Daniel; Huynh, Monica; Levin, Ethan C; Leon, Argentina; Hoffman, William Y; McGrath, Mary H; Alvarado, Michael D; Ludwig, Connor H; Truong, Hong-An; Maurano, Megan M; Gratz, Iris K; Abbas, Abul K; Rosenblum, Michael D

    2014-03-01

    Regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3, are a dynamic and heterogeneous population of cells that control immune responses and prevent autoimmunity. We recently identified a subset of Tregs in murine skin with properties typical of memory cells and defined this population as memory Tregs (mTregs). Due to the importance of these cells in regulating tissue inflammation in mice, we analyzed this cell population in humans and found that almost all Tregs in normal skin had an activated memory phenotype. Compared with mTregs in peripheral blood, cutaneous mTregs had unique cell surface marker expression and cytokine production. In normal human skin, mTregs preferentially localized to hair follicles and were more abundant in skin with high hair density. Sequence comparison of TCRs from conventional memory T helper cells and mTregs isolated from skin revealed little homology between the two cell populations, suggesting that they recognize different antigens. Under steady-state conditions, mTregs were nonmigratory and relatively unresponsive; however, in inflamed skin from psoriasis patients, mTregs expanded, were highly proliferative, and produced low levels of IL-17. Taken together, these results identify a subset of Tregs that stably resides in human skin and suggest that these cells are qualitatively defective in inflammatory skin disease.

  9. Stable radioresistance in ataxia-telangiectasia cells containing DNA from normal human cells

    Energy Technology Data Exchange (ETDEWEB)

    Kapp, L.N.; Painter, R.B. (California Univ., San Francisco, CA (USA). Lab. of Radiobiology)

    1989-11-01

    SV40-transformed ataxia-telangiectasia (AT) cells were transfected with a cosmid containing a normal human DNA library and selectable marker, the neo gene, which endows successfully transformed mammalian cells with resistance to the antibiotic G418. Cells from this line were irradiated with 50 Gy of X-rays and fused with non-transfected AT cells. Among the G418-resistant colonies recovered was one stably resistant to radiation. Resistance to ionizing radiation of both primary transfectant line and its fusion derivative was intermediate between that of AT cells and normal cells, as assayed by colony-forming ability and measurement of radiation-induced G{sub 2} chromatic aberrations; both cell lines retained AT-like radioresistant DNA synthesis. Results suggest that, because radioresistance in transfected cells was not as great as in normal human cells, two hallmarks of AT, radiosensitivity and radioresistant DNA synthesis, may still be the result of a single defective AT gene. (author).

  10. Neoplastic human embryonic stem cells as a model of radiation resistance of human cancer stem cells.

    Science.gov (United States)

    Dingwall, Steve; Lee, Jung Bok; Guezguez, Borhane; Fiebig, Aline; McNicol, Jamie; Boreham, Douglas; Collins, Tony J; Bhatia, Mick

    2015-09-08

    Studies have implicated that a small sub-population of cells within a tumour, termed cancer stem cells (CSCs), have an enhanced capacity for tumour formation in multiple cancers and may be responsible for recurrence of the disease after treatment, including radiation. Although comparisons have been made between CSCs and bulk-tumour, the more important comparison with respect to therapy is between tumour-sustaining CSC versus normal stem cells that maintain the healthy tissue. However, the absence of normal known counterparts for many CSCs has made it difficult to compare the radiation responses of CSCs with the normal stem cells required for post-radiotherapy tissue regeneration and the maintenance of tissue homeostasis. Here we demonstrate that transformed human embryonic stem cells (t-hESCs), showing features of neoplastic progression produce tumours resistant to radiation relative to their normal counterpart upon injection into immune compromised mice. We reveal that t-hESCs have a reduced capacity for radiation induced cell death via apoptosis and exhibit altered cell cycle arrest relative to hESCs in vitro. t-hESCs have an increased expression of BclXL in comparison to their normal counterparts and re-sensitization of t-hESCs to radiation upon addition of BH3-only mimetic ABT737, suggesting that overexpression of BclXL underpins t-hESC radiation insensitivity. Using this novel discovery platform to investigate radiation resistance in human CSCs, our study indicates that chemotherapy targeting Bcl2-family members may prove to be an adjuvant to radiotherapy capable of targeting CSCs.

  11. Regulatory T Cells in Human Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Dong-Jun Peng

    2012-01-01

    Full Text Available Multiple layers of suppressive components including regulatory T (TReg cells, suppressive antigen-presenting cells, and inhibitory cytokines form suppressive networks in the ovarian cancer microenvironment. It has been demonstrated that as a major suppressive element, TReg cells infiltrate tumor, interact with several types of immune cells, and mediate immune suppression through different molecular and cellular mechanisms. In this paper, we focus on human ovarian cancer and will discuss the nature of TReg cells including their subsets, trafficking, expansion, and function. We will briefly review the development of manipulation of TReg cells in preclinical and clinical settings.

  12. Generation of pancreatic islet cells from human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG DongHui; JIANG Wei; SHI Yan; DENG HongKui

    2009-01-01

    Efficiently obtaining functional pancreaUc islet cells derived from human embryonic stem (hES) cells not only provides great potential to solve the shortage of islets sources for type I diabetes cell therapy,but also benefits the study of the development of the human pancreas and diabetes pathology. In 2001,hES cells were reported to have the capacity to generate insulin-producing cells by spontaneous differentiation in vitro. Since then, many strategies (such as overexpression of key transcription factors,delivery of key proteins for pancreatic development, co-transplantation of differentiated hES cells along with fetal pancreas, stepwise differentiation by mimicking in vivo pancreatic development) have been employed in order to induce the differentiation of pancreatic islet cells from hES cells. Moreover, patient-specific induced pluripotent stem (iPS) cells can be generated by reprogramming somatic cells.iPS cells have characteristics similar to those of ES cells and offer a new cell source for type I diabetes cell therapy that reduces the risk of immunologic rejection. In this review, we summarize the recent progress made in the differentiation of hES and iPS cells into functional pancreatic islet cells and discuss the challenges for their future study.

  13. Generation of pancreatic islet cells from human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Efficiently obtaining functional pancreatic islet cells derived from human embryonic stem(hES) cells not only provides great potential to solve the shortage of islets sources for type I diabetes cell therapy,but also benefits the study of the development of the human pancreas and diabetes pathology.In 2001,hES cells were reported to have the capacity to generate insulin-producing cells by spontaneous differentiation in vitro.Since then,many strategies(such as overexpression of key transcription factors,delivery of key proteins for pancreatic development,co-transplantation of differentiated hES cells along with fetal pancreas,stepwise differentiation by mimicking in vivo pancreatic development) have been employed in order to induce the differentiation of pancreatic islet cells from hES cells.Moreover,patient-specific induced pluripotent stem(iPS) cells can be generated by reprogramming somatic cells.iPS cells have characteristics similar to those of ES cells and offer a new cell source for type I diabetes cell therapy that reduces the risk of immunologic rejection.In this review,we summarize the recent progress made in the differentiation of hES and iPS cells into functional pancreatic islet cells and discuss the challenges for their future study.

  14. Differentiation of human menstrual blood-derived endometrial mesenchymal stem cells into oocyte-like cells.

    Science.gov (United States)

    Lai, Dongmei; Guo, Ying; Zhang, Qiuwan; Chen, Yifei; Xiang, Charlie

    2016-11-01

    Human endometrial mesenchymal stem cells (EnSCs) derived from menstrual blood are a unique stem cell source. Evidence suggests that EnSCs exhibit a multi-lineage potential and have attracted extensive attention in regenerative medicine. However, the potential of EnSCs to differentiate into germline cells in vitro remains unclear. In this study, EnSCs were induced to differentiate into germ cells in a differentiation medium supplemented with 20% human follicular fluid. Our results demonstrated that EnSCs derived from human menstrual blood form oocyte-like cells and express germ cell markers. The induced cell aggregates contained not only oocyte-like structures but also cells expressing follicle stimulating hormone receptor and luteotropic hormone receptor, and produced estrogen and progesterone regulated by gonodatropin, suggesting that granulosa-like and theca-like cells were also induced. We further found that granulosa cells promote the development of oocyte-like cells and activate the induction of blastocyst-like structures derived from EnSCs. In conclusion, EnSCs may potentially represent an in vitro system for the investigation of human folliculogenesis.

  15. Immune and Inflammatory Cell Composition of Human Lung Cancer Stroma.

    Directory of Open Access Journals (Sweden)

    G-Andre Banat

    Full Text Available Recent studies indicate that the abnormal microenvironment of tumors may play a critical role in carcinogenesis, including lung cancer. We comprehensively assessed the number of stromal cells, especially immune/inflammatory cells, in lung cancer and evaluated their infiltration in cancers of different stages, types and metastatic characteristics potential. Immunohistochemical analysis of lung cancer tissue arrays containing normal and lung cancer sections was performed. This analysis was combined with cyto-/histomorphological assessment and quantification of cells to classify/subclassify tumors accurately and to perform a high throughput analysis of stromal cell composition in different types of lung cancer. In human lung cancer sections we observed a significant elevation/infiltration of total-T lymphocytes (CD3+, cytotoxic-T cells (CD8+, T-helper cells (CD4+, B cells (CD20+, macrophages (CD68+, mast cells (CD117+, mononuclear cells (CD11c+, plasma cells, activated-T cells (MUM1+, B cells, myeloid cells (PD1+ and neutrophilic granulocytes (myeloperoxidase+ compared with healthy donor specimens. We observed all of these immune cell markers in different types of lung cancers including squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma, small cell carcinoma, papillary adenocarcinoma, metastatic adenocarcinoma, and bronchioloalveolar carcinoma. The numbers of all tumor-associated immune cells (except MUM1+ cells in stage III cancer specimens was significantly greater than those in stage I samples. We observed substantial stage-dependent immune cell infiltration in human lung tumors suggesting that the tumor microenvironment plays a critical role during lung carcinogenesis. Strategies for therapeutic interference with lung cancer microenvironment should consider the complexity of its immune cell composition.

  16. Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Braam, S.R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2008-01-01

    Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired chromosom

  17. Expression analyses of human cleft palate tissue suggest a role for osteopontin and immune related factors in palatal development

    DEFF Research Database (Denmark)

    Jakobsen, L.P.; Borup, R.; Vestergaard, J.;

    2009-01-01

    . Moreover, selected differentially expressed genes were analyzed by quantitative RT-PCR, and by immunohistochemical staining of craniofacial tissue from human embryos. Osteopontin (SPP1) and other immune related genes were significantly higher expressed in palate tissue from patients with CLP compared to CP...... and palate (CLP). In order to understand the biological basis in these cleft lip and palate subgroups better we studied the expression profiles in human tissue from patients with CL/P. In each of the CL/P subgroups, samples were obtained from three patients and gene expression analysis was performed...... and immunostaining in palatal shelves against SPP1, chemokine receptor 4 (CXCR4) and serglycin (PRG1) in human embryonic craniofacial tissue were positive, supporting a role for these genes in palatal development. However, gene expression profiles are subject to variations during growth and therefore we recommend...

  18. Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro.

    Directory of Open Access Journals (Sweden)

    Holger A Russ

    Full Text Available BACKGROUND: Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT. Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells. METHODOLOGY/PRINCIPAL FINDING: Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2 using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for

  19. Suggestive evidence of a multi-cytokine resistin pathway in humans and its role on cardiovascular events in high-risk individuals

    Science.gov (United States)

    Menzaghi, Claudia; Marucci, Antonella; Antonucci, Alessandra; De Bonis, Concetta; Ortega Moreno, Lorena; Salvemini, Lucia; Copetti, Massimiliano; Trischitta, Vincenzo; Di Paola, Rosa

    2017-01-01

    In cells and tissues resistin affects IL-1β, IL-6, IL-8, IL-12 and TNF-α expression, thus suggesting the existence of a multi-cytokine “resistin pathway”. We investigated whether such pathway does exist in humans and, if so, if it is associated with cardiovascular risk factors and with major adverse cardiovascular events (MACE). Serum cytokines were measured in 280 healthy subjects from the Gargano Study 2 (GS2) whose BMI, waist circumference, HOMAIR, triglycerides, HDL-cholesterol, systolic and diastolic blood pressure data were available and in 353 patients with type 2 diabetes and coronary artery disease from the Gargano Heart Study (GHS)-prospective design (follow-up 5.4 ± 2.5 years; 71 MACE). In GS2, cytokines mRNA levels in white blood cells were also measured. In GS2, resistin mRNA was correlated with all cytokines expression (all p eRPS) and serum (sRPS) resistin pathway scores (excluding IL-12) were each other correlated (p < 0.001) and both associated with cardiovascular risk factors (all p < 0.01). In GHS, sRPS was independently associated with MACE (HR = 1.44, 95% CI = 1.10–1.90). Our data indicate the existence of a resistin pathway, which is associated with cardiovascular risk factors and which strongly and independently predicts MACE. PMID:28290549

  20. Lack of megalin expression in adult human terminal ileum suggests megalin-independent cubilin/amnionless activity during vitamin B12 absorption.

    Science.gov (United States)

    Jensen, Louise L; Andersen, Rikke K; Hager, Henrik; Madsen, Mette

    2014-07-01

    Cubilin plays an essential role in terminal ileum and renal proximal tubules during absorption of vitamin B12 and ligands from the glomerular ultrafiltrate. Cubilin is coexpressed with amnionless, and cubilin and amnionless are mutually dependent on each other for correct processing to the plasma membrane upon synthesis. Patients with defects in either protein suffer from vitamin B12-malabsorption and in some cases proteinuria. Cubilin lacks a transmembrane region and signals for endocytosis and is dependent on a transmembrane coreceptor during internalization. Amnionless has been shown to be able to mediate internalization of cubilin in a cell-based model system. Cubilin has additionally been suggested to function together with megalin, and a recent study of megalin-deficient patients indicates that uptake of cubilin ligands in the kidney is critically dependent on megalin. To further investigate the potential role of amnionless and megalin in relation to cubilin function in terminal ileum and vitamin B12 uptake, we initiated a study of CUBN/cubilin, AMN/amnionless, and LRP2/megalin expression in adult human terminal ileum. Our study is the first to reveal the expression pattern of cubilin, amnionless, and megalin in adult human terminal ileum, where cubilin and amnionless localize to the epithelial cells. Surprisingly, we did not detect any megalin protein in adult terminal ileum and consistently, only extremely low amounts of LRP2 mRNA. Our data therefore advocate that cubilin and amnionless act independently of megalin in adult terminal ileum and that the cubilin-megalin interdependency accordingly should be considered as tissue and ligand specific.

  1. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    Science.gov (United States)

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-02-03

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts.

  2. Telomere dynamics in human cells reprogrammed to pluripotency.

    Directory of Open Access Journals (Sweden)

    Steven T Suhr

    Full Text Available BACKGROUND: Human induced pluripotent stem cells (IPSCs have enormous potential in the development of cellular models of human disease and represent a potential source of autologous cells and tissues for therapeutic use. A question remains as to the biological age of IPSCs, in particular when isolated from older subjects. Studies of cloned animals indicate that somatic cells reprogrammed to pluripotency variably display telomere elongation, a common indicator of cell "rejuvenation." METHODOLOGY/PRINCIPAL FINDINGS: We examined telomere lengths in human skin fibroblasts isolated from younger and older subjects, fibroblasts converted to IPSCs, and IPSCs redifferentiated through teratoma formation and explant culture. In IPSCs analyzed at passage five (P5, telomeres were significantly elongated in 6/7 lines by >40% and approximated telomere lengths in human embryonic stem cells (hESCs. In cell lines derived from three IPSC-teratoma explants cultured to P5, two displayed telomeres shortened to lengths similar to input fibroblasts while the third line retained elongated telomeres. CONCLUSIONS/SIGNIFICANCE: While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age.

  3. Senegenin promotes in vitro proliferation of human neural progenitor cells

    Institute of Scientific and Technical Information of China (English)

    Fang Shi; Zhigang Liang; Zixuan Guo; Ran Li; Fen Yu; Zhanjun Zhang; Xuan Wang; Xiaomin Wang

    2011-01-01

    Senegenin, an effective component of Polygala tenuifolia root extract, promotes proliferation and differentiation of neural progenitor cells in the hippocampus.However, the effects of senegenin on mesencephalon-derived neural progenitor cells remain poorly understood.Cells from a ventral mesencephalon neural progenitor cell line (ReNcell VM) were utilized as models for pharmaceutical screening.The effects of various senegenin concentrations on cell proliferation were analyzed,demonstrating that high senegenin concentrations (5, 10, 50, and 100 pmo/L), particularly 50 pmol/L, significantly promoted proliferation of ReNcell VM cells.In the mitogen-activated protein kinase signal transduction pathway, senegenin significantly increased phosphorylation levels of extracellular signal-regulated kinases.Moreover, cell proliferation was suppressed by extracellular signal-regulated kinase inhibitors.Results suggested that senegenin contributed to in vitro proliferation of human neural progenitor cells by upregulating phosphorylation of extracellular signal-regulated kinase.

  4. Comparative genomics of human stem cell factor (SCF

    Directory of Open Access Journals (Sweden)

    Moein Dehbashi

    2017-03-01

    Full Text Available Stem cell factor (SCF is a critical protein with key roles in the cell such as hematopoiesis, gametogenesis and melanogenesis. In the present study a comparative analysis on nucleotide sequences of SCF was performed in Humanoids using bioinformatics tools including NCBI-BLAST, MEGA6, and JBrowse. Our analysis of nucleotide sequences to find closely evolved organisms with high similarity by NCBI-BLAST tools and MEGA6 showed that human and Chimpanzee (Pan troglodytes were placed into the same cluster. By using JBrowse, we found that SCF in Neanderthal had a single copy number similar to modern human and partly conserved nucleotide sequences. Together, the results approved the gene flow and genetics similarity of SCF among human and P. troglodytes. This may suggest that during evolution, SCF gene transferred partly intact either on the basis of sequence or function from the same ancestors to P. troglodytes, the ancient human like Neanderthal, and then to the modern human.

  5. Cybersemiotics: Suggestion for a Transdisciplinary Framework Encompassing Natural, Life, and Social Sciences as Well as Phenomenology and Humanities

    Directory of Open Access Journals (Sweden)

    Søren Brier

    2014-01-01

    Full Text Available The modern evolutionary paradigm combined with phenomenology forces us to view human consciousness as a product of evolution as well as accepting humans as observers from “within the universe”. The knowledge produced by science has first-person embodied consciousness combined with second-person meaningful communication in language as a prerequisite for third-person fallibilist scientific knowledge. Therefore, the study of consciousness forces us theoretically to encompass the natural and social sciences as well as the humanities in one framework of unrestricted or absolute naturalism. This means to view conscious quale life world with its intentionality as well as the intersubjectivity of culture as a part of nature, and therefore the whole human being as treated in modern bio-medicine. The ‘bio’ is not enough. The crucial question for a transdisciplinary theory of conscious human being is therefore: What is the role of consciousness, signs, and meaning in evolution as well as in cultural development? But this is problematic since the sciences in their present form are without concepts of qualia and meaning, and the European phenomenological-hermeneutic “sciences of meaning” does not have an evolutionary foundation. It is therefore interesting that C.S. Peirce phaneroscopic semiotics - in its modern form of a biosemiotics - was based on a phenomenological basis as well as an evolutionary thinking and ecology of sign webs at the same time drawing on knowledge from the sciences. To develop this 100 year old paradigm it is necessary to supplement it with the knowledge gained from the technologically founded information sciences, as well as systems, and cybernetics in order to produce a transdisciplinary alternative to logical positivism on the one hand and postmodern constructivism on the other. Cybersemiotics constructs such a non-reductionist naturalistic framework in order to integrate third-person knowledge from the exact sciences

  6. Mast cells and human hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Fabio Grizzi; Barbara Franceschini; Maurizio Chiriva-Internati; Young Liu; Paul L. Hermonat; Nicola Dioguardi

    2003-01-01

    AIM: To investigate the density of mast cells (MCs) in human hepatocellular carcinoma (HCC), and to determine whether the MCs density has any correlations with histopathological grading, staging or some baseline patient characteristics.METHODS: Tissue sections of 22 primary HCCs were histochemically stained with toluidine blue, in order to be able to quantify the MCs in and around the neoplasm using a computer-assisted image analysis system. HCC was staged and graded by two independent pathologists. To identify the sinusoidal capillarisation of each specimen 3μm thick sections were histochemically stained with sirius red, and semi-quantitatively evaluated by two independent observers. The data were statistically analysed using Spearman′s correlation and Student′s t-test when appropriate.RESULTS: MCs density did not correlate with the age or sex of the patients, the serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels, or the stage or grade of the HCC. No significant differences were found between the MCs density of the patients with and without hepatitis C virus infection, but they were significantly higher in the specimens showing marked sinusoidal capillarisation.CONCLUSION: The lack of any significant correlation between MCs density and the stage or grade of the neoplastic lesions suggests that there is no causal relationship between MCs recruitment and HCC. However, as capillarisation proceeds concurrently with arterial blood supply during hepatocarcinogenesis, MCs may be considered of primary importance in the transition from sinusoidal to capillary-type endothelial cells and the HCC growth.

  7. Hepatic progenitor cells in human liver tumor development

    Institute of Scientific and Technical Information of China (English)

    Louis Libbrecht

    2006-01-01

    In recent years, the results of several studies suggest that human liver tumors can be derived from hepatic progenitor cells rather than from mature cell types.The available data indeed strongly suggest that most combined hepatocellular-cholangiocarcinomas arise from hepatic progenitor cells that retained their potential to differentiate into the hepatocytic and biliary lineages.Hepatic progenitor cells could also be the basis for some hepatocellular carcinomas and hepatocellular adenomas, although it is very difficult to determine the origin of an individual hepatocellular carcinoma. There is currently not enough data to make statements regarding a hepatic progenitor cell origin of cholangiocarcinoma.The presence of hepatic progenitor cell markers and the presence and extent of the cholangiocellular component are factors that are related to the prognosis of hepatocellular carcinomas and combined hepatocellularcholangiocarcinomas, respectively.

  8. Expression analyses of human cleft palate tissue suggest a role for osteopontin and immune related factors in palatal development

    DEFF Research Database (Denmark)

    Jakobsen, Linda P; Borup, Rehannah; Vestergaard, Janni

    2009-01-01

    Cleft lip and/or palate (CL/P) is a common congenital malformation with a complex etiology which is not fully elucidated yet. Epidemiological studies point to different etiologies in the cleft lip and palate subgroups, isolated cleft lip (CL), isolated cleft palate (CP) and combined cleft lip...... and palate (CLP). In order to understand the biological basis in these cleft lip and palate subgroups better we studied the expression profiles in human tissue from patients with CL/P. In each of the CL/P subgroups, samples were obtained from three patients and gene expression analysis was performed....... Moreover, selected differentially expressed genes were analyzed by quantitative RT-PCR, and by immunohistochemical staining of craniofacial tissue from human embryos. Osteopontin (SPP1) and other immune related genes were significantly higher expressed in palate tissue from patients with CLP compared to CP...

  9. Hypofractionation in non-small cell lung cancer (NSCLC): suggestions from modelling both acute and chronic hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Ruggieri, Ruggero [Department of Medical Physics, Az. Osp. Bianchi Melacrino Morelli, Via Melacrino, 89100 Reggio Calabria (Italy)

    2004-10-21

    Based on experimental estimates for acute and chronic tumour hypoxia, a speculative analysis of the therapeutic ratio dependence on the number of once-daily five-days-per-week fractions (n) for non-small cell lung cancer (NSCLC) radiotherapy is proposed. For this purpose an adapted formulation of the linear-quadratic model has been derived, including the effects of tumour repopulation, inter-tumour {alpha}-heterogeneity and oxygen enhancement ratio dependence on the dose per fraction. The relation between the curative dose D{sub 50}, assuring 50% tumour control probability, and n has been computed: for (n, D{sub 50}) fractionation schemes, the therapeutic ratios have been compared in terms of effective normalized total doses to the lungs (NTD{sup eff}{sub L}), estimated by a few supposed fractions of the normalized total dose to the tumour. Results suggest that D{sub 50} is dominated by chronic hypoxia for shortly hypofractionated treatments and by acute hypoxia for multifractionated treatments. Furthermore, the optimum number of fractions depends on the rapidity of the reoxygenation from chronically hypoxic cells, almost independently of the extent of both chronic and acute hypoxia. For NSCLC, both the reduction of n until about 20 fractions in hypofractionated dose-escalation trials, and the extension of extra-cranial stereotactic radiotherapy schedules to include at least 5-10 fractions, seem to be supported by this model.

  10. Largescale Transcriptomics Analysis Suggests Over-Expression of BGH3, MMP9 and PDIA3 in Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    He, Yuan; Shao, Fangyang; Pi, Weidong; Shi, Cong; Chen, Yujia; Gong, Diping; Wang, Bingjie; Cao, Zhiwei; Tang, Kailin

    2016-01-01

    Oral squamous cell carcinoma (OSCC) has been reported as the most prevalent cancer of the head and neck region, while early diagnosis remains challenging. Here we took a comprehensive bioinformatics study on microarray data of 326 OSCC clinical samples with control of 165 normal tissues. The cell interaction pathways of ECM-receptor interaction and focal adhesion were found to be significantly regulated in OSCC samples. Further analysis of the topological properties and expression consistency identified that three hub genes in the gene interaction network, MMP9, PDIA3 and BGH3, were consistently up-expressed in OSCC samples. When being validated on additional microarray datasets of 41 OSCC samples, the validation rate of over-expressed BGH3, MMP9, and PDIA3 reached 90%, 90% and 84% respectively. At last, immuno-histochemical assays were done to test the protein expression of the three genes on newly collected clinical samples of 35 OSCC, 20 samples of pre-OSCC stage, and 12 normal oral mucosa specimens. Their protein expression levels were also found to progressively increase from normal mucosa to pre-OSCC stage and further to OSCC (ANOVA p = 0.000), suggesting their key roles in OSCC pathogenesis. Based on above solid validation, we propose BGH3, MMP9 and PDIA3 might be further explored as potential biomarkers to aid OSCC diagnosis.

  11. Largescale Transcriptomics Analysis Suggests Over-Expression of BGH3, MMP9 and PDIA3 in Oral Squamous Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Yuan He

    Full Text Available Oral squamous cell carcinoma (OSCC has been reported as the most prevalent cancer of the head and neck region, while early diagnosis remains challenging. Here we took a comprehensive bioinformatics study on microarray data of 326 OSCC clinical samples with control of 165 normal tissues. The cell interaction pathways of ECM-receptor interaction and focal adhesion were found to be significantly regulated in OSCC samples. Further analysis of the topological properties and expression consistency identified that three hub genes in the gene interaction network, MMP9, PDIA3 and BGH3, were consistently up-expressed in OSCC samples. When being validated on additional microarray datasets of 41 OSCC samples, the validation rate of over-expressed BGH3, MMP9, and PDIA3 reached 90%, 90% and 84% respectively. At last, immuno-histochemical assays were done to test the protein expression of the three genes on newly collected clinical samples of 35 OSCC, 20 samples of pre-OSCC stage, and 12 normal oral mucosa specimens. Their protein expression levels were also found to progressively increase from normal mucosa to pre-OSCC stage and further to OSCC (ANOVA p = 0.000, suggesting their key roles in OSCC pathogenesis. Based on above solid validation, we propose BGH3, MMP9 and PDIA3 might be further explored as potential biomarkers to aid OSCC diagnosis.

  12. Chestnut extract induces apoptosis in AGS human gastric cancer cells.

    Science.gov (United States)

    Lee, Hyun Sook; Kim, Eun Ji; Kim, Sun Hyo

    2011-06-01

    In Korea, chestnut production is increasing each year, but consumption is far below production. We investigated the effect of chestnut extracts on antioxidant activity and anticancer effects. Ethanol extracts of raw chestnut (RCE) or chestnut powder (CPE) had dose-dependent superoxide scavenging activity. Viable numbers of MDA-MD-231 human breast cancer cells, DU145 human prostate cancer cells, and AGS human gastric cancer cells decreased by 18, 31, and 69%, respectively, following treatment with 200 µg/mL CPE for 24 hr. CPE at various concentrations (0-200 µg/mL) markedly decreased AGS cell viability and increased apoptotic cell death dose and time dependently. CPE increased the levels of cleaved caspase-8, -7, -3, and poly (ADP-ribose) polymerase in a dose-dependent manner but not cleaved caspase-9. CPE exerted no effects on Bcl-2 and Bax levels. The level of X-linked inhibitor of apoptosis protein decreased within a narrow range following CPE treatment. The levels of Trail, DR4, and Fas-L increased dose-dependently in CPE-treated AGS cells. These results show that CPE decreases growth and induces apoptosis in AGS gastric cancer cells and that activation of the death receptor pathway contributes to CPE-induced apoptosis in AGS cells. In conclusion, CPE had more of an effect on gastric cancer cells than breast or prostate cancer cells, suggesting that chestnuts would have a positive effect against gastric cancer.

  13. Opiate receptor blockade on human granulosa cells inhibits VEGF release.

    Science.gov (United States)

    Lunger, Fabian; Vehmas, Anni P; Fürnrohr, Barbara G; Sopper, Sieghart; Wildt, Ludwig; Seeber, Beata

    2016-03-01

    The objectives of this study were to determine whether the main opioid receptor (OPRM1) is present on human granulosa cells and if exogenous opiates and their antagonists can influence granulosa cell vascular endothelial growth factor (VEGF) production via OPRM1. Granulosa cells were isolated from women undergoing oocyte retrieval for IVF. Complementary to the primary cells, experiments were conducted using COV434, a well-characterized human granulosa cell line. Identification and localization of opiate receptor subtypes was carried out using Western blot and flow cytometry. The effect of opiate antagonist on granulosa cell VEGF secretion was assessed by enzyme-linked immunosorbent assay. For the first time, the presence of OPRM1 on human granulosa cells is reported. Blocking of opiate signalling using naloxone, a specific OPRM1 antagonist, significantly reduced granulosa cell-derived VEGF levels in both COV434 and granulosa-luteal cells (P opiate receptors and opiate signalling in granulosa cells suggest a possible role in VEGF production. Targeting this signalling pathway could prove promising as a new clinical option in the prevention and treatment of ovarian hyperstimulation syndrome.

  14. Novel synthetic organosulfur compounds induce apoptosis of human leukemic cells.

    Science.gov (United States)

    Wong, W W; Macdonald, S; Langler, R F; Penn, L Z

    2000-01-01

    It has been well documented that natural organosulfur compounds (OSCs) derived from plants such as garlic, onions and mahogany trees possess antiproliferative properties; however, the essential chemical features of the active OSC compounds remain unclear. To investigate the association between OSC structure and growth inhibitory activity, we synthesized novel relatives of dysoxysulfone, a natural OSC derived from the Fijian medicinal plant, Dysoxylum richii. In this study, we have examined the antiproliferative effects of these novel OSCs on a model human leukemic cell system and show that the compounds segregate into three groups. Group I, consisting of compounds A, B, G and J, did not affect either cell proliferation or the cell cycle profile of the leukemic cell lines. Group II, consisting of compounds F and H, induced the cells to undergo apoptosis from the G2/M phase of the cell cycle. Group III, consisting of compounds C, D, E and I, decreased cell proliferation and induced apoptosis throughout the cell cycle. The apoptotic agonists of Group II and III shared a common disulfide moiety, essential for leukemic cell cytotoxicity. Interestingly, Group II compounds did not affect cell viability of normal human diploid cells, suggesting the regions flanking the disulfide group contributes to the specificity of cell killing. Thus, we provide evidence that structure-activity analysis of natural products can identify novel compounds for the development of new therapeutics that can trigger apoptosis in a tumor-specific manner.

  15. Mitotic spindle orientation predicts outer radial glial cell generation in human neocortex.

    Science.gov (United States)

    LaMonica, Bridget E; Lui, Jan H; Hansen, David V; Kriegstein, Arnold R

    2013-01-01

    The human neocortex is increased in size and complexity as compared with most other species. Neocortical expansion has recently been attributed to protracted neurogenesis by outer radial glial cells in the outer subventricular zone, a region present in humans but not in rodents. The mechanisms of human outer radial glial cell generation are unknown, but are proposed to involve division of ventricular radial glial cells; neural stem cells present in all developing mammals. Here we show that human ventricular radial glial cells produce outer radial glial cells and seed formation of the outer subventricular zone via horizontal divisions, which occur more frequently in humans than in rodents. We further find that outer radial glial cell mitotic behaviour is cell intrinsic, and that the basal fibre, inherited by outer radial glial cells after ventricular radial glial division, determines cleavage angle. Our results suggest that altered regulation of mitotic spindle orientation increased outer radial glial cell number, and ultimately neuronal number, during human brain evolution.

  16. Genome-wide association study in multiple human prion diseases suggests genetic risk factors additional to PRNP

    OpenAIRE

    Mead, Simon; Uphill, James; Beck, John; Poulter, Mark; Campbell, Tracy; Lowe, Jessica; Adamson, Gary; Hummerich, Holger; Klopp, Norman; Rückert, Ina-Maria; Wichmann, H-Erich; Azazi, Dhoyazan; Plagnol, Vincent; Pako, Wandagi H.; Whitfield, Jerome

    2011-01-01

    Prion diseases are fatal neurodegenerative diseases of humans and animals caused by the misfolding and aggregation of prion protein (PrP). Mammalian prion diseases are under strong genetic control but few risk factors are known aside from the PrP gene locus (PRNP). No genome-wide association study (GWAS) has been done aside from a small sample of variant Creutzfeldt–Jakob disease (CJD). We conducted GWAS of sporadic CJD (sCJD), variant CJD (vCJD), iatrogenic CJD, inherited prion disease, kuru...

  17. Characterizing human herpes virus 6 following hematopoietic stem cell transplantation.

    Science.gov (United States)

    Perissinotti, Anthony J; Gulbis, Alison; Shpall, Elizabeth J; Howell, Joshua

    2015-04-01

    Human herpes virus 6 reactivation occurs in approximately 50% of patients following hematopoietic stem cell transplant, however, the significance of human herpes virus 6 reactivation remains uncertain. A retrospective study was conducted analyzing clinical data of patients testing positive for human herpes virus 6 by quantitative polymerase chain reaction following hematopoietic stem cell transplant from 1 January 1998 to 1 October 2011. Data retrieved were used to describe the clinical course and outcome of human herpes virus 6 positive hematopoietic stem cell transplant patients. Sixty patients were identified who tested positive for human herpes virus 6 by polymerase chain reaction following hematopoietic stem cell transplant. A high proportion of patients were identified in this cohort with acute myeloid leukemia (28.3%), active disease (65%), transplanted with a matched unrelated donor (30%), ≥ 1 antigen mismatched (28.3%) matched unrelated donor, or an umbilical cord graft (25%), and those who received antithymocyte globulin (42.4%). Thirty-eight (63.3%) patients were treated for human herpes virus 6 with foscarnet alone or in combination with intravenous immunoglobulin, whereas 18 (30%) did not require treatment survival at Day 100 was 73.3%. This study suggests human herpes virus 6 reactivation occurs shortly after hematopoietic stem cell transplant (median of 25 days (interquartile range, 20-31.75) after hematopoietic stem cell transplant). Many potential risk factors are described in this report. Treatment of human herpes virus 6 predominately consisted of foscarnet with or without intravenous immunoglobulin; however, treatment of human herpes virus 6 was not always warranted. Furthermore, the effect of treatment on patient outcomes is uncertain. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  18. Structural and Sequence Similarities of Hydra Xeroderma Pigmentosum A Protein to Human Homolog Suggest Early Evolution and Conservation

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    Apurva Barve

    2013-01-01

    Full Text Available Xeroderma pigmentosum group A (XPA is a protein that binds to damaged DNA, verifies presence of a lesion, and recruits other proteins of the nucleotide excision repair (NER pathway to the site. Though its homologs from yeast, Drosophila, humans, and so forth are well studied, XPA has not so far been reported from protozoa and lower animal phyla. Hydra is a fresh-water cnidarian with a remarkable capacity for regeneration and apparent lack of organismal ageing. Cnidarians are among the first metazoa with a defined body axis, tissue grade organisation, and nervous system. We report here for the first time presence of XPA gene in hydra. Putative protein sequence of hydra XPA contains nuclear localization signal and bears the zinc-finger motif. It contains two conserved Pfam domains and various characterized features of XPA proteins like regions for binding to excision repair cross-complementing protein-1 (ERCC1 and replication protein A 70 kDa subunit (RPA70 proteins. Hydra XPA shows a high degree of similarity with vertebrate homologs and clusters with deuterostomes in phylogenetic analysis. Homology modelling corroborates the very close similarity between hydra and human XPA. The protein thus most likely functions in hydra in the same manner as in other animals, indicating that it arose early in evolution and has been conserved across animal phyla.

  19. INTERACTIONS BETWEEN THE HUMAN GASTRIC CARCINOMA CELL AND THE HUMAN VASCULAR ENDOTHELIAL CELL

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To definite the interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the establishment and maintenance of the tumor vascular system and the tumor hematogenous metastasis.Methods We prepared the conditioned mediums of each cell so as to study the effect of the conditioned medium on itself or others by MTT colorimetry. The comprehensive effect of interactions between two cells was determined by stratified transfilter co-culture or direct contact co-culture.Results The conditioned medium of human gastric carcinoma cell can stimulate the proliferation of the human vascular endothelial cell, but the CM of HVEC can inhibit the growth of HGCC. Both kinds of cells can inhibit the growth of itself. The ultimate comprehensive effect of the interactions between two kinds of cells was increase of total cell numbers.Conclusion There exist the complicated interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the tumor angiogenesis and the tumor hematogenous metastasis. The ultimate comprehensive effect of the interactions is increase of total cells numbers and tumor volume.

  20. Acquired resistance to auranofin in cultured human cells.

    Science.gov (United States)

    Glennås, A; Rugstad, H E

    1985-01-01

    A substrain (HEAF) of cultured human epithelial cells, grown as monolayers, was selected for resistance to auranofin (AF), a gold-containing anti-arthritic drug, by growing the parental HE cells with stepwise increased concentrations of AF in the medium. HEAF cells acquired resistance to 2 mumol AF/l, twice the concentration tolerated by the sensitive HE cells. Resistance to AF was also demonstrated in another substrain (HE100) originally selected for by its cadmium resistance, and characterized by a high cytosolic metallothionein (MT) content. Following continuous exposure to 2 mumol AF/l for 4 days, 58% of the HEAF cells, 67% of the HE100 cells, and 16% of the HE cells remained adherent to the flasks, compared with non-treated controls. Following 24 h AF exposure to living cells, HEAF cells had one-half and HE100 cells twice the cellular and cytosolic gold concentration per mg protein, as compared with HE cells. Gel filtration of cell cytosols revealed gold-binding proteins with a mol. wt. of about 10 000 apparently occurring on AF exposure in HEAF and HE cells. They bound 10-15% of cytosolic gold. MT in HE100 cells bound AF-gold to about the same extent. We suggest that the ability of cells to maintain the gold concentration at a low level (HEAF) and trapping of gold by MT (HE100) or low molecular weight proteins occurring on AF treatment (HEAF) may be mechanisms contributing to the observed cellular resistance to AF.

  1. Teplizumab induces human gut-tropic regulatory cells in humanized mice and patients.

    Science.gov (United States)

    Waldron-Lynch, Frank; Henegariu, Octavian; Deng, Songyan; Preston-Hurlburt, Paula; Tooley, James; Flavell, Richard; Herold, Kevan C

    2012-01-25

    The development and optimization of immune therapies in patients has been hampered by the lack of preclinical models in which their effects on human immune cells can be studied. As a result, observations that have been made in preclinical studies have suggested mechanisms of drug action in murine models that have not been confirmed in clinical studies. Here, we used a humanized mouse reconstituted with human hematopoietic stem cells to study the mechanism of action of teplizumab, an Fc receptor nonbinding humanized monoclonal antibody to CD3 being tested in clinical trials for the treatment of patients with type 1 diabetes mellitus. In this model, human gut-tropic CCR6(+) T cells exited the circulation and secondary lymph organs and migrated to the small intestine. These cells then produced interleukin-10 (IL-10), a regulatory cytokine, in quantities that could be detected in the peripheral circulation. Blocking T cell migration to the small intestine with natalizumab, which prevents cellular adhesion by inhibiting α(4) integrin binding, abolished the treatment effects of teplizumab. Moreover, IL-10 expression by CD4(+)CD25(high)CCR6(+)FoxP3 cells returning to the peripheral circulation was increased in patients with type 1 diabetes treated with teplizumab. These findings demonstrate that humanized mice may be used to identify novel immunologic mechanisms that occur in patients treated with immunomodulators.

  2. Fibronectin production by human mammary cells

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    Stampfer, M.R. (Univ. of California, Berkeley); Vlodavsky, I.; Smith, H.S.; Ford, R.; Becker, F.F.; Riggs, J.

    1981-01-01

    Human mammary cells were examined for the presence of the high-molecular-weight surface glycoprotein fibronectin. Early passage mammary epithelial cell and fibroblast cultures from both carcinomas and normal tissues were tested for the presence of cell-associated fibronectin by immunofluorescence microscopy and for the synthesis and secretion of fibronectin by specific immunoprecipitation of metabolically labeled protein. In vivo frozen sections of primary carcinomas and normal tissues were tested for the localization of fibronectin by immunofluorescence microscopy. In contrast to the extensive fibrillar networks of fibronectin found in the fibroblast cultures, the epithelial cell cultures from both tissue sources displayed a pattern of cell-associated fibronectin characterizd by powdery, punctate staining. However, the cultured epithelial cells, as well as the fibroblasts, secreted large quantities of fibronectin into the medium. Putative myoepithelial cells also displayed extensive fibrillar networks of fibronectin. The difference in cell-associated fibronectin distribution between the epithelial cells and the fibroblasts and putative myoepithelial cells provided a simple means of quantitating stromal and myoepithelial cell contamination of the mammary epithelial cells in culture. In vivo, normal tissues showed fibronectin primarily localized in the basement membrane surrounding the epithelial cells and in the stroma. Most primary carcinomas displayed powdery, punctate staining on the epithelial cells in addition to the fibronectin present in the surrounding stroma.

  3. Genome-wide association study in multiple human prion diseases suggests genetic risk factors additional to PRNP

    Science.gov (United States)

    Mead, Simon; Uphill, James; Beck, John; Poulter, Mark; Campbell, Tracy; Lowe, Jessica; Adamson, Gary; Hummerich, Holger; Klopp, Norman; Rückert, Ina-Maria; Wichmann, H-Erich; Azazi, Dhoyazan; Plagnol, Vincent; Pako, Wandagi H.; Whitfield, Jerome; Alpers, Michael P.; Whittaker, John; Balding, David J.; Zerr, Inga; Kretzschmar, Hans; Collinge, John

    2012-01-01

    Prion diseases are fatal neurodegenerative diseases of humans and animals caused by the misfolding and aggregation of prion protein (PrP). Mammalian prion diseases are under strong genetic control but few risk factors are known aside from the PrP gene locus (PRNP). No genome-wide association study (GWAS) has been done aside from a small sample of variant Creutzfeldt–Jakob disease (CJD). We conducted GWAS of sporadic CJD (sCJD), variant CJD (vCJD), iatrogenic CJD, inherited prion disease, kuru and resistance to kuru despite attendance at mortuary feasts. After quality control, we analysed 2000 samples and 6015 control individuals (provided by the Wellcome Trust Case Control Consortium and KORA-gen) for 491032-511862 SNPs in the European study. Association studies were done in each geographical and aetiological group followed by several combined analyses. The PRNP locus was highly associated with risk in all geographical and aetiological groups. This association was driven by the known coding variation at rs1799990 (PRNP codon 129). No non-PRNP loci achieved genome-wide significance in the meta-analysis of all human prion disease. SNPs at the ZBTB38–RASA2 locus were associated with CJD in the UK (rs295301, P = 3.13 × 10−8; OR, 0.70) but these SNPs showed no replication evidence of association in German sCJD or in Papua New Guinea-based tests. A SNP in the CHN2 gene was associated with vCJD [P = 1.5 × 10−7; odds ratio (OR), 2.36], but not in UK sCJD (P = 0.049; OR, 1.24), in German sCJD or in PNG groups. In the overall meta-analysis of CJD, 14 SNPs were associated (P < 10−5; two at PRNP, three at ZBTB38–RASA2, nine at nine other independent non-PRNP loci), more than would be expected by chance. None of the loci recently identified as genome-wide significant in studies of other neurodegenerative diseases showed any clear evidence of association in prion diseases. Concerning common genetic variation, it is likely that the PRNP locus contains the only

  4. Laser printing of skin cells and human stem cells.

    Science.gov (United States)

    Koch, Lothar; Kuhn, Stefanie; Sorg, Heiko; Gruene, Martin; Schlie, Sabrina; Gaebel, Ralf; Polchow, Bianca; Reimers, Kerstin; Stoelting, Stephanie; Ma, Nan; Vogt, Peter M; Steinhoff, Gustav; Chichkov, Boris

    2010-10-01

    Laser printing based on laser-induced forward transfer (LIFT) is a new biofabrication technique for the arrangement of biological materials or living cells in well-defined patterns. In the current study, skin cell lines (fibroblasts/keratinocytes) and human mesenchymal stem cells (hMSC) were chosen for laser printing experiments due to their high potential in regeneration of human skin and new application possibilities of stem cell therapy. To evaluate the influence of LIFT on the cells, their survival rate, their proliferation and apoptotic activity, and the DNA damages and modifications of their cell surface markers were assessed and statistically evaluated over several days. The cells survived the transfer procedure with a rate of 98%  +/- 1% standard error of the mean (skin cells) and 90%  +/- 10% (hMSC), respectively. All used cell types maintain their ability to proliferate after LIFT. Further, skin cells and hMSC did not show an increase of apoptosis or DNA fragmentation. In addition, the hMSC keep their phenotype as proven by fluorescence activated cell sorting (FACS) analysis. This study demonstrates LIFT as a suitable technique for unharmed computer-controlled positioning of different cell types and a promising tool for future applications in the ex vivo generation of tissue replacements.

  5. Genetic engineering of human embryonic stem cells with lentiviral vectors.

    Science.gov (United States)

    Xiong, Chen; Tang, Dong-Qi; Xie, Chang-Qing; Zhang, Li; Xu, Ke-Feng; Thompson, Winston E; Chou, Wayne; Gibbons, Gary H; Chang, Lung-Ji; Yang, Li-Jun; Chen, Yuqing E

    2005-08-01

    Human embryonic stem (hES) cells present a valuable source of cells with a vast therapeutic potential. However, the low efficiency of directed differentiation of hES cells remains a major obstacle in their uses for regenerative medicine. While differentiation may be controlled by the genetic manipulation, effective and efficient gene transfer into hES cells has been an elusive goal. Here, we show stable and efficient genetic manipulations of hES cells using lentiviral vectors. This method resulted in the establishment of stable gene expression without loss of pluripotency in hES cells. In addition, lentiviral vectors were effective in conveying the expression of an U6 promoter-driven small interfering RNA (siRNA), which was effective in silencing its specific target. Taken together, our results suggest that lentiviral gene delivery holds great promise for hES cell research and application.

  6. Human spleen and red blood cells

    Science.gov (United States)

    Pivkin, Igor; Peng, Zhangli; Karniadakis, George; Buffet, Pierre; Dao, Ming

    2016-11-01

    Spleen plays multiple roles in the human body. Among them is removal of old and altered red blood cells (RBCs), which is done by filtering cells through the endothelial slits, small micron-sized openings. There is currently no experimental technique available that allows us to observe RBC passage through the slits. It was previously noticed that people without a spleen have less deformable red blood cells, indicating that the spleen may play a role in defining the size and shape of red blood cells. We used detailed RBC model implemented within the Dissipative Particle Dynamics (DPD) simulation framework to study the filter function of the spleen. Our results demonstrate that spleen indeed plays major role in defining the size and shape of the healthy human red blood cells.

  7. Rotating cell culture systems for human cell culture: human trophoblast cells as a model.

    Science.gov (United States)

    Zwezdaryk, Kevin J; Warner, Jessica A; Machado, Heather L; Morris, Cindy A; Höner zu Bentrup, Kerstin

    2012-01-18

    The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines support our understanding of invasion of the uterine wall and remodeling of uterine spiral arteries by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS. The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies. The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS

  8. Microenvironmental modulation of asymmetric cell division in human lung cancer cells.

    Science.gov (United States)

    Pine, Sharon R; Ryan, Bríd M; Varticovski, Lyuba; Robles, Ana I; Harris, Curtis C

    2010-02-02

    Normal tissue homeostasis is maintained through asymmetric cell divisions that produce daughter cells with differing self-renewal and differentiation potentials. Certain tumor cell subfractions can self-renew and repopulate the heterogeneous tumor bulk, suggestive of asymmetric cell division, but an equally plausible explanation is that daughter cells of a symmetric division subsequently adopt differing cell fates. Cosegregation of template DNA during mitosis is one mechanism by which cellular components are segregated asymmetrically during cell division in fibroblast, muscle, mammary, intestinal, and neural cells. Asymmetric cell division of template DNA in tumor cells has remained elusive, however. Through pulse-chase experiments with halogenated thymidine analogs, we determined that a small population of cells within human lung cancer cell lines and primary tumor cell cultures asymmetrically divided their template DNA, which could be visualized in single cells and in real time. Template DNA cosegregation was enhanced by cell-cell contact. Its frequency was density-dependent and modulated by environmental changes, including serum deprivation and hypoxia. In addition, we found that isolated CD133(+) lung cancer cells were capable of tumor cell repopulation. Strikingly, during cell division, CD133 cosegregated with the template DNA, whereas the differentiation markers prosurfactant protein-C and pan-cytokeratins were passed to the opposing daughter cell, demonstrating that segregation of template DNA correlates with lung cancer cell fate. Our results demonstrate that human lung tumor cell fate decisions may be regulated during the cell division process. The characterization and modulation of asymmetric cell division in lung cancer can provide insight into tumor initiation, growth, and maintenance.

  9. Neural progenitor cells from human induced pluripotent stem cells generated less autogenous immune response.

    Science.gov (United States)

    Huang, Ke; Liu, PengFei; Li, Xiang; Chen, ShuBin; Wang, LiHui; Qin, Li; Su, ZhengHui; Huang, WenHao; Liu, Juli; Jia, Bei; Liu, Jie; Cai, JingLei; Pei, DuanQing; Pan, GuangJin

    2014-02-01

    The breakthrough development of induced pluripotent stem cells (iPSCs) raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells. However, whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear. In this study, we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with autogenous peripheral blood mononuclear cells (PBMCs), we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation. However, a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs. Furthermore, no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells (CD3(+)CD8(-) T cells, CD3(+)CD8(+) T cells or CD3(-)CD56(+) NK cells) by NPCs in both PBMC and T cell co-culture systems. These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants, and thus set a base for further preclinical evaluation of human iPSCs.

  10. Autophagy in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Thien Tra

    Full Text Available Autophagy (macroautophagy is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.

  11. Lack of megalin expression in adult human terminal ileum suggests megalin‐independent cubilin/amnionless activity during vitamin B12 absorption

    Science.gov (United States)

    Jensen, Louise L.; Andersen, Rikke K.; Hager, Henrik; Madsen, Mette

    2014-01-01

    Abstract Cubilin plays an essential role in terminal ileum and renal proximal tubules during absorption of vitamin B12 and ligands from the glomerular ultrafiltrate. Cubilin is coexpressed with amnionless, and cubilin and amnionless are mutually dependent on each other for correct processing to the plasma membrane upon synthesis. Patients with defects in either protein suffer from vitamin B12‐malabsorption and in some cases proteinuria. Cubilin lacks a transmembrane region and signals for endocytosis and is dependent on a transmembrane coreceptor during internalization. Amnionless has been shown to be able to mediate internalization of cubilin in a cell‐based model system. Cubilin has additionally been suggested to function together with megalin, and a recent study of megalin‐deficient patients indicates that uptake of cubilin ligands in the kidney is critically dependent on megalin. To further investigate the potential role of amnionless and megalin in relation to cubilin function in terminal ileum and vitamin B12 uptake, we initiated a study of CUBN/cubilin, AMN/amnionless, and LRP2/megalin expression in adult human terminal ileum. Our study is the first to reveal the expression pattern of cubilin, amnionless, and megalin in adult human terminal ileum, where cubilin and amnionless localize to the epithelial cells. Surprisingly, we did not detect any megalin protein in adult terminal ileum and consistently, only extremely low amounts of LRP2 mRNA. Our data therefore advocate that cubilin and amnionless act independently of megalin in adult terminal ileum and that the cubilin‐megalin interdependency accordingly should be considered as tissue and ligand specific. PMID:25052491

  12. Human BLyS facilitates engraftment of human PBL derived B cells in immunodeficient mice.

    Directory of Open Access Journals (Sweden)

    Madelyn R Schmidt

    Full Text Available The production of fully immunologically competent humanized mice engrafted with peripheral lymphocyte populations provides a model for in vivo testing of new vaccines, the durability of immunological memory and cancer therapies. This approach is limited, however, by the failure to efficiently engraft human B lymphocytes in immunodeficient mice. We hypothesized that this deficiency was due to the failure of the murine microenvironment to support human B cell survival. We report that while the human B lymphocyte survival factor, B lymphocyte stimulator (BLyS/BAFF enhances the survival of human B cells ex vivo, murine BLyS has no such protective effect. Although human B cells bound both human and murine BLyS, nuclear accumulation of NF-kappaB p52, an indication of the induction of a protective anti-apoptotic response, following stimulation with human BLyS was more robust than that induced with murine BLyS suggesting a fundamental disparity in BLyS receptor signaling. Efficient engraftment of both human B and T lymphocytes in NOD rag1(-/- Prf1(-/- immunodeficient mice treated with recombinant human BLyS is observed after adoptive transfer of human PBL relative to PBS treated controls. Human BLyS treated recipients had on average 40-fold higher levels of serum Ig than controls and mounted a de novo antibody response to the thymus-independent antigens in pneumovax vaccine. The data indicate that production of fully immunologically competent humanized mice from PBL can be markedly facilitated by providing human BLyS.

  13. Human embryonic stem cells for neuronal repair.

    Science.gov (United States)

    Ben-Hur, Tamir

    2006-02-01

    Human embryonic stem cells may serve as a potentially endeless source of transplantable cells to treat various neurologic disorders. Accumulating data have shown the therapeutic value of various neural precursor cell types in experimental models of neurologic diseases. Tailoring cell therapy for specific disorders requires the generation of cells that are committed to specific neural lineages. To this end, protocols were recently developed for the derivation of dopaminergic neurons, spinal motor neurons and oligodendrocytes from hESC. These protocols recapitulate normal development in culture conditions. However, a novel concept emerging from these studies is that the beneficial effect of transplanted stem cells is not only via cell replacement in damaged host tissue, but also by trophic and protective effects, as well as by an immunomodulatory effect that down-regulates detrimental brain inflammation.

  14. Novel agents inhibit human leukemic cells

    Institute of Scientific and Technical Information of China (English)

    Wei-ping YU; Juan LI

    2012-01-01

    Ouabain (OUA) and pyrithione zinc (PZ) have been proved as the potential drugs for treating acute myeloid leukemia (AML).Selected from a screening among 1040 Food and Drug Administration-approved pharmacological agents,both drugs showability to induce apoptosis of the culturing AML cells,exhibiting the poisoning effect on the cells.Studies also reveal the efficiency of the drugs in inhibiting the growth of human AML cells injected into the mice lacking of immunity and killing primary AML cells from the peripheral blood of AML patients[1].

  15. Expression of zebrafish GATA 3 (gta3) during gastrulation and neurulation suggests a role in the specification of cell fate.

    Science.gov (United States)

    Neave, B; Rodaway, A; Wilson, S W; Patient, R; Holder, N

    1995-06-01

    In order to understand the role of the transcription factor GATA 3 in vertebrate development, we have examined its expression and some aspects of its regulation during gastrulation and neurulation in the zebrafish. The complete coding sequence of the cDNA encoding the zebrafish GATA 3 homologue, termed gta3, is described. Analysis of expression patterns by in situ hybridisation shows the gene to be expressed during gastrulation in the ventral region of the embryo which includes tissue fated to form the non-neural ectoderm. By the end of gastrulation, there is a clear border to the gta3 expression domain that is close to the edge of the neural plate. Subsequently, gta3 expresses in the pronephric duct and in defined regions of the central nervous system which include specific cells in each segment of the spinal cord and nuclei in the brain. Double labelling embryos with a probe for gta3 and antibodies which identify differentiated neurons suggest that gta3 is dynamically expressed during the early differentiation phase of a subset of neurons but not in the terminal phase. Analysis of gta3 expression in dorsalised embryos and in cyc and spt mutant embryos indicates that the neural expression of the gene is subject to control by signals from the mesoderm, including both the notochord and the somites, which influence the segmental organisation of expression in the spinal cord.

  16. Molecular Signatures of Cardiac Defects in Down Syndrome Lymphoblastoid Cell Lines Suggest Altered Ciliome and Hedgehog Pathways

    Science.gov (United States)

    Ripoll, Clémentine; Rivals, Isabelle; Ait Yahya-Graison, Emilie; Dauphinot, Luce; Paly, Evelyne; Mircher, Clothilde; Ravel, Aimé; Grattau, Yann; Bléhaut, Henri; Mégarbane, André; Dembour, Guy; de Fréminville, Bénédicte; Touraine, Renaud; Créau, Nicole; Potier, Marie Claude; Delabar, Jean Maurice

    2012-01-01

    Forty percent of people with Down syndrome exhibit heart defects, most often an atrioventricular septal defect (AVSD) and less frequently a ventricular septal defect (VSD) or atrial septal defect (ASD). Lymphoblastoid cell lines (LCLs) were established from lymphocytes of individuals with trisomy 21, the chromosomal abnormality causing Down syndrome. Gene expression profiles generated from DNA microarrays of LCLs from individuals without heart defects (CHD−; n = 22) were compared with those of LCLs from patients with cardiac malformations (CHD+; n = 21). After quantile normalization, principal component analysis revealed that AVSD carriers could be distinguished from a combined group of ASD or VSD (ASD+VSD) carriers. From 9,758 expressed genes, we identified 889 and 1,016 genes differentially expressed between CHD− and AVSD and CHD− and ASD+VSD, respectively, with only 119 genes in common. A specific chromosomal enrichment was found in each group of affected genes. Among the differentially expressed genes, more than 65% are expressed in human or mouse fetal heart tissues (GEO dataset). Additional LCLs from new groups of AVSD and ASD+VSD patients were analyzed by quantitative PCR; observed expression ratios were similar to microarray results. Analysis of GO categories revealed enrichment of genes from pathways regulating clathrin-mediated endocytosis in patients with AVSD and of genes involved in semaphorin-plexin-driven cardiogenesis and the formation of cytoplasmic microtubules in patients with ASD-VSD. A pathway-oriented search revealed enrichment in the ciliome for both groups and a specific enrichment in Hedgehog and Jak-stat pathways among ASD+VSD patients. These genes or related pathways are therefore potentially involved in normal cardiogenesis as well as in cardiac malformations observed in individuals with trisomy 21. PMID:22912673

  17. Molecular signatures of cardiac defects in Down syndrome lymphoblastoid cell lines suggest altered ciliome and Hedgehog pathways.

    Directory of Open Access Journals (Sweden)

    Clémentine Ripoll

    Full Text Available Forty percent of people with Down syndrome exhibit heart defects, most often an atrioventricular septal defect (AVSD and less frequently a ventricular septal defect (VSD or atrial septal defect (ASD. Lymphoblastoid cell lines (LCLs were established from lymphocytes of individuals with trisomy 21, the chromosomal abnormality causing Down syndrome. Gene expression profiles generated from DNA microarrays of LCLs from individuals without heart defects (CHD(-; n = 22 were compared with those of LCLs from patients with cardiac malformations (CHD(+; n = 21. After quantile normalization, principal component analysis revealed that AVSD carriers could be distinguished from a combined group of ASD or VSD (ASD+VSD carriers. From 9,758 expressed genes, we identified 889 and 1,016 genes differentially expressed between CHD(- and AVSD and CHD(- and ASD+VSD, respectively, with only 119 genes in common. A specific chromosomal enrichment was found in each group of affected genes. Among the differentially expressed genes, more than 65% are expressed in human or mouse fetal heart tissues (GEO dataset. Additional LCLs from new groups of AVSD and ASD+VSD patients were analyzed by quantitative PCR; observed expression ratios were similar to microarray results. Analysis of GO categories revealed enrichment of genes from pathways regulating clathrin-mediated endocytosis in patients with AVSD and of genes involved in semaphorin-plexin-driven cardiogenesis and the formation of cytoplasmic microtubules in patients with ASD-VSD. A pathway-oriented search revealed enrichment in the ciliome for both groups and a specific enrichment in Hedgehog and Jak-stat pathways among ASD+VSD patients. These genes or related pathways are therefore potentially involved in normal cardiogenesis as well as in cardiac malformations observed in individuals with trisomy 21.

  18. Structures of the human Pals1 PDZ domain with and without ligand suggest gated access of Crb to the PDZ peptide-binding groove

    Energy Technology Data Exchange (ETDEWEB)

    Ivanova, Marina E.; Fletcher, Georgina C.; O’Reilly, Nicola; Purkiss, Andrew G.; Thompson, Barry J. [Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3LY (United Kingdom); McDonald, Neil Q., E-mail: neil.mcdonald@cancer.org.uk [Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3LY (United Kingdom); Birkbeck College, University of London, Malet Street, London WC1E 7HX (United Kingdom)

    2015-03-01

    This study characterizes the interaction between the carboxy-terminal (ERLI) motif of the essential polarity protein Crb and the Pals1/Stardust PDZ-domain protein. Structures of human Pals1 PDZ with and without a Crb peptide are described, explaining the highly conserved nature of the ERLI motif and revealing a sterically blocked peptide-binding groove in the absence of ligand. Many components of epithelial polarity protein complexes possess PDZ domains that are required for protein interaction and recruitment to the apical plasma membrane. Apical localization of the Crumbs (Crb) transmembrane protein requires a PDZ-mediated interaction with Pals1 (protein-associated with Lin7, Stardust, MPP5), a member of the p55 family of membrane-associated guanylate kinases (MAGUKs). This study describes the molecular interaction between the Crb carboxy-terminal motif (ERLI), which is required for Drosophila cell polarity, and the Pals1 PDZ domain using crystallography and fluorescence polarization. Only the last four Crb residues contribute to Pals1 PDZ-domain binding affinity, with specificity contributed by conserved charged interactions. Comparison of the Crb-bound Pals1 PDZ structure with an apo Pals1 structure reveals a key Phe side chain that gates access to the PDZ peptide-binding groove. Removal of this side chain enhances the binding affinity by more than fivefold, suggesting that access of Crb to Pals1 may be regulated by intradomain contacts or by protein–protein interaction.

  19. Lipopolysaccharide induces IFN-γ production in human NK cells

    Directory of Open Access Journals (Sweden)

    Leonid M Kanevskiy

    2013-01-01

    Full Text Available NK cells have been shown to play a regulatory role in sepsis. According to the current view, NK cells become activated via macrophages or dendritic cells primed by lipopolysaccharide (LPS. Recently TLR4 gene expression was detected in human NK cells suggesting the possibility of a direct action of LPS on NK cells. In this study, effects of LPS on NK cell cytokine production and cytotoxicity were studied using highly purified human NK cells. LPS induced IFN-γ production in the presence of IL-2 in cell populations containing >98% CD56+ cells. Surprisingly, in the same experiments LPS decreased NK cell degranulation. No significant expression of markers related to blood dendritic cells, monocytes or T or B lymphocytes in the NK cell preparations was observed; the portions of HLA-DRbright, CD14+, CD3+ and CD20+ cells amounted to less than 0.1% within the cell populations. No more than 0.2% of NK cells were shown to be slightly positive for surface TLR4 in our experimental system, although intracellular staining revealed moderate amounts of TLR4 inside the NK cell population. These cells were negative for surface CD14, the receptor participating in LPS recognition by TLR4. Incubation of NK cells with IL-2 or/and LPS did not lead to an increase in TLR4 surface expression. TLR4–CD56+ NK cells isolated by cell sorting secreted IFN-γ in response to LPS. Antibody to TLR4 did not block the LPS-induced increase in IFN-γ production. We have also shown that Re-form of LPS lacking outer core oligosaccharide and O-antigen induces less cytokine production in NK cells than full length LPS. We speculate that the polysaccharide fragments of LPS molecule may take part in LPS-induced IFN-γ production by NK cells. Collectively our data suggest the existence of a mechanism of LPS direct action on NK cells distinct from established TLR4-mediated signaling.

  20. Limited gene expression variation in human embryonic stem cell and induced pluripotent stem cell-derived endothelial cells.

    Science.gov (United States)

    White, Mark P; Rufaihah, Abdul J; Liu, Lei; Ghebremariam, Yohannes T; Ivey, Kathryn N; Cooke, John P; Srivastava, Deepak

    2013-01-01

    Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes, yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified, homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here, we report a differentiation protocol based on embryonic development that consistently yields large numbers of endothelial cells (ECs) derived from multiple hESCs or iPS cells. Mesoderm differentiation of embryoid bodies was maximized, and defined growth factors were used to generate KDR(+) EC progenitors. Magnetic purification of a KDR(+) progenitor subpopulation resulted in an expanding, homogeneous pool of ECs that expressed EC markers and had functional properties of ECs. Comparison of the transcriptomes revealed limited gene expression variability between multiple lines of human iPS-derived ECs or between lines of ES- and iPS-derived ECs. These results demonstrate a method to generate large numbers of pure human EC progenitors and differentiated ECs from pluripotent stem cells and suggest individual lineages derived from human iPS cells may have significantly less variance than their pluripotent founders.

  1. Mapping of HPV transcripts in four human cervical lesions using RNAseq suggests quantitative rearrangements during carcinogenic progression.

    Science.gov (United States)

    Chen, Jinmiao; Xue, Yuezhen; Poidinger, Michael; Lim, Thimothy; Chew, Sung Hock; Pang, Chai Ling; Abastado, Jean-Pierre; Thierry, Françoise

    2014-08-01

    Two classes of Human papillomaviruses (HPV) infect the anogenital track: high risk viruses that are associated with risk of cervical cancer and low risk types that drive development of benign lesions, such as condylomas. In the present study, we established quantitative transcriptional maps of the viral genome in clinical lesions associated with high risk HPV16 or low risk HPV6b. Marked qualitative and quantitative changes in the HPV16 transcriptome were associated with progression from low to high grade lesions. Specific transcripts encoding essential regulatory proteins such as E7, E2, E1^E4 and E5 were identified. We also identified intrinsic differences between the HPV6b-associated condyloma transcript map and that of the HPV16-associated low grade CIN specifically regarding promoter usage. Characterization and quantification of HPV transcripts in patient samples thus establish the impact of viral transcriptional regulation on the status of HPV-associated lesions and may therefore help in defining new biologically-relevant prognosis markers. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Epitope Mapping of Antibodies Suggests the Novel Membrane Topology of B-Cell Receptor Associated Protein 31 on the Cell Surface of Embryonic Stem Cells: The Novel Membrane Topology of BAP31.

    Directory of Open Access Journals (Sweden)

    Won-Tae Kim

    Full Text Available When located in the endoplasmic reticulum (ER membrane, B-cell receptor associated protein 31 (BAP31 is involved in the export of secreted proteins from the ER to the plasma membrane. In a previous study, we generated two monoclonal antibodies (mAbs, 297-D4 and 144-A8, that bound to surface molecules on human embryonic stem cells (hESCs, but not to surface molecules on mouse embryonic stem cells (mESCs. Subsequent studies revealed that the mAbs recognized BAP31 on the surface of hESCs. To investigate the membrane topology of BAP31 on the cell surface, we first examined the epitope specificity of 297-D4 and 144-A8, as well as a polyclonal anti-BAP31 antibody (α-BAP31. We generated a series of GST-fused BAP31 mutant proteins in which BAP31 was serially deleted at the C- terminus. GST-fused BAP31 mutant proteins were then screened to identify the epitopes targeted by the antibodies. Both 297-D4 and 144-A8 recognized C-terminal residues 208-217, while α-BAP31 recognized C-terminal residues 165-246, of BAP31 on hESCs, suggesting that the C-terminal domain of BAP31 is exposed on the cell surface. The polyclonal antibody α-BAP31 bound to mESCs, which confirmed that the C-terminal domain of BAP31 is also exposed on the surface of these cells. Our results show for the first time the novel membrane topology of cell surface-expressed BAP31 as the extracellular exposure of the BAP31 C-terminal domain was not predicted from previous studies.

  3. Epitope Mapping of Antibodies Suggests the Novel Membrane Topology of B-Cell Receptor Associated Protein 31 on the Cell Surface of Embryonic Stem Cells: The Novel Membrane Topology of BAP31.

    Science.gov (United States)

    Kim, Won-Tae; Choi, Hong Seo; Hwang, Hyo Jeong; Jung, Han-Sung; Ryu, Chun Jeih

    2015-01-01

    When located in the endoplasmic reticulum (ER) membrane, B-cell receptor associated protein 31 (BAP31) is involved in the export of secreted proteins from the ER to the plasma membrane. In a previous study, we generated two monoclonal antibodies (mAbs), 297-D4 and 144-A8, that bound to surface molecules on human embryonic stem cells (hESCs), but not to surface molecules on mouse embryonic stem cells (mESCs). Subsequent studies revealed that the mAbs recognized BAP31 on the surface of hESCs. To investigate the membrane topology of BAP31 on the cell surface, we first examined the epitope specificity of 297-D4 and 144-A8, as well as a polyclonal anti-BAP31 antibody (α-BAP31). We generated a series of GST-fused BAP31 mutant proteins in which BAP31 was serially deleted at the C- terminus. GST-fused BAP31 mutant proteins were then screened to identify the epitopes targeted by the antibodies. Both 297-D4 and 144-A8 recognized C-terminal residues 208-217, while α-BAP31 recognized C-terminal residues 165-246, of BAP31 on hESCs, suggesting that the C-terminal domain of BAP31 is exposed on the cell surface. The polyclonal antibody α-BAP31 bound to mESCs, which confirmed that the C-terminal domain of BAP31 is also exposed on the surface of these cells. Our results show for the first time the novel membrane topology of cell surface-expressed BAP31 as the extracellular exposure of the BAP31 C-terminal domain was not predicted from previous studies.

  4. Interleukin-24 inhibits the plasma cell differentiation program in human germinal center B cells.

    Science.gov (United States)

    Maarof, Ghyath; Bouchet-Delbos, Laurence; Gary-Gouy, Hélène; Durand-Gasselin, Ingrid; Krzysiek, Roman; Dalloul, Ali

    2010-03-04

    Complex molecular mechanisms control B-cell fate to become a memory or a plasma cell. Interleukin-24 (IL-24) is a class II family cytokine of poorly understood immune function that regulates the cell cycle. We previously observed that IL-24 is strongly expressed in leukemic memory-type B cells. Here we show that IL-24 is also expressed in human follicular B cells; it is more abundant in CD27(+) memory B cells and CD5-expressing B cells, whereas it is low to undetectable in centroblasts and plasma cells. Addition of IL-24 to B cells, cultured in conditions shown to promote plasma cell differentiation, strongly inhibited plasma cell generation and immunoglobulin G (IgG) production. By contrast, IL-24 siRNA increased terminal differentiation of B cells into plasma cells. IL-24 is optimally induced by BCR triggering and CD40 engagement; IL-24 increased CD40-induced B-cell proliferation and modulated the transcription of key factors involved in plasma cell differentiation. It also inhibited activation-induced tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT-3), and inhibited the transcription of IL-10. Taken together, our results indicate that IL-24 is a novel cytokine involved in T-dependent antigen (Ag)-driven B-cell differentiation and suggest its physiologic role in favoring germinal center B-cell maturation in memory B cells at the expense of plasma cells.

  5. Characterization of Side Cell Populations Obtained from Human Amnion Mesenchymal Cells

    Institute of Scientific and Technical Information of China (English)

    LI Ning; PIAO Zhengfu; Mamoru Kobayashi; Koji Sasaki; DING Shu-qin; Aiko Kikuchi; Isao Kamo; Norio Sakuragawa

    2009-01-01

    Human amnion mesenchymal cells (AMCs) contain multipotent cells. To enrich such multipotent stem cells, we applied to AMCs the new method for the isolation of side population (SP) cells used for the enrichment of multipotent stem cells from many tissues. We succeeded in obtaining SP cells from AMCs (AMC-SP cells). AMC-SP cells were found in 0.2% of AMCs, irrespective of the length of pregnant period, ranging from 37 to 40 weeks. Cell cycle analyses uggested that AMC-SP cells belonged to a cell population that proliferated very slowly and/or was in a quiescent state in the amniotic membrane. Upon culturing, they proliferated with 40 to 80 cell doublings. However, they did not form colonies in a soft agarose culture, whereas HepG2 cells, representative human hepatoma cells formed many large colonies. These results suggest that AMC-SP cells that have considerable value for the use of regenerative medicine can be managed safely in vitro.

  6. 3 CFR - Guidelines for Human Stem Cell Research

    Science.gov (United States)

    2010-01-01

    ... 3 The President 1 2010-01-01 2010-01-01 false Guidelines for Human Stem Cell Research Presidential Documents Other Presidential Documents Memorandum of July 30, 2009 Guidelines for Human Stem Cell Research..., scientifically worthy human stem cell research, including human embryonic stem cell research, to the extent...

  7. Myristoylation profiling in human cells and zebrafish

    Directory of Open Access Journals (Sweden)

    Malgorzata Broncel

    2015-09-01

    Full Text Available Human cells (HEK 293, HeLa, MCF-7 and zebrafish embryos were metabolically tagged with an alkynyl myristic acid probe, lysed with an SDS buffer and tagged proteomes ligated to multifunctional capture reagents via copper-catalyzed alkyne azide cycloaddition (CuAAC. This allowed for affinity enrichment and high-confidence identification, by delivering direct MS/MS evidence for the modification site, of 87 and 61 co-translationally myristoylated proteins in human cells and zebrafish, respectively. The data have been deposited to ProteomeXchange Consortium (Vizcaíno et al., 2014 Nat. Biotechnol., 32, 223–6 (PXD001863 and PXD001876 and are described in detail in Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic protein lipidation during vertebrate development׳ by Broncel et al., Angew. Chem. Int. Ed.

  8. Bone formation induced in mouse thigh by cultured human cells.

    Science.gov (United States)

    Anderson, H C; Coulter, P R

    1967-04-01

    Cultured FL human amnion cells injected intramuscularly into cortisone-conditioned mice proliferate to form discrete nodules which become surrounded by fibroblasts. Within 12 days, fibroblastic zones differentiate into cartilage which calcifies to form bone. Experiments were conducted to test the hypothesis that FL cells behave as an inductor of bone formation. In the electron microscope, FL cells were readily distinguished from surrounding fibroblasts. Transitional forms between the two cell types were not recognized. Stains for acid mucopolysaccharides emphasized the sharp boundary between metachromatic fibroblastic and cartilaginous zones and nonmetachromatic FL cells. (35)S was taken up preferentially by fibroblasts and chondrocytes and then deposited extracellularly in a manner suggesting active secretion of sulfated mucopolysaccharides. FL cells showed negligible (35)S utilization and secretion. FL cells, labeled in vitro with thymidine-(3)H, were injected and followed radioautographically, during bone formation. Nuclear label of injected FL cells did not appear in adjacent fibroblasts in quantities sufficient to indicate origin of the latter from FL cells. The minimal fibroblast nuclear labeling seen may represent reutilization of label from necrotic FL cells. It is suggested that FL cells injected into the mouse thigh induced cartilage and bone formation by host fibroblasts.

  9. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins

    Science.gov (United States)

    Fukusumi, Hayato; Shofuda, Tomoko; Bamba, Yohei; Yamamoto, Atsuyo; Kanematsu, Daisuke; Handa, Yukako; Okita, Keisuke; Nakamura, Masaya; Yamanaka, Shinya; Okano, Hideyuki; Kanemura, Yonehiro

    2016-01-01

    Human neural progenitor cells (hNPCs) have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC) clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB) formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi). Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes. PMID:27212953

  10. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins.

    Science.gov (United States)

    Fukusumi, Hayato; Shofuda, Tomoko; Bamba, Yohei; Yamamoto, Atsuyo; Kanematsu, Daisuke; Handa, Yukako; Okita, Keisuke; Nakamura, Masaya; Yamanaka, Shinya; Okano, Hideyuki; Kanemura, Yonehiro

    2016-01-01

    Human neural progenitor cells (hNPCs) have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC) clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB) formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi). Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes.

  11. Hematopoietic development from human induced pluripotent stem cells.

    Science.gov (United States)

    Lengerke, Claudia; Grauer, Matthias; Niebuhr, Nina I; Riedt, Tamara; Kanz, Lothar; Park, In-Hyun; Daley, George Q

    2009-09-01

    A decade of research on human embryonic stem cells (ESC) has paved the way for the discovery of alternative approaches to generating pluripotent stem cells. Combinatorial overexpression of a limited number of proteins linked to pluripotency in ESC was recently found to reprogram differentiated somatic cells back to a pluripotent state, enabling the derivation of isogenic (patient-specific) pluripotent stem cell lines. Current research is focusing on improving reprogramming protocols (e.g., circumventing the use of retroviral technology and oncoproteins), and on methods for differentiation into transplantable tissues of interest. In mouse ESC, we have previously shown that the embryonic morphogens BMP4 and Wnt3a direct blood formation via activation of Cdx and Hox genes. Ectopic expression of Cdx4 and HoxB4 enables the generation of mouse ESC-derived hematopoietic stem cells (HSC) capable of multilineage reconstitution of lethally irradiated adult mice. Here, we explore hematopoietic development from human induced pluripotent stem (iPS) cells generated in our laboratory. Our data show robust differentiation of iPS cells to mesoderm and to blood lineages, as shown by generation of CD34(+)CD45(+) cells, hematopoietic colony activity, and gene expression data, and suggest conservation of blood patterning pathways between mouse and human hematopoietic development.

  12. Homo-trimeric Structure of the Type IVb Minor Pilin CofB Suggests Mechanism of CFA/III Pilus Assembly in Human Enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Kawahara, Kazuki; Oki, Hiroya; Fukakusa, Shunsuke; Yoshida, Takuya; Imai, Tomoya; Maruno, Takahiro; Kobayashi, Yuji; Motooka, Daisuke; Iida, Tetsuya; Ohkubo, Tadayasu; Nakamura, Shota

    2016-03-27

    In gram-negative bacteria, the assembly of type IV pilus (T4P) and the evolutionally related pseudopilus of type II secretion system involves specialized structural proteins called pilins and pseudopilins, respectively, and is dynamically regulated to promote bacterial pathogenesis. Previous studies have suggested that a structural "tip"-like hetero-complex formed through the interaction of at least three minor (pseudo) pilins plays an important role in this process, while some members of the pathogenic type IVb subfamily are known to have only one such minor pilin subunit whose function is still unknown. Here, we determined the crystal structure of the type IVb minor pilin CofB of colonization factor antigen/III from human enterotoxigenic Escherichia coli at 1.88-Å resolution. The crystal structure, in conjunction with physicochemical analysis in solution, reveals a symmetrical homo-trimeric arrangement distinct from the hetero-complexes of minor (pseudo) pilins observed in other T4P and type II secretion systems. Each CofB monomer adopts a unique three-domain architecture, in which the C-terminal β-sheet-rich lectin domain can effectively initiate trimer association of its pilin-like N-terminal domain through extensive hydrophobic interactions followed by domain swapping at the central hinge-like domain. Deletion of cofB produces a phenotype with no detectable pili formation on the cell surface, while molecular modeling indicates that the characteristic homo-trimeric structure of CofB is well situated at the pilus tip of colonization factor antigen/III formed by the major pilin CofA, suggesting a role for the minor pilin in the efficient initiation of T4P assembly. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Differences in incidence and co-occurrence of vaccine and nonvaccine human papillomavirus types in Finnish population before human papillomavirus mass vaccination suggest competitive advantage for HPV33.

    Science.gov (United States)

    Merikukka, Marko; Kaasila, Marjo; Namujju, Proscovia B; Palmroth, Johanna; Kirnbauer, Reinhard; Paavonen, Jorma; Surcel, Heljä-Marja; Lehtinen, Matti

    2011-03-01

    To understand likelihood of type replacement after vaccination against the high-risk human papillomavirus (HPV) types, we evaluated competition of the seven most common genital HPV types in a population sample of unvaccinated, fertile-aged Finnish women. First trimester sera from two consecutive pregnancies were retrieved from 3,183 Finnish women (mean age, 23.1 years) of whom 42.3% had antibodies to at least one HPV type (6/11/16/18/31/33/45) at the baseline. Antibody positivity to more than one HPV types by the second pregnancy was common among the baseline HPV seropositives. However, compared to baseline HPV-seronegative women, significantly increased incidence rate ratios (IRRs), indicating an increased risk to seroconvert for another HPV type, were consistently noted only for HPV33 among baseline HPV16 or HPV18 antibody (ab)-positive women: HPV(16ab only) (→) (16&33ab) IRR 2.9 [95% confidence interval (CI) 1.6-5.4] and HPV(18ab only) (→) (18&33ab) IRR 2.5 (95% CI 1.1-6.0), irrespectively of the presence of antibodies to other HPV types at baseline: HPV(16ab) (→) (16&33ab) IRR 3.2 (95% CI 2.0-5.2) and HPV(18ab) (→) (18&33ab) IRR 3.6 (95% CI 2.1-5.9). Our findings suggest a possible competitive advantage for HPV33 over other genital HPV types in the unvaccinated population. HPV33 should be monitored for type replacement after HPV mass vaccination. Copyright © 2010 UICC.

  14. Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines.

    Science.gov (United States)

    Zou, Chunlin; Chou, Bin-Kuan; Dowey, Sarah N; Tsang, Kitman; Huang, Xiaosong; Liu, Cyndi F; Smith, Cory; Yen, Jonathan; Mali, Prashant; Zhang, Yu Alex; Cheng, Linzhao; Ye, Zhaohui

    2012-08-10

    Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.

  15. Th1-like human T-cell clones recognizing Leishmania gp63 inhibit Leishmania major in human macrophages

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Bendtzen, K

    1994-01-01

    The major surface protease of Leishmania major, gp63, has been suggested as a vaccine candidate for cutaneous leishmaniasis. In this study gp63 was purified from L. major promastigotes. A panel of human T-cell clones recognizing this protein were generated from individuals who had previously had...... self-healing cutaneous leishmaniasis. The T-cell clones expressed CD4, and the alpha chain of the T-cell antigen receptor. GP63 reactive T-cell clones activated by antigen or by immobilized anti-CD3 antibody released relative large amounts of interferon-gamma and no or little interleukin-4, thereby...... resembling Th1 cells. Autologous mononuclear cells and Epstein-Barr virus-transformed B cell lines were equally efficient in presenting the antigen to the T cells. The gp63 reactive T cells induced resistance to infection in cultured human macrophages by L. major. The data confirm that human CD4+ T cells...

  16. Imaging Proteolysis by Living Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Mansoureh Sameni

    2000-01-01

    Full Text Available Malignant progression is accompanied by degradation of extracellular matrix proteins. Here we describe a novel confocal assay in which we can observe proteolysis by living human breast cancer cells (BT20 and BT549 through the use of quenchedfluorescent protein substrates. Degradation thus was imaged, by confocal optical sectioning, as an accumulation of fluorescent products. With the BT20 cells, fluorescence was localized to pericellular focal areas that coincide with pits in the underlying matrix. In contrast, fluorescence was localized to intracellular vesicles in the BT549 cells, vesicles that also label for lysosomal markers. Neither intracellular nor pericellular fluorescence was observed in the BT549 cells in the presence of cytochalasin B, suggesting that degradation occurred intracellularly and was dependent on endocytic uptake of substrate. In the presence of a cathepsin 13-selective cysteine protease inhibitor, intracellular fluorescence was decreased ~90% and pericellular fluorescence decreased 67% to 96%, depending on the protein substrate. Matrix metallo protease inhibitors reduced pericellular fluorescence ~50%, i.e., comparably to a serine and a broad spectrum cysteine protease inhibitor. Our results suggest that: 1 a proteolytic cascade participates in pericellular digestion of matrix proteins by living human breast cancer cells, and 2 the cysteine protease cathepsin B participates in both pericellular and intracellular digestion of matrix proteins by living human breast cancer cells.

  17. Application of human amniotic mesenchymal cells as an allogeneic transplantation cell source in bone regenerative therapy

    Energy Technology Data Exchange (ETDEWEB)

    Tsuno, Hiroaki [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Yoshida, Toshiko [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Nogami, Makiko [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Department of Orthopedic Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Koike, Chika; Okabe, Motonori [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Noto, Zenko [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Arai, Naoya; Noguchi, Makoto [Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Nikaido, Toshio, E-mail: tnikaido@med.u-toyama.ac.jp [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan)

    2012-12-01

    Autogenous mesenchymal stem cells (MSCs) have therapeutic applications in bone regenerative therapy due to their pluripotency. However, the ability of MSCs to proliferate and differentiate varies between donors. Furthermore, alternative sources of MSCs are required for patients with contraindications to autogenous cell therapy. The aim of this study was to evaluate the potential of mesenchymal cells from the human amniotic membrane (HAM) as a source of cells for allogeneic transplantation in bone regenerative therapy. Cells that retained a proliferative capacity of more than 50 population doubling level were distinguished from other HAM cells as HAM{alpha} cells and induced to osteogenic status-their in vivo osteogenesis was subsequently investigated in rats. It was found that HAM{alpha} cells were spindle shaped and were positive for MSC markers and negative for hematopoietic stem cell markers. Alkaline phosphatase activity and calcium deposition increased with osteogenic status of HAM{alpha} cells. The expression of osteocalcin mRNA was increased in HAM{alpha} cells cultured on calcium phosphate scaffolds. Moreover, xenografted HAM{alpha} cells remained viable and produced extracellular matrix for several weeks. Thus, this study suggests that human amniotic mesenchymal cells possess osteogenic differentiation potential and could be applied to allogeneic transplantation in bone regenerative therapy. - Highlights: Black-Right-Pointing-Pointer Human amniotic mesenchymal cells include cells (HAM{alpha} cells) that have the properties of MSCs. Black-Right-Pointing-Pointer HAM{alpha} cells have excellent osteogenic differentiation potential. Black-Right-Pointing-Pointer Osteogenic differentiation ability of HAM{alpha} was amplified by calcium phosphate scaffolds. Black-Right-Pointing-Pointer HAM{alpha} cells can be applicable to allogeneic cell transplantation in bone regenerative therapy.

  18. [Human pluripotent stem cell and neural differentiation].

    Science.gov (United States)

    Wataya, Takafumi; Muguruma, Keiko; Sasai, Yoshiki

    2008-10-01

    Recovery of lost brain function is an important issue in medical studies because neurons of the central nervous system (CNS) have poor potential for regeneration. Since few CNS diseases can be treated completely by medicines, regenerative therapy by using stem cells should be studied as a new type of therapeutic intervention. The efficacy of cell replacement therapy in Parkinson's disease has been well investigated. Several studies on fetal tissue transplantation have revealed that quantity and purity of transplanted cells are necessary for recovery of symptoms. SFEB (Serum-free floating culture of embryoid body-like aggregates) method is capable of inducing multi-potential CNS progenitors that can be steered to differentiate into region-specific tissues. On the basis of the existing knowledge of embryology, we have succeeded in the generating of various types of neurons such as telencephalic, cerebeller (Purkinje and granule cells), retinal (photoreceptor cells) and hypothalamic neurons. Application of this culture method to human ES (hES) cells is necessary for clinical purpose: however, poor survival of hES cells in SFEB culture might limit the possibility of using these cells for future medical applications. We found that a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, markedly diminished the dissociation-induced apoptosis of hES cells and enabled the cells to form aggregates in SFEB culture. For both mouse and human ES cells, SFEB culture is a favorable method that can generate large amounts of region-specific neurons. However, stem cell-based therapy continues to face several obstacles. It is important that researchers in the basic sciences and clinical medicine should discuss these problems together to overcome both scientific and ethical issues related to stem cells.

  19. Characterization of human pluripotent stem cells.

    Science.gov (United States)

    Gokhale, Paul J; Andrews, Peter W

    2013-12-18

    Human pluripotent stem cells (PSCs), whether embryonic stem cells or induced PSCs, offer enormous opportunities for regenerative medicine and other biomedical applications once we have developed the ability to harness their capacity for extensive differentiation. Central to this is our ability to identify and characterize such PSCs, but this is fraught with potential difficulties that arise from a tension between functional definitions of pluripotency and the more convenient use of 'markers', a problem exacerbated by ethical issues, our lack of knowledge of early human embryonic development, and differences from the mouse paradigm.

  20. CLOSTRIDIUM SPORE ATTACHMENT TO HUMAN CELLS

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    PANESSA-WARREN,B.; TORTORA,G.; WARREN,J.

    1997-08-10

    This paper uses high resolution scanning electron microscopy (SEM) with a LaB6 gun and the newest commercial field emission guns, to obtain high magnification images of intact clostridial spores throughout the activation/germination/outgrowth process. By high resolution SEM, the clostridial exosporial membrane can be seen to produce numerous delicate projections (following activation), that extend from the exosporial surface to a nutritive substrate (agar), or cell surface when anaerobically incubated in the presence of human cells (embryonic fibroblasts and colon carcinoma cells). Magnifications of 20,000 to 200,000Xs at accelerating voltages low enough to minimize or eliminate specimen damage (1--5 kV) have permitted the entire surface of C.sporogenes and C.difficile endospores to be examined during all stages of germination. The relationships between the spore and the agar or human cell surface were also clearly visible.

  1. Human pluripotent stem cells in contemporary medicine

    Directory of Open Access Journals (Sweden)

    S. A. Rodin

    2015-01-01

    Full Text Available Human pluripotent stem cells (hPSCs are capable of indefinite proliferation and can be differentiated into any cell type of the human body. Therefore, they are a promising source of cells for treatment of numerous degenerative diseases and injuries. Pluripotent stem cells are also associated with a number of ethical, safety and technological issues. In this review, we describe various types of hPSCs, safety issues that concern all or some types of hPSCs and methods of clinical-grade hPSC line development. Also, we discuss current and past clinical trials involving hPSCs, their outcomes and future perspectives of hPSC-based therapy. 

  2. Nucleosome Organization in Human Embryonic Stem Cells.

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    Puya G Yazdi

    Full Text Available The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a "ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this "ground state" by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational

  3. Nucleosome Organization in Human Embryonic Stem Cells.

    Science.gov (United States)

    Yazdi, Puya G; Pedersen, Brian A; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E; Wang, Ping H

    2015-01-01

    The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a "ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this "ground state" by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states) are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational analysis of nucleosome

  4. Immunosurveillance function of human mast cell?

    Institute of Scientific and Technical Information of China (English)

    (O)ner (O)zdemir

    2005-01-01

    Mast cell (MC) is so widely recognized as a critical effector in allergic disorders that it can be difficult to think of MC in any other context. Indeed, MCs are multifunctional and recently shown that MCs can also act as antigen presenters as well as effector elements of human immune system. First observations of their possible role as anti-tumor cells in peri- or intra-tumoral tissue were mentioned five decades ago and a high content of MCs is considered as a favorable prognosis,consistent with this study. Believers of this hypothesis assumed them to be inhibitors of tumor development through their pro-apoptotic and -necrolytic granules e.g.,granzymes and TNF-α. However, some still postulate them to be enhancers of tumor development through their effects on angiogenesis due to mostly tryptase.There are also some data suggesting increased MC density causes tumor development and indicates bad prognosis. Furthermore, since MC-associated mediators have shown to influence various aspects of tumor biology, the net effect of MCs on the development/progression of tumors has been difficult to evaluate. For instance, chymase induces apoptosis in targets; yet,tryptase, another MC protease, is a well-known mitogen.MCs with these various enzyme expression patterns may mediate different functions and the predominant MC type in tissues may be determined by the environmental needs. The coexistence of tryptase-expressing MCs(MCT) and chymase and tryptase-expressing MCs (MCTC)in physiological conditions reflects a naturally occurring balance that contributes to tissue homeostasis. We have recently discussed the role and relevance of MC serine proteases in different bone marrow diseases.

  5. Cancer genes hypermethylated in human embryonic stem cells.

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    Vincenzo Calvanese

    Full Text Available Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.

  6. Triazole fungicide tebuconazole disrupts human placental trophoblast cell functions.

    Science.gov (United States)

    Zhou, Jinghua; Zhang, Jianyun; Li, Feixue; Liu, Jing

    2016-05-05

    Triazole fungicides are one of the top ten classes of current-use pesticides. Although exposure to triazole fungicides is associated with reproductive toxicity in mammals, limited information is available regarding the effects of triazole fungicides on human placental trophoblast function. Tebuconazole (TEB) is a common triazole fungicide that has been extensively used for fungi control. In this work, we showed that TEB could reduce cell viability, disturb normal cell cycle distribution and induce apoptosis of human placental trophoblast cell line HTR-8/SVneo (HTR-8). Bcl-2 protein expression decreased and the level of Bax protein increased after TEB treatment in HTR-8 cells. The results demonstrated that this fungicide induced apoptosis of trophoblast cells via mitochondrial pathway. Importantly, we found that the invasive and migratory capacities of HTR-8 cells decreased significantly after TEB administration. TEB altered the expression of key regulatory genes involved in the modulation of trophoblast functions. Taken together, TEB suppressed human trophoblast invasion and migration through affecting the expression of protease, hormones, angiogenic factors, growth factors and cytokines. As the invasive and migratory abilities of trophoblast are essential for successful placentation and fetus development, our findings suggest a potential risk of triazole fungicides to human pregnancy.

  7. Cell pattern in adult human corneal endothelium.

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    Carlos H Wörner

    Full Text Available A review of the current data on the cell density of normal adult human endothelial cells was carried out in order to establish some common parameters appearing in the different considered populations. From the analysis of cell growth patterns, it is inferred that the cell aging rate is similar for each of the different considered populations. Also, the morphology, the cell distribution and the tendency to hexagonallity are studied. The results are consistent with the hypothesis that this phenomenon is analogous with cell behavior in other structures such as dry foams and grains in polycrystalline materials. Therefore, its driving force may be controlled by the surface tension and the mobility of the boundaries.

  8. Merkel cell distribution in the human eyelid

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    C.A. May

    2013-10-01

    Full Text Available Although Merkel cell carcinoma of the eye lid is reported frequently in the literature, only limited information exists about the distribution of Merkel cells in this tissue. Therefore, serial sections of 18 human cadaver eye lids (donors ages ranging between 63 and 97 years were stained for cytokeratin 20 in various planes. The overall appearance of Merkel cells in these samples was low and mainly located in the outer root layer of the cilia hair follicles. Merkel cells were more frequent in the middle, and almost not detectable at the nasal and temporal edges. The localization is in accordance with that of Merkel cell carcinoma, but concerning the scarce appearance within this adulthood group, a specific physiological role of these cells in the eye lid is difficult to establish.

  9. Natural killer cells in human autoimmune disorders

    Science.gov (United States)

    2013-01-01

    Natural killer (NK) cells are innate lymphocytes that play a critical role in early host defense against viruses. Through their cytolytic capacity and generation of cytokines and chemokines, NK cells modulate the activity of other components of the innate and adaptive immune systems and have been implicated in the initiation or maintenance of autoimmune responses. This review focuses on recent research elucidating a potential immunoregulatory role for NK cells in T-cell and B-cell-mediated autoimmune disorders in humans, with a particular focus on multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematous. A better understanding of the contributions of NK cells to the development of autoimmunity may lead to novel therapeutic targets in these diseases. PMID:23856014

  10. Human Colon Cancer Cells Cultivated in Space

    Science.gov (United States)

    1995-01-01

    Within five days, bioreactor cultivated human colon cancer cells (shown) grown in Microgravity on the STS-70 mission in 1995, had grown 30 times the volume of the control specimens on Earth. The samples grown in space had a higher level of cellular organization and specialization. Because they more closely resemble tumors found in the body, microgravity grown cell cultures are ideal for research purposes.

  11. Localizing transcripts to single cells suggests an important role of uncultured deltaproteobacteria in the termite gut hydrogen economy.

    Science.gov (United States)

    Rosenthal, Adam Z; Zhang, Xinning; Lucey, Kaitlyn S; Ottesen, Elizabeth A; Trivedi, Vikas; Choi, Harry M T; Pierce, Niles A; Leadbetter, Jared R

    2013-10-01

    Identifying microbes responsible for particular environmental functions is challenging, given that most environments contain an uncultivated microbial diversity. Here we combined approaches to identify bacteria expressing genes relevant to catabolite flow and to locate these genes within their environment, in this case the gut of a "lower," wood-feeding termite. First, environmental transcriptomics revealed that 2 of the 23 formate dehydrogenase (FDH) genes known in the system accounted for slightly more than one-half of environmental transcripts. FDH is an essential enzyme of H2 metabolism that is ultimately important for the assimilation of lignocellulose-derived energy by the insect. Second, single-cell PCR analysis revealed that two different bacterial types expressed these two transcripts. The most commonly transcribed FDH in situ is encoded by a previously unappreciated deltaproteobacterium, whereas the other FDH is spirochetal. Third, PCR analysis of fractionated gut contents demonstrated that these bacteria reside in different spatial niches; the spirochete is free-swimming, whereas the deltaproteobacterium associates with particulates. Fourth, the deltaproteobacteria expressing FDH were localized to protozoa via hybridization chain reaction-FISH, an approach for multiplexed, spatial mapping of mRNA and rRNA targets. These results underscore the importance of making direct vs. inference-based gene-species associations, and have implications in higher termites, the most successful termite lineage, in which protozoa have been lost from the gut community. Contrary to expectations, in higher termites, FDH genes related to those from the protozoan symbiont dominate, whereas most others were absent, suggesting that a successful gene variant can persist and flourish after a gut perturbation alters a major environmental niche.

  12. Safrole induces apoptosis in human oral cancer HSC-3 cells.

    Science.gov (United States)

    Yu, F-S; Yang, J-S; Yu, C-S; Lu, C-C; Chiang, J-H; Lin, C-W; Chung, J-G

    2011-02-01

    Phytochemicals have been used as potential chemopreventive or chemotherapeutic agents. However, there are data suggesting a mutagenic effect of some phytochemicals. We hypothesized that safrole would have anticancer effects on human oral squamous cell carcinoma HSC-3 cells. Safrole decreased the percentage of viable HSC-3 cells via induction of apoptosis by an increased level of cytosolic Ca(2+) and a reduction in the mitochondrial membrane potential (ΔΨ(m)). Changes in the membrane potential were associated with changes in the Bax, release of cytochrome c from mitochondria, and activation of downstream caspases-9 and -3, resulting in apoptotic cell death. In vivo studies also showed that safrole reduced the size and volume of an HSC-3 solid tumor on a xenograft athymic nu/nu mouse model. Western blotting and flow cytometric analysis studies confirmed that safrole-mediated apoptotic cell death of HSC-3 cells is regulated by cytosolic Ca(2+) and by mitochondria- and Fas-dependent pathways.

  13. Normal human pluripotent stem cell lines exhibit pervasive mosaic aneuploidy.

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    Suzanne E Peterson

    Full Text Available Human pluripotent stem cell (hPSC lines have been considered to be homogeneously euploid. Here we report that normal hPSC--including induced pluripotent--lines are karyotypic mosaics of euploid cells intermixed with many cells showing non-clonal aneuploidies as identified by chromosome counting, spectral karyotyping (SKY and fluorescent in situ hybridization (FISH of interphase/non-mitotic cells. This mosaic aneuploidy resembles that observed in progenitor cells of the developing brain and preimplantation embryos, suggesting that it is a normal, rather than pathological, feature of stem cell lines. The karyotypic heterogeneity generated by mosaic aneuploidy may contribute to the reported functional and phenotypic heterogeneity of hPSCs lines, as well as their therapeutic efficacy and safety following transplantation.

  14. Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

    Science.gov (United States)

    Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

    2015-03-01

    The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

  15. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

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    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  16. Smooth muscles and stem cells of embryonic guts express KIT, PDGFRRA, CD34 and many other stem cell antigens: suggestion that GIST arise from smooth muscles and gut stem cells.

    Science.gov (United States)

    Terada, Tadashi

    2013-01-01

    Gastrointestinal stromal tumor (GIST) is believed to original from interstitial cells of (ICC) present in Auerbach's nerve plexus. GIST frequently shows gain-of-function mutations of KIT and PDGFRA. In practical pathology, GIST is diagnosed by positive immunostaining or KIT and/or CD34. The author herein demonstrates that human embryonic gastrointestinal tract smooth muscles (HEGITSM) and human embryonic stem gastrointestinal cells (HEGISC) consistently express KIT, CD34, NCAM, PDGFRA and other stem cell (SC) antigens NSE, synaptophysin, chromogranin, bcl-2, ErbB, and MET throughout the embryonic development of 7-40 gestational week (GW). CK14 was negative. The author examines 42 cases (7-40 GW) of embryonic GI tract (EGI). The HEGISM, HEGIST, and gall bladder smooth muscles (SM) were consistently positive for KIT, CD34, NCAM, PDGFRA, synaptophysin, chromogranin, NSE, bcl-2, ErbB2, and MET in foregut, stomach, GB, midgut, and hindgut throughout the fetal life (7-40 GW). The stem cells (SC) were seen to create the SM, nerves, ICC, and other all structures of GI tract. In adult gastrointestinal walls (n=30), KIT, CD34, PDGFRA, and S100 proteins were expressed in Auerbach's nerve plexus and ICC. The bronchial and vascular SM of embryos did not express these molecules. In GIST, frequent expressions of KIT (100%, 30/30), CD34 (90%, 27/30), and PDGFRA (83%, 25/30) were seen. In general, characteristics of tumors recapitulate their embryonic life. Therefore, it is strongly suggested that GIST may be originated from GI SM and/or GI SC in addition to ICC.

  17. Genetic Manipulation of Human Embryonic Stem Cells.

    Science.gov (United States)

    Eiges, Rachel

    2016-01-01

    One of the great advantages of embryonic stem (ES) cells over other cell types is their accessibility to genetic manipulation. They can easily undergo genetic modifications while remaining pluripotent, and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Since the first isolation of ES cells in mice, many effective techniques have been developed for gene delivery and manipulation of ES cells. These include transfection, electroporation, and infection protocols, as well as different approaches for inserting, deleting, or changing the expression of genes. These methods proved to be extremely useful in mouse ES cells, for monitoring and directing differentiation, discovering unknown genes, and studying their function, and are now being extensively implemented in human ES cells (HESCs). This chapter describes the different approaches and methodologies that have been applied for the genetic manipulation of HESCs and their applications. Detailed protocols for generating clones of genetically modified HESCs by transfection, electroporation, and infection will be described, with special emphasis on the important technical details that are required for this purpose. All protocols are equally effective in human-induced pluripotent stem (iPS) cells.

  18. RNA Directed Modulation of Phenotypic Plasticity in Human Cells.

    Science.gov (United States)

    Trakman, Laura; Hewson, Chris; Burdach, Jon; Morris, Kevin V

    2016-01-01

    Natural selective processes have been known to drive phenotypic plasticity, which is the emergence of different phenotypes from one genome following environmental stimulation. Long non-coding RNAs (lncRNAs) have been observed to modulate transcriptional and epigenetic states of genes in human cells. We surmised that lncRNAs are governors of phenotypic plasticity and drive natural selective processes through epigenetic modulation of gene expression. Using heat shocked human cells as a model we find several differentially expressed transcripts with the top candidates being lncRNAs derived from retro-elements. One particular retro-element derived transcripts, Retro-EIF2S2, was found to be abundantly over-expressed in heat shocked cells. Over-expression of Retro-EIF2S2 significantly enhanced cell viability and modulated a predisposition for an adherent cellular phenotype upon heat shock. Mechanistically, we find that this retro-element derived transcript interacts directly with a network of proteins including 40S ribosomal protein S30 (FAU), Eukaryotic translation initiation factor 5A (EIF5A), and Ubiquitin-60S ribosomal protein L40 (UBA52) to affect protein modulated cell adhesion pathways. We find one motif in Retro-EIF2S2 that exhibits binding to FAU and modulates phenotypic cell transitions from adherent to suspension states. The observations presented here suggest that retroviral derived transcripts actively modulate phenotypic plasticity in human cells in response to environmental selective pressures and suggest that natural selection may play out through the action of retro-elements in human cells.

  19. Mercury induces inflammatory mediator release from human mast cells

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    Peterson Erika

    2010-03-01

    Full Text Available Abstract Background Mercury is known to be neurotoxic, but its effects on the immune system are less well known. Mast cells are involved in allergic reactions, but also in innate and acquired immunity, as well as in inflammation. Many patients with Autism Spectrum Disorders (ASD have "allergic" symptoms; moreover, the prevalence of ASD in patients with mastocytosis, characterized by numerous hyperactive mast cells in most tissues, is 10-fold higher than the general population suggesting mast cell involvement. We, therefore, investigated the effect of mercuric chloride (HgCl2 on human mast cell activation. Methods Human leukemic cultured LAD2 mast cells and normal human umbilical cord blood-derived cultured mast cells (hCBMCs were stimulated by HgCl2 (0.1-10 μM for either 10 min for beta-hexosaminidase release or 24 hr for measuring vascular endothelial growth factor (VEGF and IL-6 release by ELISA. Results HgCl2 induced a 2-fold increase in β-hexosaminidase release, and also significant VEGF release at 0.1 and 1 μM (311 ± 32 pg/106 cells and 443 ± 143 pg/106 cells, respectively from LAD2 mast cells compared to control cells (227 ± 17 pg/106 cells, n = 5, p 2 (0.1 μM to the proinflammatory neuropeptide substance P (SP, 0.1 μM had synergestic action in inducing VEGF from LAD2 mast cells. HgCl2 also stimulated significant VEGF release (360 ± 100 pg/106 cells at 1 μM, n = 5, p 6 cells, and IL-6 release (466 ± 57 pg/106 cells at 0.1 μM compared to untreated cells (13 ± 25 pg/106 cells, n = 5, p 2 (0.1 μM to SP (5 μM further increased IL-6 release. Conclusions HgCl2 stimulates VEGF and IL-6 release from human mast cells. This phenomenon could disrupt the blood-brain-barrier and permit brain inflammation. As a result, the findings of the present study provide a biological mechanism for how low levels of mercury may contribute to ASD pathogenesis.

  20. Human ES cells: starting culture from frozen cells.

    Science.gov (United States)

    Trish, Erin; Dimos, John; Eggan, Kevin

    2006-11-09

    Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock. First, a one to two day old ten cm plate of approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator. A frozen vial of cells from long term liquid nitrogen storage or a -80 C freezer is sourced and quickly submerged in a 37 C water bath for quick thawing. Cells in freezing media are then removed from the vial and placed in a large volume of HuES media. The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate. Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate. It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate. The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

  1. Defining the nature of human pluripotent stem cell progeny

    Institute of Scientific and Technical Information of China (English)

    Michaela Patterson; David N Chan; Iris Ha; Dana Case; Yongyan Cui; Ben Van Handel; Hanna KA Mikkola; William E Lowry

    2012-01-01

    While it is clear that human pluripotent stem cells (hPSCs) can differentiate to generate a panoply of various cell types,it is unknown how closely in vitro development mirrors that which occurs in vivo.To determine whether human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) make equivalent progeny,and whether either makes cells that are analogous to tissue-derived cells,we performed comprehensive transcriptome profiling of purified PSC derivatives and their tissue-derived counterparts.Expression profiling demonstrated that hESCs and hiPSCs make nearly identical progeny for the neural,hepatic,and mesenchymal lineages,and an absence of re-expression from exogenous reprogramming factors in hiPSC progeny.However,when compared to a tissuederived counterpart,the progeny of both hESCs and hiPSCs maintained expression of a subset of genes normally associated with early mammalian development,regardless of the type of cell generated.While pluripotent genes (OCT4,SOX2,REX1,and NANOG) appeared to be silenced immediately upon differentiation from hPSCs,genes normally unique to early embryos (LIN28A,LIN28B,DPPA4,and others) were not fully silenced in hPSC derivatives.These data and evidence from expression patterns in early human fetal tissue (3-16 weeks of development) suggest that the differentiated progeny of hPSCs are reflective of very early human development (< 6 weeks).These findings provide support for the idea that hPSCs can serve as useful in vitro models of early human development,but also raise important issues for disease modeling and the clinical application of hPSC derivatives.

  2. Human embryonic stem cell lines model experimental human cytomegalovirus latency.

    Science.gov (United States)

    Penkert, Rhiannon R; Kalejta, Robert F

    2013-05-28

    Herpesviruses are highly successful pathogens that persist for the lifetime of their hosts primarily because of their ability to establish and maintain latent infections from which the virus is capable of productively reactivating. Human cytomegalovirus (HCMV), a betaherpesvirus, establishes latency in CD34(+) hematopoietic progenitor cells during natural infections in the body. Experimental infection of CD34(+) cells ex vivo has demonstrated that expression of the viral gene products that drive productive infection is silenced by an intrinsic immune defense mediated by Daxx and histone deacetylases through heterochromatinization of the viral genome during the establishment of latency. Additional mechanistic details about the establishment, let alone maintenance and reactivation, of HCMV latency remain scarce. This is partly due to the technical challenges of CD34(+) cell culture, most notably, the difficulty in preventing spontaneous differentiation that drives reactivation and renders them permissive for productive infection. Here we demonstrate that HCMV can establish, maintain, and reactivate in vitro from experimental latency in cultures of human embryonic stem cells (ESCs), for which spurious differentiation can be prevented or controlled. Furthermore, we show that known molecular aspects of HCMV latency are faithfully recapitulated in these cells. In total, we present ESCs as a novel, tractable model for studies of HCMV latency.

  3. Semicarbazone EGA Inhibits Uptake of Diphtheria Toxin into Human Cells and Protects Cells from Intoxication

    Directory of Open Access Journals (Sweden)

    Leonie Schnell

    2016-07-01

    Full Text Available Diphtheria toxin is a single-chain protein toxin that invades human cells by receptor-mediated endocytosis. In acidic endosomes, its translocation domain inserts into endosomal membranes and facilitates the transport of the catalytic domain (DTA from endosomal lumen into the host cell cytosol. Here, DTA ADP-ribosylates elongation factor 2 inhibits protein synthesis and leads to cell death. The compound 4-bromobenzaldehyde N-(2,6-dimethylphenylsemicarbazone (EGA has been previously shown to protect cells from various bacterial protein toxins which deliver their enzymatic subunits from acidic endosomes to the cytosol, including Bacillus anthracis lethal toxin and the binary clostridial actin ADP-ribosylating toxins C2, iota and Clostridium difficile binary toxin (CDT. Here, we demonstrate that EGA also protects human cells from diphtheria toxin by inhibiting the pH-dependent translocation of DTA across cell membranes. The results suggest that EGA might serve for treatment and/or prevention of the severe disease diphtheria.

  4. Arsenic trioxide inhibits cell proliferation and human papillomavirus oncogene expression in cervical cancer cells.

    Science.gov (United States)

    Wang, Hongtao; Gao, Peng; Zheng, Jie

    2014-09-05

    Arsenic trioxide (As2O3) has shown therapeutic effects in some leukemias and solid cancers. However, the molecular mechanisms of its anticancer efficacy have not been clearly elucidated, particularly in solid cancers. Our previous data showed that As2O3 induced apoptosis of human papillomavirus (HPV) 16 DNA-immortalized human cervical epithelial cells and cervical cancer cells and inhibited the expression of HPV oncogenes in these cells. In the present study, we systemically examined the effects of As2O3 on five human cervical cancer cell lines and explored the possible molecular mechanisms. MTT assay showed that HPV-negative C33A cells were more sensitive to growth inhibition induced by As2O3 than HPV-positive cervical cancer cells, and HPV 18-positive HeLa and C4-I cells were more sensitive to As2O3 than HPV 16-positive CaSki and SiHa cells. After As2O3 treatment, both mRNA and protein levels of HPV E6 and E7 obviously decreased in all HPV positive cell lines. In contrast, p53 and Rb protein levels increased in all tested cell lines. Transcription factor AP-1 protein expression decreased significantly in HeLa, CaSki and C33A cells with ELISA method. These results suggest that As2O3 is a potential anticancer drug for cervical cancer.

  5. Nuclear distribution of claudin-2 increases cell proliferation in human lung adenocarcinoma cells.

    Science.gov (United States)

    Ikari, Akira; Watanabe, Ryo; Sato, Tomonari; Taga, Saeko; Shimobaba, Shun; Yamaguchi, Masahiko; Yamazaki, Yasuhiro; Endo, Satoshi; Matsunaga, Toshiyuki; Sugatani, Junko

    2014-09-01

    Claudin-2 is expressed in human lung adenocarcinoma tissue and cell lines, although it is absent in normal lung tissue. However, the role of claudin-2 in cell proliferation and the regulatory mechanism of intracellular distribution remain undefined. Proliferation of human adenocarcinoma A549 cells was decreased by claudin-2 knockdown together with a decrease in the percentage of S phase cells. This knockdown decreased the expression levels of ZONAB and cell cycle regulators. Claudin-2 was distributed in the nucleus in human adenocarcinoma tissues and proliferating A549 cells. The nuclear distribution of ZONAB and percentage of S phase cells were higher in cells exogenously expressing claudin-2 with a nuclear localization signal than in cells expressing claudin-2 with a nuclear export signal. Nuclear claudin-2 formed a complex with ZO-1, ZONAB, and cyclin D1. Nuclear distribution of S208A mutant, a dephosphorylated form of claudin-2, was higher than that of wild type. We suggest that nuclear distribution of claudin-2 is up-regulated by dephosphorylation and claudin-2 serves to retain ZONAB and cyclin D1 in the nucleus, resulting in the enhancement of cell proliferation in lung adenocarcinoma cells.

  6. Xanthine oxidase activity regulates human embryonic brain cells growth

    Directory of Open Access Journals (Sweden)

    Kevorkian G. A.

    2011-10-01

    Full Text Available Aim. Involvement of Xanthine Oxidase (XO; EC1.1.3.22 in cellular proliferation and differentiation has been suggested by the numerous investigations. We have proposed that XO might have undoubtedly important role during the development, maturation as well as the death of human embryos brain cells. Methods. Human abortion material was utilized for the cultivation of brain cells (E90. XO activity was measured by the formation of uric acid in tissue. Cell death was detected by the utility of Trypan Blue dye. Results. Allopurinol suppressed the XO activity in the brain tissue (0.12 ± 0.02; 0.20 ± 0.03 resp., p < 0.05. On day 12th the number of cells in the culture treated with the Allopurinol at the early stage of development was higher in comparison with the Control (2350.1 ± 199.0 vs 2123 ± 96 and higher in comparison with the late period of treatment (1479.6 ± 103.8, p < < 0.05. In all groups, the number of the dead cells was less than in Control, indicating the protective nature of Allopurinol as an inhibitor of XO. Conclusions. Allopurinol initiates cells proliferation in case of the early treatment of the human brain derived cell culture whereas at the late stages it has an opposite effect.

  7. Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Kim, Jong Hyun; Kim, Hyung Woo; Cha, Kyoung Je; Han, Jiyou; Jang, Yu Jin; Kim, Dong Sung; Kim, Jong-Hoon

    2016-03-22

    Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.

  8. Mechanism of mast cell adhesion to human tenocytes in vitro.

    Science.gov (United States)

    Behzad, Hayedeh; Tsai, Shu-Huei; Nassab, Paulina; Mousavizadeh, Rouhollah; McCormack, Robert G; Scott, Alex

    2015-01-01

    Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell-fibroblast co-culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5β1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte-conditioned media; the tenocyte-derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5β1 integrin and tenocyte fibronectin. Additionally, tenocyte-derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5β1 integrin mediates binding of mast cells to human tenocyte and to tenocyte-derived ECM proteins, in particular fibronectin.

  9. [Immune system evolution. (From cells to humans)].

    Science.gov (United States)

    Belek, A S

    1992-01-01

    The great variety of cells and molecules observed in the mammalian immune system can be explained by stepwise acquisition of them during phylogeny. Self/nonself discrimination and cell-mediated immunity have been present since the early stages of evolution. Although some inducible antimicrobial molecules have been demonstrated in invertebrates, immunoglobulins appear in vertebrates. T and B cell diversity, development of the lymphoid organs, MHC molecules, complement and cytokines are the characteristics that appear through the evolution of vertebrates. Further knowledge that will be obtained from phylogenetic studies will improve our understanding of the immune system of human.

  10. Centre for human development, stem cells & regeneration.

    Science.gov (United States)

    Oreffo, Richard O C

    2014-01-01

    The Centre for Human Development, Stem Cells and Regeneration (CHDSCR) was founded in 2004 as a cross-disciplinary research and translational program within the Faculty of Medicine at the University of Southampton. The Centre undertakes fundamental research into early development and stem cells together with applied translational research for patient benefit. The Centre has vibrant and thriving multidisciplinary research programs that harness the translational strength of the Faculty together with an innovative Stem Cell PhD program, outstanding clinical infrastructure and enterprise to deliver on this vision.

  11. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy...... are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  12. Distinct human stem cell populations in small and large intestine.

    Science.gov (United States)

    Cramer, Julie M; Thompson, Timothy; Geskin, Albert; LaFramboise, William; Lagasse, Eric

    2015-01-01

    The intestine is composed of an epithelial layer containing rapidly proliferating cells that mature into two regions, the small and the large intestine. Although previous studies have identified stem cells as the cell-of-origin for intestinal epithelial cells, no studies have directly compared stem cells derived from these anatomically distinct regions. Here, we examine intrinsic differences between primary epithelial cells isolated from human fetal small and large intestine, after in vitro expansion, using the Wnt agonist R-spondin 2. We utilized flow cytometry, fluorescence-activated cell sorting, gene expression analysis and a three-dimensional in vitro differentiation assay to characterize their stem cell properties. We identified stem cell markers that separate subpopulations of colony-forming cells in the small and large intestine and revealed important differences in differentiation, proliferation and disease pathways using gene expression analysis. Single cells from small and large intestine cultures formed organoids that reflect the distinct cellular hierarchy found in vivo and respond differently to identical exogenous cues. Our characterization identified numerous differences between small and large intestine epithelial stem cells suggesting possible connections to intestinal disease.

  13. Distinct human stem cell populations in small and large intestine.

    Directory of Open Access Journals (Sweden)

    Julie M Cramer

    Full Text Available The intestine is composed of an epithelial layer containing rapidly proliferating cells that mature into two regions, the small and the large intestine. Although previous studies have identified stem cells as the cell-of-origin for intestinal epithelial cells, no studies have directly compared stem cells derived from these anatomically distinct regions. Here, we examine intrinsic differences between primary epithelial cells isolated from human fetal small and large intestine, after in vitro expansion, using the Wnt agonist R-spondin 2. We utilized flow cytometry, fluorescence-activated cell sorting, gene expression analysis and a three-dimensional in vitro differentiation assay to characterize their stem cell properties. We identified stem cell markers that separate subpopulations of colony-forming cells in the small and large intestine and revealed important differences in differentiation, proliferation and disease pathways using gene expression analysis. Single cells from small and large intestine cultures formed organoids that reflect the distinct cellular hierarchy found in vivo and respond differently to identical exogenous cues. Our characterization identified numerous differences between small and large intestine epithelial stem cells suggesting possible connections to intestinal disease.

  14. Enterococcus faecalis internalization in human umbilical vein endothelial cells (HUVEC).

    Science.gov (United States)

    Millán, Diana; Chiriboga, Carlos; Patarroyo, Manuel A; Fontanilla, Marta R

    2013-04-01

    Initial Enterococcus faecalis-endothelial cell molecular interactions which lead to enterococci associating in the host endothelial tissue, colonizing it and proliferating there can be assessed using in vitro models. Cultured human umbilical vein endothelial cells (HUVEC) have been used to study other Gram-positive bacteria-cell interactions; however, few studies have been aimed at establishing the relationship of E. faecalis with endothelial cells. The aggregation substance (AS) family of adhesins represents an E. faecalis virulence factor which has been implicated in endocarditis severity and bacterial persistence. The Asc10 protein (a member of this family) promotes bacterium-bacterium aggregation and bacterium-host cell binding. Evaluating Asc10 role in bacterial internalization by cultured enterocytes has shown that this adhesin facilitates E. faecalis endocytosis by HT-29 cells. A few eukaryotic cell structural components, such as cytoskeletal proteins, have been involved in E. faecalis entry into cell-lines; it is thus relevant to determine whether Asc10, as well as microtubules and actin microfilaments, play a role in E. faecalis internalization by cultured endothelial cells. The role of Asc10 and cytoskeleton proteins in E. faecalis ability to enter HUVEC was assessed in the present study, as well as cell apoptosis induction by enterococcal internalization by HUVEC; the data indicated increased cell apoptosis and that cytoskeleton components were partially involved in E. faecalis entry to endothelial cells, thereby suggesting that E. faecalis Asc10 protein would not be a critical factor for bacterial entry to cultured HUVEC.

  15. Chronic cadmium exposure in vitro induces cancer cell characteristics in human lung cells

    Energy Technology Data Exchange (ETDEWEB)

    Person, Rachel J.; Tokar, Erik J.; Xu, Yuanyuan; Orihuela, Ruben; Ngalame, Ntube N. Olive; Waalkes, Michael P., E-mail: waalkes@niehs.nih.gov

    2013-12-01

    Cadmium is a known human lung carcinogen. Here, we attempt to develop an in vitro model of cadmium-induced human lung carcinogenesis by chronically exposing the peripheral lung epithelia cell line, HPL-1D, to a low level of cadmium. Cells were chronically exposed to 5 μM cadmium, a noncytotoxic level, and monitored for acquired cancer characteristics. By 20 weeks of continuous cadmium exposure, these chronic cadmium treated lung (CCT-LC) cells showed marked increases in secreted MMP-2 activity (3.5-fold), invasion (3.4-fold), and colony formation in soft agar (2-fold). CCT-LC cells were hyperproliferative, grew well in serum-free media, and overexpressed cyclin D1. The CCT-LC cells also showed decreased expression of the tumor suppressor genes p16 and SLC38A3 at the protein levels. Also consistent with an acquired cancer cell phenotype, CCT-LC cells showed increased expression of the oncoproteins K-RAS and N-RAS as well as the epithelial-to-mesenchymal transition marker protein Vimentin. Metallothionein (MT) expression is increased by cadmium, and is typically overexpressed in human lung cancers. The major MT isoforms, MT-1A and MT-2A were elevated in CCT-LC cells. Oxidant adaptive response genes HO-1 and HIF-1A were also activated in CCT-LC cells. Expression of the metal transport genes ZNT-1, ZNT-5, and ZIP-8 increased in CCT-LC cells culminating in reduced cadmium accumulation, suggesting adaptation to the metal. Overall, these data suggest that exposure of human lung epithelial cells to cadmium causes acquisition of cancer cell characteristics. Furthermore, transformation occurs despite the cell's ability to adapt to chronic cadmium exposure. - Highlights: • Chronic cadmium exposure induces cancer cell characteristics in human lung cells. • This provides an in vitro model of cadmium-induced human lung cell transformation. • This occurred with general and lung specific changes typical for cancer cells. • These findings add insight to the

  16. Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution

    Directory of Open Access Journals (Sweden)

    Wen-Yang Hu

    2017-08-01

    Full Text Available Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland.

  17. Advances in human B cell phenotypic profiling

    Directory of Open Access Journals (Sweden)

    Denise A Kaminski

    2012-10-01

    Full Text Available To advance our understanding and treatment of disease, research immunologists have been called-upon to place more centralized emphasis on impactful human studies. Such endeavors will inevitably require large-scale study execution and data management regulation (Big Biology, necessitating standardized and reliable metrics of immune status and function. A well-known example setting this large-scale effort in-motion is identifying correlations between eventual disease outcome and T lymphocyte phenotype in large HIV-patient cohorts using multiparameter flow cytometry. However, infection, immunodeficiency, and autoimmunity are also characterized by correlative and functional contributions of B lymphocytes, which to-date have received much less attention in the human Big Biology enterprise. Here, we review progress in human B cell phenotyping, analysis, and bioinformatics tools that constitute valuable resources for the B cell research community to effectively join in this effort.

  18. Human plasma cells express granzyme B.

    Science.gov (United States)

    Xu, Wei; Narayanan, Priya; Kang, Ning; Clayton, Sandra; Ohne, Yoichiro; Shi, Peiqing; Herve, Marie-Cecile; Balderas, Robert; Picard, Capucine; Casanova, Jean-Laurent; Gorvel, Jean-Pierre; Oh, Sangkon; Pascual, Virginia; Banchereau, Jacques

    2014-01-01

    While studying the plasma cell (PC) compartment in human tonsils, we identified that immunoglobulin kappa or lambda chain-expressing PCs are the main cells expressing granzyme B (GrzB). In vitro studies revealed that activated B cells differentiated into GrzB-expressing PCs when co-cultured with macrophages and follicular helper T cells. This effect could be reproduced on combined stimulation of IL-15 (produced by macrophages) and IL-21 (produced by T follicular helper cells) in a STAT3-dependent manner. Whereas IL-21 triggers the transcription of mRNA of GrzB, IL-15 synergizes the translation of GrzB proteins. The precise role of GrzB in PC biology remains to be understood and studies in mice will not help as their PCs do not express GrzB.

  19. Human CD56bright NK Cells

    DEFF Research Database (Denmark)

    Michel, Tatiana; Poli, Aurélie; Cuapio, Angelica

    2016-01-01

    Human NK cells can be subdivided into various subsets based on the relative expression of CD16 and CD56. In particular, CD56(bright)CD16(-/dim) NK cells are the focus of interest. They are considered efficient cytokine producers endowed with immunoregulatory properties, but they can also become...... cytotoxic upon appropriate activation. These cells were shown to play a role in different disease states, such as cancer, autoimmunity, neuroinflammation, and infection. Although their phenotype and functional properties are well known and have been extensively studied, their lineage relationship with other...... NK cell subsets is not fully defined, nor is their precise hematopoietic origin. In this article, we summarize recent studies about CD56(bright) NK cells in health and disease and briefly discuss the current controversies surrounding them....

  20. Human embryonic stem cells: preclinical perspectives

    Directory of Open Access Journals (Sweden)

    Sarda Kanchan

    2008-01-01

    Full Text Available Abstract Human embryonic stem cells (hESCs have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic.

  1. Generation of human/rat xenograft animal model for the study of human donor stem cell behaviors in vivo

    Institute of Scientific and Technical Information of China (English)

    Yan Sun; Dong Xiao; Xing-Hua Pan; Ruo-Shuang Zhang; Guang-Hui Cui; Xi-Gu Chen

    2007-01-01

    AIM: To accurately and realistically elucidate human stem cell behaviors In vivo and the fundamental mechanisms controlling human stem cell fates in vivo, which is urgently required in regenerative medicine and treatments for some human diseases, a surrogate human-rat chimera model was developed.METHODS: Human-rat chimeras were achieved by in utero transplanting low-density mononuclear cells from human umbilical cord blood into the fetal rats at 9-11 d of gestation, and subsequently, a variety of methods, including flow cytometry, PCR as well as immunohistochemical assay, were used to test the human donor contribution in the recipients.RESULTS: Of 29 live-born recipients, 19 had the presence of human CD45+ cells in peripheral blood (PB) detected by flow cytometry, while PCR analysis on genomic DNA from 11 different adult tissues showed that 14 selected from flow cytometry-positive 19 animals possessed of donor-derived human cell engraftment in multiple tissues (i.e. liver, spleen, thymus, heart, kidney, blood, lung, muscle, gut and skin) examined at the time of tissue collection, as confirmed by detecting human β2-microglobulin expression using immunohistochemistry.In this xenogeneic system, the engrafted donor-derived human cells persisted in multiple tissues for at least 6 mo after birth. Moreover, transplanted human donor cells underwent site-specific differentiation into CK18-positive human cells in chimeric liver and CD45-positive human cells in chimeric spleen and thymus of recipients.CONCLUSION: Taken together, these findings suggest that we successfully developed human-rat chimeras, in which xenogeneic human cells exist up to 6 mo later. This humanized small animal model, which offers an in vivo environment more closely resembling to the situations in human, provides an invaluable and effective approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future

  2. Human Liver Cells Expressing Albumin and Mesenchymal Characteristics Give Rise to Insulin-Producing Cells

    Directory of Open Access Journals (Sweden)

    Irit Meivar-Levy

    2011-01-01

    Full Text Available Activation of the pancreatic lineage in the liver has been suggested as a potential autologous cell replacement therapy for diabetic patients. Transcription factors-induced liver-to-pancreas reprogramming has been demonstrated in numerous species both in vivo and in vitro. However, human-derived liver cells capable of acquiring the alternate pancreatic repertoire have never been characterized. It is yet unknown whether hepatic-like stem cells or rather adult liver cells give rise to insulin-producing cells. Using an in vitro experimental system, we demonstrate that proliferating adherent human liver cells acquire mesenchymal-like characteristics and a considerable level of cellular plasticity. However, using a lineage-tracing approach, we demonstrate that insulin-producing cells are primarily generated in cells enriched for adult hepatic markers that coexpress both albumin and mesenchymal markers. Taken together, our data suggest that adult human hepatic tissue retains a substantial level of developmental plasticity, which could be exploited in regenerative medicine approaches.

  3. Mechanisms of ploidy increase in human cancers: a new role for cell cannibalism

    Science.gov (United States)

    Krajcovic, Matej; Overholtzer, Michael

    2011-01-01

    Aneuploidy is a hallmark of human cancers that originates from abnormal mitoses. Many aneuploid cancer cells also have greater-than-diploid DNA content, suggesting that polyploidy is a common precursor to aneuploidy during tumor progression. Polyploid cells can originate from cell fusion, endoreplication, and cytokinesis failure. Recently we found that cell cannibalism by entosis, a form of cell engulfment involving live cells, also leads to polyploidy, as internalized cells disrupt cytokinesis of their engulfing cell hosts. By this mechanism, cannibalistic cell behavior could promote tumor progression by leading to aneuploidy. Here we discuss cell cannibalism in cancer and other mechanisms that result in the formation of polyploid cancer cells. PMID:22447569

  4. Transcriptional Dynamics of Immortalized Human Mesenchymal Stem Cells during Transformation.

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    Masao Takeuchi

    Full Text Available Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type 16 E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN; these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent growth and tumorigenicity in nude mice, (sarcoma and exhibited increased expression of genes encoding growth factor and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5 (GPC5, which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1, the cell surface receptor for hedgehog (Hh signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in growth in U3-DT cells (transformants derived from UE6E7T-3 cells through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional

  5. Human lung natural killer cells are predominantly comprised of highly differentiated hypofunctional CD69(-)CD56(dim) cells.

    Science.gov (United States)

    Marquardt, Nicole; Kekäläinen, Eliisa; Chen, Puran; Kvedaraite, Egle; Wilson, Jennifer N; Ivarsson, Martin A; Mjösberg, Jenny; Berglin, Lena; Säfholm, Jesper; Manson, Martijn L; Adner, Mikael; Al-Ameri, Mamdoh; Bergman, Per; Orre, Ann-Charlotte; Svensson, Mattias; Dahlén, Barbro; Dahlén, Sven-Erik; Ljunggren, Hans-Gustaf; Michaëlsson, Jakob

    2017-04-01

    In contrast to the extensive knowledge about human natural killer (NK) cells in peripheral blood, relatively little is known about NK cells in the human lung. Knowledge about the composition, differentiation, and function of human lung NK cells is critical to better understand their role in diseases affecting the lung, including asthma, chronic obstructive pulmonary disease, infections, and cancer. We sought to analyze and compare the phenotypic and functional characteristics of NK cells in the human lung and peripheral blood at the single-cell level. NK cells in human lung tissue and matched peripheral blood from 132 subjects were analyzed by using 16-color flow cytometry and confocal microscopy. CD56(dim)CD16(+) NK cells made up the vast majority of NK cells in human lungs, had a more differentiated phenotype, and more frequently expressed educating killer cell immunoglobulin-like receptors compared with NK cells in peripheral blood. Despite this, human lung NK cells were hyporesponsive toward target cell stimulation, even after priming with IFN-α. Furthermore, we detected a small subset of NK cells expressing CD69, a marker of tissue residency. These CD69(+) NK cells in the lung consisted predominantly of immature CD56(bright)CD16(-) NK cells and less differentiated CD56(dim)CD16(+) NK cells. Here, we characterize the major NK cell populations in the human lung. Our data suggest a model in which the majority of NK cells in the human lung dynamically move between blood and the lung rather than residing in the lung as bona fide tissue-resident CD69(+) NK cells. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. Stem cell factor and c-Kit in human primordial germ cells and fetal ovaries

    DEFF Research Database (Denmark)

    Høyer, Poul Erik; Byskov, Anne Grete; Møllgård, Kjeld

    2005-01-01

    Prenatal ovary (human), Primordial germ cells, Folliculogenesis, c-Kit, Stem cell factor, immunohistochemistry......Prenatal ovary (human), Primordial germ cells, Folliculogenesis, c-Kit, Stem cell factor, immunohistochemistry...

  7. Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPα.

    Science.gov (United States)

    Bueno, C; Sardina, J L; Di Stefano, B; Romero-Moya, D; Muñoz-López, A; Ariza, L; Chillón, M C; Balanzategui, A; Castaño, J; Herreros, A; Fraga, M F; Fernández, A; Granada, I; Quintana-Bustamante, O; Segovia, J C; Nishimura, K; Ohtaka, M; Nakanishi, M; Graf, T; Menendez, P

    2016-03-01

    B cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphoblastoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus. Co-expression of C/EBPα with OSKM facilitates iPSC generation from both CB- and PB-derived B cells. We also demonstrate that myeloid cells are much easier to reprogram than B and T lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B cells because biased reprogramming was avoided by using freshly isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B cells offers an unprecedented opportunity for studying developmental B lymphopoiesis and modeling B-cell malignancies.

  8. Electrostimulation of rat callus cells and human lymphocytes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Aro, H.; Eerola, E.; Aho, A.J.; Penttinen, R.

    1984-01-01

    Asymmetrical pulsing low voltage current was supplied via electrodes to cultured rat fracture callus cells and human peripheral blood lymphocytes. The (/sup 3/H)thymidine incorporation of the callus cells and 5-(/sup 125/I)iodo-2'-deoxyuridine incorporation of the lymphocytes were determined. The growth pattern of callus cells (estimated by cellular density) did not respond to electrical stimulation. However, the uptake of (/sup 3/H)thymidine was increased at the early phase of cell proliferation and inhibited at later phases of proliferation. The (/sup 3/H)thymidine uptake of confluent callus cell cultures did not respond to electrical stimulation. Lymphocytes reacted in a similar way; stimulated cells took up more DNA precursor than control cells at the early phase of stimulation. During cell division, induced by the mitogens phytohemagglutinin and Concanavalin-A, the uptake of DNA precursor by stimulated cells was constantly inhibited. The results suggest that electrical stimuli affect the uptake mechanisms of cell membranes. The duality of the effect seems to be dependent on the cell cycle.

  9. Generation of mature hematopoietic cells from human pluripotent stem cells.

    Science.gov (United States)

    Togarrati, Padma Priya; Suknuntha, Kran

    2012-06-01

    A number of malignant and non-malignant hematological disorders are associated with the abnormal production of mature blood cells or primitive hematopoietic precursors. Their capacity for continuous self-renewal without loss of pluripotency and the ability to differentiate into adult cell types from all three primitive germ layers make human embryonic stem cells and induced pluripotent stem cells (hiPSCs) attractive complementary cell sources for large-scale production of transfusable mature blood cell components in cell replacement therapies. The generation of patient-specific hematopoietic stem/precursor cells from iPSCs by the regulated manipulation of various factors involved in reprograming to ensure complete pluripotency, and developing innovative differentiation strategies for generating unlimited supply of clinically safe, transplantable, HLA-matched cells from hiPSCs to outnumber the inadequate source of hematopoietic stem cells obtained from cord blood, bone marrow and peripheral blood, would have a major impact on the field of regenerative and personalized medicine leading to translation of these results from bench to bedside.

  10. Exploiting human memory B cell heterogeneity for improved vaccine efficacy

    Directory of Open Access Journals (Sweden)

    Noel Thomas Pauli

    2011-12-01

    Full Text Available The major goal in vaccination is establishment of long-term, prophylactic humoral memory to a pathogen. Two major components to long-lived humoral memory are plasma cells for the production of specific immunoglobulin and memory B cells that survey for their specific antigen in the periphery for later affinity maturation, proliferation, and differentiation. The study of human B cell memory has been aided by the discovery of a general marker for B cell memory, expression of CD27; however, new data suggests the existence of CD27- memory B cells as well. These recently described non-canonical memory populations have increasingly pointed to the heterogeneity of the memory compartment. The novel B memory subsets in humans appear to have unique origins, localization, and functions compared to what was considered to be a classical memory B cell. In this article, we review the known B cell memory subsets, the establishment of B cell memory in vaccination and infection, and how understanding these newly described subsets can inform vaccine design and disease treatment.

  11. Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines.

    Science.gov (United States)

    Bock, Christoph; Kiskinis, Evangelos; Verstappen, Griet; Gu, Hongcang; Boulting, Gabriella; Smith, Zachary D; Ziller, Michael; Croft, Gist F; Amoroso, Mackenzie W; Oakley, Derek H; Gnirke, Andreas; Eggan, Kevin; Meissner, Alexander

    2011-02-04

    The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines.

  12. Triazole fungicide tebuconazole disrupts human placental trophoblast cell functions

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jinghua [Key Laboratory of Environmental Remediation and Ecological Health, Ministry of Education, Zhejiang University, Hangzhou 310058 (China); Zhang, Jianyun [Research Center for Air Pollution and Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058 (China); Li, Feixue [Zhejiang Key Laboratory of Organ Development and Regeneration, Institute of Developmental and Regenerative Biology, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036 (China); Liu, Jing, E-mail: jliue@zju.edu.cn [Key Laboratory of Environmental Remediation and Ecological Health, Ministry of Education, Zhejiang University, Hangzhou 310058 (China); Research Center for Air Pollution and Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058 (China)

    2016-05-05

    Highlights: • Tebuconazole (TEB) inhibited the proliferation of human placental trophoblasts. • TEB changed cell cycle distribution of G1 and G2 phases of trophoblasts. • TEB induced apoptosis of trophoblasts via mitochondrial pathway. • TEB decreased the invasive and migratory capacities of trophoblasts. • TEB altered the mRNA levels of key regulatory genes in trophoblasts - Abstract: Triazole fungicides are one of the top ten classes of current-use pesticides. Although exposure to triazole fungicides is associated with reproductive toxicity in mammals, limited information is available regarding the effects of triazole fungicides on human placental trophoblast function. Tebuconazole (TEB) is a common triazole fungicide that has been extensively used for fungi control. In this work, we showed that TEB could reduce cell viability, disturb normal cell cycle distribution and induce apoptosis of human placental trophoblast cell line HTR-8/SVneo (HTR-8). Bcl-2 protein expression decreased and the level of Bax protein increased after TEB treatment in HTR-8 cells. The results demonstrated that this fungicide induced apoptosis of trophoblast cells via mitochondrial pathway. Importantly, we found that the invasive and migratory capacities of HTR-8 cells decreased significantly after TEB administration. TEB altered the expression of key regulatory genes involved in the modulation of trophoblast functions. Taken together, TEB suppressed human trophoblast invasion and migration through affecting the expression of protease, hormones, angiogenic factors, growth factors and cytokines. As the invasive and migratory abilities of trophoblast are essential for successful placentation and fetus development, our findings suggest a potential risk of triazole fungicides to human pregnancy.

  13. Human colostral cells. I. Separation and characterization.

    Science.gov (United States)

    Crago, S S; Prince, S J; Pretlow, T G; McGhee, J R; Mestecky, J

    1979-12-01

    Analyses of the cells present in human colostrum obtained from fifty-four healthy donors during the first four days of lactation revealed that there were 3.3 x 10(6) (range 1.1 x 10(5)--1.2 x 10(7)) cells per ml of colostrum. Based on histochemical examinations, it was found that this population consisted of 30--47% macrophages, 40--60% polymorphonuclear leucocytes, 5.2--8.9% lymphocytes, and 1.3--2.8% colostral corpuscles; epithelial cells were rarely encountered. The identity of various cell types was confirmed by Wright's stain and by a series of histochemical techniques which disclosed the presence of non-specific esterase, peroxidase, and lipids. For further characterization, the different types of cells were separated by various methods, such as Ficoll-Hypaque density centrifugation, isokinetic centrifugation on a linear Ficoll gradient, adherence to glass or plastic, and phagocytosis of carbonyl iron. Immunohistochemical staining with FITC- and/or TRITC-labelled reagents to IgA, IgM, IgG, K- and lambda-chains, secretory component, lactoferrin, and alpha-lactalbumin were applied to unseparated as well as separated colostral cells. Polymorphonuclear leucocytes (staining for peroxidase) as well as macrophages and colostral corpuscles (staining for non-specific esterase) exhibited numerous intracellular vesicles that contained lipids as well as various combinations of milk proteins. Lymphoid cells did not stain with any of these reagents and plasma cells were not detected among the colostral cells. Individual phagocytic cells contained immunoglobulins of the IgA and IgM classes, both K and lambda light chains, secretory component, lactoferrin, and alpha-lactalbumin. The coincidental appearance of these proteins in single, phagocytic cells but not in lymphoid cells indicate that the cells acquired these proteins by ingestion from the environment. Markers commonly used for the identification of B lymphocytes (surface immunoglobulins) and T lymphocytes (receptors

  14. Cell lineage analysis in human brain using endogenous retroelements.

    Science.gov (United States)

    Evrony, Gilad D; Lee, Eunjung; Mehta, Bhaven K; Benjamini, Yuval; Johnson, Robert M; Cai, Xuyu; Yang, Lixing; Haseley, Psalm; Lehmann, Hillel S; Park, Peter J; Walsh, Christopher A

    2015-01-07

    Somatic mutations occur during brain development and are increasingly implicated as a cause of neurogenetic disease. However, the patterns in which somatic mutations distribute in the human brain are unknown. We used high-coverage whole-genome sequencing of single neurons from a normal individual to identify spontaneous somatic mutations as clonal marks to track cell lineages in human brain. Somatic mutation analyses in >30 locations throughout the nervous system identified multiple lineages and sublineages of cells marked by different LINE-1 (L1) retrotransposition events and subsequent mutation of poly-A microsatellites within L1. One clone contained thousands of cells limited to the left middle frontal gyrus, whereas a second distinct clone contained millions of cells distributed over the entire left hemisphere. These patterns mirror known somatic mutation disorders of brain development and suggest that focally distributed mutations are also prevalent in normal brains. Single-cell analysis of somatic mutation enables tracing of cell lineage clones in human brain. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells

    Directory of Open Access Journals (Sweden)

    Yukari Komuta

    2016-06-01

    Full Text Available Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration.

  16. 21 CFR 864.2280 - Cultured animal and human cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro...

  17. Light- and electron microscopical studies of interstitial cells of Cajal and muscle cells at the submucosal border of human colon

    DEFF Research Database (Denmark)

    Rumessen, J J; Peters, S; Thuneberg, L

    1993-01-01

    It has been suggested that interstitial cells of Cajal (ICC) at the submucosal border of the colonic circular muscle are pacemaker cells. We studied smooth muscle cells and ICC at the submucosal surface of the circular muscle layer of the normal human colon....

  18. The effect of stem cell factor on proliferation of human endometrial CD146+ cells

    Directory of Open Access Journals (Sweden)

    Mehri Fayazi

    2016-07-01

    Full Text Available Background: Stem cell factor (SCF is a transcriptional factor which plays crucial roles in normal proliferation, differentiation and survival in a range of stem cells. Objective: The aim of the present study was to examine the proliferation effect of different concentrations of SCF on expansion of human endometrial CD146+ cells. Materials and Methods: In this experimental study, total populations of isolated human endometrial suspensions after fourth passage were isolated by magnetic activated cell sorting (MACS into CD146+ cells. Human endometrial CD146+ cells were karyotyped and tested for the effect of SCF on proliferation of CD146+ cells, then different concentrations of 0, 12.5, 25, 50 and 100 ng/ml was carried out and mitogens-stimulated endometrial CD146+ cells proliferation was assessed by MTT assay. Results: Chromosomal analysis showed a normal metaphase spread and 46XX karyotype. The proliferation rate of endometrial CD146P + P cells in the presence of 0, 12.5, 25, 50 and 100 ng/ml SCF were 0.945±0.094, 0.962±0.151, 0.988±0.028, 1.679±0.012 and 1.129±0.145 respectively. There was a significant increase in stem/ stromal cell proliferation following in vitro treatment by 50 ng/ml than other concentrations of SCF (p=0.01. Conclusion: The present study suggests that SCF could have effect on the proliferation and cell survival of human endometrial CD146P+P cells and it has important implications for medical sciences and cell therapies

  19. Human T Cell Memory: A Dynamic View

    Directory of Open Access Journals (Sweden)

    Derek C. Macallan

    2017-02-01

    Full Text Available Long-term T cell-mediated protection depends upon the formation of a pool of memory cells to protect against future pathogen challenge. In this review we argue that looking at T cell memory from a dynamic viewpoint can help in understanding how memory populations are maintained following pathogen exposure or vaccination. For example, a dynamic view resolves the apparent paradox between the relatively short lifespans of individual memory cells and very long-lived immunological memory by focussing on the persistence of clonal populations, rather than individual cells. Clonal survival is achieved by balancing proliferation, death and differentiation rates within and between identifiable phenotypic pools; such pools correspond broadly to sequential stages in the linear differentiation pathway. Each pool has its own characteristic kinetics, but only when considered as a population; single cells exhibit considerable heterogeneity. In humans, we tend to concentrate on circulating cells, but memory T cells in non-lymphoid tissues and bone marrow are increasingly recognised as critical for immune defence; their kinetics, however, remain largely unexplored. Considering vaccination from this viewpoint shifts the focus from the size of the primary response to the survival of the clone and enables identification of critical system pinch-points and opportunities to improve vaccine efficacy.

  20. Biological impact of human embryonic stem cells.

    Science.gov (United States)

    Martín, Miguel; Menéndez, Pablo

    2012-01-01

    Research on human embryonic stem cells (hESCs) and induced pluripotent (iPS) stem cells is currently a field of great potential in biomedicine. These cells represent a highly valuable tool for developmental biology studies, disease models, and drug screening and toxicity. The ultimate goal of hESCs and iPS cell research is the treatment of diseases or disorders for which there is currently no treatment or existing therapies are only partially effective. Despite the disproportionate short-term hopes generated, which are putting too much pressure on scientists, the international scientific community is making rapid progress in understanding hESCs and iPS cells. Nonetheless, great efforts have to be made to provide an answer to still quite basic questions concerning their biology. Moreover, translation to clinical applications in cell replacement therapy requires prior solution to ethical barriers. The recent development of iPS cells has provided a strong alternative to overcome ethical issues concerning hESCs. However, an in-depth characterization of their genetic and epigenetic features, as well as their differentiation potential still remains to be undertaken. This chapter will describe, precisely, what the critical issues are, where scientific and ethical barriers stand, and how we are to overcome them. Only then, we shall finally discover whether hESCs and iPS cells will allow building reproducible disease models, and whether they really are a safe tool, with great potential for regenerative medicine.

  1. Comparative studies of placentation and immunology in non-human primates suggest a scenario for the evolution of deep trophoblast invasion and an explanation for human pregnancy disorders

    DEFF Research Database (Denmark)

    Carter, Anthony Michael

    2011-01-01

    Deep trophoblast invasion in the placental bed has been considered the hallmark of human pregnancy. It occurs by two routes, interstitial and endovascular, and results in transformation of the walls of the spiral arteries as they traverse the decidua and the inner third of the myometrium. Disturb...

  2. Propionibacterium acnes inhibits FOXM1 and induces cell cycle alterations in human primary prostate cells

    DEFF Research Database (Denmark)

    Sayanjali, Behnam; Christensen, Gitte J M; Al-Zeer, Munir A;

    2016-01-01

    Propionibacterium acnes has been detected in diseased human prostate tissue, and cell culture experiments suggest that the bacterium can establish a low-grade inflammation. Here, we investigated its impact on human primary prostate epithelial cells. Microarray analysis confirmed the inflammation......-inducing capability of P. acnes but also showed deregulation of genes involved in the cell cycle. qPCR experiments showed that viable P. acnes downregulates a master regulator of cell cycle progression, FOXM1. Flow cytometry experiments revealed that P. acnes increases the number of cells in S-phase. We tested...... the hypothesis that a P. acnes-produced berninamycin-like thiopeptide is responsible for this effect, since it is related to the FOXM1 inhibitor siomycin. The thiopeptide biosynthesis gene cluster was strongly expressed; it is present in subtype IB of P. acnes, but absent from type IA, which is most abundant...

  3. Human fetal liver stromal cells expressing erythropoietin promote hematopoietic development from human embryonic stem cells.

    Science.gov (United States)

    Yang, Chao; Ji, Lei; Yue, Wen; Shi, Shuang-Shuang; Wang, Ruo-Yong; Li, Yan-Hua; Xie, Xiao-Yan; Xi, Jia-Fei; He, Li-Juan; Nan, Xue; Pei, Xue-Tao

    2012-02-01

    Blood cells transfusion and hematopoietic stem cells (HSCs) transplantation are important methods for cell therapy. They are widely used in the treatment of incurable hematological disorder, infectious diseases, genetic diseases, and immunologic deficiency. However, their availability is limited by quantity, capacity of proliferation and the risk of blood transfusion complications. Recently, human embryonic stem cells (hESCs) have been shown to be an alternative resource for the generation of hematopoietic cells. In the current study, we describe a novel method for the efficient production of hematopoietic cells from hESCs. The stable human fetal liver stromal cell lines (hFLSCs) expressing erythropoietin (EPO) were established using the lentiviral system. We observed that the supernatant from the EPO transfected hFLSCs could induce the hESCs differentiation into hematopoietic cells, especially erythroid cells. They not only expressed fetal and embryonic globins but also expressed the adult-globin chain on further maturation. In addition, these hESCs-derived erythroid cells possess oxygen-transporting capacity, which indicated hESCs could generate terminally mature progenies. This should be useful for ultimately developing an animal-free culture system to generate large numbers of erythroid cells from hESCs and provide an experimental model to study early human erythropoiesis.

  4. Effect of passage number on electrophoretic mobility distributions of cultured human embryonic kidney cells

    Science.gov (United States)

    Kunze, M. E.

    1985-01-01

    A systematic investigation was undertaken to characterize population shifts that occur in cultured human embryonic kidney cells as a function of passage number in vitro after original explantation. This approach to cell population shift analysis follows the suggestion of Mehreshi, Klein and Revesz that perturbed cell populations can be characterized by electrophoretic mobility distributions if they contain subpopulations with different electrophoretic mobilities. It was shown that this is the case with early passage cultured human embryo cells.

  5. Passive diffusion of naltrexone into human and animal cells and upregulation of cell proliferation.

    Science.gov (United States)

    Cheng, Fan; McLaughlin, Patricia J; Banks, William A; Zagon, Ian S

    2009-09-01

    Naltrexone (NTX) is a potent opioid antagonist that promotes cell proliferation by upregulating DNA synthesis through displacement of the tonically active inhibitory peptide, opioid growth factor (OGF) from its receptor (OGFr). To investigate how NTX enters cells, NTX was fluorescently labeled [1-(N)-fluoresceinyl NTX thiosemicarbazone; FNTX] to study its uptake by living cultured cells. When human head and neck squamous cell carcinoma cell line (SCC-1) was incubated with FNTX for as little as 1 min, cells displayed nuclear and cytoplasmic staining of FNTX as determined by fluorescent deconvolution microscopy, with enrichment of fluorescent signal in the nucleus and nucleolus. The same temporal-spatial distribution of FNTX was detected in a human pancreatic cancer cell line (MIA PaCa-2), African green monkey kidney cell line (COS-7), and human mesenchymal stem cells (hMSCs). FNTX remained in cells for as long as 48 h. FNTX was internalized in SCC-1 cells when incubation occurred at 4 degrees C, with the signal being comparable to that recorded at 37 degrees C. A 100-fold excess of NTX or a variety of other opioid ligands did not alter the temporal-spatial distribution of FNTX. Neither fluorescein-labeled dextran nor fluorescein alone entered the cells. To study the effect of FNTX on DNA synthesis, cells incubated with FNTX at concentrations ranging from 10(-5) to 10(-8) M had a 5-bromo-2'-deoxyuridine index that was 39-82% greater than for vehicle-treated cells and was comparable to that of unlabeled NTX (37-70%). Taken together, these results suggested that NTX enters cells by passive diffusion in a nonsaturable manner.

  6. Cell phoney: human cloning after Quintavalle.

    Science.gov (United States)

    Morgan, Derek; Ford, Mary

    2004-12-01

    Reproductive cloning has thrown up new scientific possibilities, ethical conundrums, and legal challenges. An initial question, considered by the English courts in 2003, was whether the technique presently available, that of cell nucleus replacement, falls outside the provisions of the Human Fertilisation and Embryology Act 1990. If it does, the creation and use, including use in research protocols, of human embryos would be unregulated, disclosing a need to consider remedial legislation. The resolution by the courts of this legal question dramatically engages them in a resolution of fundamental ethical dilemmas, and discloses the possibilities and limitation of negotiating science policy through the processes of litigation.

  7. Neural Progenitor Cells Derived from Human Embryonic Stem Cells as an Origin of Dopaminergic Neurons

    Directory of Open Access Journals (Sweden)

    Parinya Noisa

    2015-01-01

    Full Text Available Human embryonic stem cells (hESCs are able to proliferate in vitro indefinitely without losing their ability to differentiate into multiple cell types upon exposure to appropriate signals. Particularly, the ability of hESCs to differentiate into neuronal subtypes is fundamental to develop cell-based therapies for several neurodegenerative disorders, such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In this study, we differentiated hESCs to dopaminergic neurons via an intermediate stage, neural progenitor cells (NPCs. hESCs were induced to neural progenitor cells by Dorsomorphin, a small molecule that inhibits BMP signalling. The resulting neural progenitor cells exhibited neural bipolarity with high expression of neural progenitor genes and possessed multipotential differentiation ability. CBF1 and bFGF responsiveness of these hES-NP cells suggested their similarity to embryonic neural progenitor cells. A substantial number of dopaminergic neurons were derived from hES-NP cells upon supplementation of FGF8 and SHH, key dopaminergic neuron inducers. Importantly, multiple markers of midbrain neurons were detected, including NURR1, PITX3, and EN1, suggesting that hESC-derived dopaminergic neurons attained the midbrain identity. Altogether, this work underscored the generation of neural progenitor cells that retain the properties of embryonic neural progenitor cells. These cells will serve as an unlimited source for the derivation of dopaminergic neurons, which might be applicable for treating patients with Parkinson’s disease.

  8. Lymphoid Cell-Glioma Cell Interaction Enhances Cell Coat Production by Human Gliomas: Novel Suppressor Mechanism

    Science.gov (United States)

    Dick, Steven J.; Macchi, Beatrice; Papazoglou, Savvas; Oldfield, Edward H.; Kornblith, Paul L.; Smith, Barry H.; Gately, Maurice K.

    1983-05-01

    Certain human glioma lines produce mucopolysaccharide coats that impair the generation of cytolytic lymphocytes in response to these lines in vitro. Coat production is substantially enhanced by the interaction of glioma cells with a macromolecular factor released by human peripheral blood mononuclear cells in culture. This interaction thus constitutes an unusual mechanism by which inflammatory cells may nonspecifically suppress the cellular immune response to at least one class of solid tumors in humans.

  9. Human somatic cell nuclear transfer and cloning.

    Science.gov (United States)

    2012-10-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Contact-independent cell death of human microglial cells due to pathogenic Naegleria fowleri trophozoites.

    Science.gov (United States)

    Kim, Jong-Hyun; Kim, Daesik; Shin, Ho-Joon

    2008-12-01

    Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increase of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death.

  11. [Neuronal differentiation of human small cell lung cancer cell line PC-6 by Solcoseryl].

    Science.gov (United States)

    Shimizu, T

    1997-11-01

    Solcoseryl is composed of extracts from calf blood, and is a drug known to activate tissue respiration. In the present study, I demonstrated the cell biological effects of Solcoseryl on a human small cell lung cancer cell line, PC-6, by analyzing cell morphology, cell growth, expression of neuronal differentiation markers, and the ras proto-oncogene product(ras p21). Exposure of PC-6 cells to Solcoseryl at the concentration of 200 microliters/ml induced (1) cell morphological changes, including neurodendrite-like projections from the cell surface, and (2) complete inhibition of cell growth, that was shown by the loss of Ki-67 expression. Solcoseryl also induced the expression of neurofilament protein and acetylcholinesterase, both of which are markers of neuronal differentiation. Moreover, it upregulated the expression of the ras proto-oncogene product, ras p21. Taken together, these data suggest that Solcoseryl is composed of component(s) which can induce neuronal differentiation of the human small cell lung cancer cell line, PC-6.

  12. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells.

    Science.gov (United States)

    Katz, Adam J; Tholpady, Ashok; Tholpady, Sunil S; Shang, Hulan; Ogle, Roy C

    2005-03-01

    Adult human subcutaneous adipose tissue contains cells with intriguing multilineage developmental plasticity, much like marrow-derived mesenchymal stem cells. Putative stem or progenitor cells from fat have been given many different names in the literature, reflecting an early and evolving consensus regarding their phenotypic characterization. The study reported here used microarrays to evaluate over 170 genes relating to angiogenesis and extracellular matrix in undifferentiated, early-passage human adipose-derived adherent stromal (hADAS) cells isolated from three separate donors. The hADAS populations unanimously transcribed 66% of the screened genes, and 83% were transcribed by at least two of the three populations. The most highly transcribed genes relate to functional groupings such as cell adhesion, matrix proteins, growth factors and receptors, and proteases. The transcriptome of hADAS cells demonstrated by this work reveals many similarities to published profiles of bone marrow mesenchymal stem cells (MSCs). In addition, flow analysis of over 24 hADAS cell surface proteins (n = 7 donors) both confirms and expands on the existing literature and reveals strong intergroup correlation, despite an inconsistent nomenclature and the lack of standardized protocols for cell isolation and culture. Finally, based on flow analysis and reverse transcription polymerase chain reaction studies, our results suggest that hADAS cells do not express several proteins that are implicated as markers of "stemness" in other stem cell populations, including telomerase, CD133, and the membrane transporter ABCG2.

  13. Establishment of human cell type-specific iPS cells with enhanced chondrogenic potential.

    Science.gov (United States)

    Guzzo, Rosa M; Scanlon, Vanessa; Sanjay, Archana; Xu, Ren-He; Drissi, Hicham

    2014-12-01

    The propensity of induced pluripotent stem (iPS) cells to differentiate into specific lineages may be influenced by a number of factors, including the selection of the somatic cell type used for reprogramming. Herein we report the generation of new iPS cells, which we derived from human articular chondrocytes and from cord blood mononucleocytes via lentiviral-mediated delivery of Oct4, Klf4, Sox2, and cMyc. Molecular, cytochemical, and cytogenic analyses confirmed the acquisition of hallmark features of pluripotency, as well as the retention of normal karyotypes following reprogramming of both the human articular chondrocytes (AC) and the cord blood (CB) cells. In vitro and in vivo functional analyses formally established the pluripotent differentiation capacity of all cell lines. Chondrogenic differentiation assays comparing iPS cells derived from AC, CB, and a well established dermal fibroblast cell line (HDFa-Yk26) identified enhanced proteoglycan-rich matrix formation and cartilage-associated gene expression from AC-derived iPS cells. These findings suggest that the tissue of origin may impact the fate potential of iPS cells for differentiating into specialized cell types, such as chondrocytes. Thus, we generated new cellular tools for the identification of inherent features driving high chondrogenic potential of reprogrammed cells.

  14. Suppression of HIV-1 Infectivity by Human Glioma Cells.

    Science.gov (United States)

    Hoque, Sheikh Ariful; Tanaka, Atsushi; Islam, Salequl; Ahsan, Gias Uddin; Jinno-Oue, Atsushi; Hoshino, Hiroo

    2016-05-01

    HIV-1 infection to the central nervous system (CNS) is very common in AIDS patients. The predominant cell types infected in the brain are monocytes and macrophages, which are surrounded by several HIV-1-resistant cell types, such as astrocytes, oligodendrocytes, neurons, and microvascular cells. The effect of these HIV-1-resistant cells on HIV-1 infection is largely unknown. In this study, we examined the stability of HIV-1 cultured with several human glioblastoma cell lines, for example, NP-2, U87MG, T98G, and A172, to determine whether these HIV-1-resistant brain cells could enhance or suppress HIV-1 infection and thus modulate HIV-1 infection in the CNS. The HIV-1 titer was determined using the MAGIC-5A indicator cell line as well as naturally occurring CD4(+) T cells. We found that the stability of HIV-1 incubated with NP-2 or U87MG cells at 37°C was significantly shorter (half-life, 2.5-4 h) compared to that of HIV-1 incubated with T98G or A172 cells or in culture medium without cells (half-life, 8-18 h). The spent culture media (SCM) of NP-2 and U87MG cells had the ability to suppress both R5- and X4-HIV-1 infection by inhibiting HIV-1 attachment to target cells. This inhibitory effect was eliminated by the treatment of the SCM with chondroitinase ABC but not heparinase, suggesting that the inhibitory factor(s) secreted by NP-2 and U87MG cells was chiefly mediated by chondroitin sulfate (CS) or CS-like moiety. Thus, this study reveals that some but not all glioma cells secrete inhibitory molecules to HIV-1 infection that may contribute in lowering HIV-1 infection in the CNS in vivo.

  15. Combined gene expression and proteomic analysis of EGF induced apoptosis in A431 cells suggests multiple pathways trigger apoptosis.

    Science.gov (United States)

    Alanazi, Ibrahim; Ebrahimie, Esmaeil; Hoffmann, Peter; Adelson, David L

    2013-11-01

    A431 cells, derived from epidermoid carcinoma, overexpress the epidermal growth factor receptor (EGFR) and when treated with a high dose of EGF will undergo apoptosis. We exploited microarray and proteomics techniques and network prediction to study the regulatory mechanisms of EGF-induced apoptosis in A431 cells. We observed significant changes in gene expression in 162 genes, approximately evenly split between pro-apoptotic and anti-apoptotic genes and identified 30 proteins from the proteomic data that had either pro or anti-apoptotic annotation. Our correlation analysis of gene expression and proteome modeled a number of distinct sub-networks that are associated with the onset of apoptosis, allowing us to identify specific pathways and components. These include components of the interferon signalling pathway, and down stream components, including cytokines and suppressors of cytokine signalling. A central component of almost all gene expression sub-networks identified was TP53, which is mutated in A431 cells, and was down regulated. This down regulation of TP53 appeared to be correlated with proteomic sub-networks of cytoskeletal or cell adhesion components that might induce apoptosis by triggering cytochrome C release. Of the only three genes also differentially expressed as proteins, only serpinb1 had a known association with apoptosis. We confirmed that up regulation and cleavage of serpinb1 into L-DNAaseII was correlated with the induction of apoptosis. It is unlikely that a single pathway, but more likely a combination of pathways is needed to trigger EGF induced apoptosis in A431cells.

  16. HCMV Infection Depress NGF Expression in Human Glioma Cells

    Institute of Scientific and Technical Information of China (English)

    Hai-tao WANG; Bin WANG; Zhi-jun LIU; Zhi-qiang BAI; Ling LI; Dong-meng QIAN; Zhi-yong YAN; Xu-xia SONG

    2009-01-01

    Human cytomegalovirus (HCMV) is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, RT-PCR and Western Blotting were performed to quantify the regulation of endogenic nerve growth factor expression in neuroglia cells by HCMV infection. The results showed that basal, endogenous NGF expression in U251 was unchanged during early HCMV infection. NGF expression is strongly down-regulated during the latent phase of infection. These results suggest that HCMV can depress the NGF expression in U251 cells.

  17. Cathepsin G, a Neutrophil Protease, Induces Compact Cell-Cell Adhesion in MCF-7 Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Tomoya Kudo

    2009-01-01

    Full Text Available Cathepsin G is a serine protease secreted by activated neutrophils that play a role in the inflammatory response. Because neutrophils are known to be invading leukocytes in various tumors, their products may influence the characteristics of tumor cells such as the growth state, motility, and the adhesiveness between cells or the extracellular matrix. Here, we demonstrate that cathepsin G induces cell-cell adhesion of MCF-7 human breast cancer cells resulting from the contact inhibition of cell movement on fibronectin but not on type IV collagen. Cathepsin G subsequently induced cell condensation, a very compact cell colony, resulting due to the increased strength of E-cadherin-mediated cell-cell adhesion. Cathepsin G action is protease activity-dependent and was inhibited by the presence of serine protease inhibitors. Cathepsin G promotes E-cadherin/catenin complex formation and Rap1 activation in MCF-7 cells, which reportedly regulates E-cadherin-based cell-cell junctions. Cathepsin G also promotes E-cadherin/protein kinase D1 (PKD1 complex formation, and Go6976, the selective PKD1 inhibitor, suppressed the cathepsin G-induced cell condensation. Our findings provide the first evidence that cathepsin G regulates E-cadherin function, suggesting that cathepsin G has a novel modulatory role against tumor cell-cell adhesion.

  18. Analysis of meiosis regulators in human gonads: a sexually dimorphic spatio-temporal expression pattern suggests involvement of DMRT1 in meiotic entry.

    Science.gov (United States)

    Jørgensen, Anne; Nielsen, John E; Blomberg Jensen, Martin; Græm, Niels; Rajpert-De Meyts, Ewa

    2012-11-01

    The mitosis-meiosis switch is a key event in the differentiation of germ cells. In humans, meiosis is initiated in fetal ovaries, whereas in testes meiotic entry is inhibited until puberty. The purpose of this study was to examine the expression pattern of meiosis regulators in human gonads and to investigate a possible role of DMRT1 in the regulation of meiotic entry. The expression pattern of DMRT1, STRA8, SCP3, DMC1, NANOS3, CYP26B1 and NANOS2 was investigated by RT-PCR and immunohistochemistry in a series of human testis samples from fetal life to adulthood, and in fetal ovaries. DMRT1 was expressed in testes throughout development but with marked spatio-temporal changes. At the early fetal period of 8-20 gestational weeks (GW) and at infantile mini-puberty, DMRT1 was predominantly expressed in Sertoli cells, whereas at later stages of gestation (22-40 GW), during childhood and in post-pubertal testes, DMRT1 was most abundant in spermatogonia, except in the A-dark type. In fetal ovaries, DMRT1 was detected in oogonia and oocytes until 20 GW, but was completely down-regulated following meiotic entry. STRA8, SCP3 and DMC1 were expressed mainly in oocytes and spermatogonia in accordance with their role in initiation and progression of meiosis. The putative meiosis inhibitors, CYP26B1 and NANOS2, were primarily expressed in Leydig cells and spermatocytes, respectively. In conclusion, the expression pattern of the investigated meiotic regulators is largely conserved in the human gonads compared with rodents, but with some minor differences, such as a stable expression of CYP26B1 in human fetal ovaries. The sexually dimorphic expression pattern of DMRT1 indicates a similar role in the mitosis-meiosis switch in human gonads as previously demonstrated in mice. The biological importance of the changes in expression of DMRT1 in Sertoli cells remains to be established, but it is consistent with DMRT1 reinforcing the inhibition of meiosis in the testis.

  19. TALEN-Induced Translocations in Human Cells.

    Science.gov (United States)

    Piganeau, Marion; Renouf, Benjamin; Ghezraoui, Hind; Brunet, Erika

    2016-01-01

    Induction of chromosomal translocations in human cells is of a great interest to study tumorigenesis and genome instability. Here, we explain in detail a method to induce translocations using the transcription activator-like effector nucleases (TALENs). We describe how to detect translocation formation by PCR, calculate translocation frequency by 96-well PCR screen, and analyze breakpoint junctions. When inducing cancer translocations, it is also possible to detect the fusion gene by FISH analysis or western blot.

  20. High concentrations of NaCl induce cell swelling leading to senescence in human cells.

    Science.gov (United States)

    Yamakami, Yoshimi; Yonekura, Ryuzo; Matsumoto, Yuko; Takauji, Yuki; Miki, Kensuke; Fujii, Michihiko; Ayusawa, Dai

    2016-01-01

    Cell swelling and retardation in DNA replication are always observed in senescent cells. When DNA replication is slowed down with RNA and protein syntheses unchanged in proliferating cells, it causes a phenomenon known as unbalanced growth. The purpose of this study is to assess the role of cell swelling in unbalanced growth in terms of senescence and investigate the mechanism underlying this phenomenon. We tried to induce cell swelling with minimum damage to cells in this study. We perturbed the osmoregulatory functions to induce cell swelling under hypotonic and hypertonic conditions in normal human fibroblasts. Addition of excess NaCl was found to induce significant cell and nuclear swelling in dose- and time-dependent manners. Excess NaCl immediately retarded DNA replication, accumulated cells at G1 phase of the cell cycle, and eventually deprived division potential of the cells. Such cells showed typical senescent cell shape followed by expression of the typical senescence-associated genes. Excess NaCl also activated ERK1/2, p38, and JNK of the mitogen activated protein kinase family. Addition of U0126, an inhibitor of ERK1/2, prevented appearance of senescent features induced by excess NaCl. These results suggest that hypertonic conditions induce cell swelling due to unbalanced growth, thereby leading to cellular senescence.

  1. Acrylamide induces accelerated endothelial aging in a human cell model.

    Science.gov (United States)

    Sellier, Cyril; Boulanger, Eric; Maladry, François; Tessier, Frédéric J; Lorenzi, Rodrigo; Nevière, Rémi; Desreumaux, Pierre; Beuscart, Jean-Baptiste; Puisieux, François; Grossin, Nicolas

    2015-09-01

    Acrylamide (AAM) has been recently discovered in food as a Maillard reaction product. AAM and glycidamide (GA), its metabolite, have been described as probably carcinogenic to humans. It is widely established that senescence and carcinogenicity are closely related. In vitro, endothelial aging is characterized by replicative senescence in which primary cells in culture lose their ability to divide. Our objective was to assess the effects of AAM and GA on human endothelial cell senescence. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as model. HUVECs were cultured over 3 months with AAM or GA (1, 10 or 100 μM) until growth arrest. To analyze senescence, β-galactosidase activity and telomere length of HUVECs were measured by cytometry and semi-quantitative PCR, respectively. At all tested concentrations, AAM or GA reduced cell population doubling compared to the control condition (p < 0.001). β-galactosidase activity in endothelial cells was increased when exposed to AAM (≥10 μM) or GA (≥1 μM) (p < 0.05). AAM (≥10 μM) or GA (100 μM) accelerated telomere shortening in HUVECs (p < 0.05). In conclusion, in vitro chronic exposure to AAM or GA at low concentrations induces accelerated senescence. This result suggests that an exposure to AAM might contribute to endothelial aging.

  2. Molecular and biochemical analysis of rainbow trout LCK suggests a conserved mechanism for T-cell signaling in gnathostomes

    Science.gov (United States)

    Laing, K.J.; Dutton, S.; Hansen, J.D.

    2007-01-01

    Two genes were identified in rainbow trout that display high sequence identity to vertebrate Lck. Both of the trout Lck transcripts are associated with lymphoid tissues and were found to be highly expressed in IgM-negative lymphocytes. In vitro analysis of trout lymphocytes indicates that trout Lck mRNA is up-regulated by T-cell mitogens, supporting an evolutionarily conserved function for Lck in the signaling pathways of T-lymphocytes. Here, we describe the generation and characterization of a specific monoclonal antibody raised against the N-terminal domains of recombinant trout Lck that can recognize Lck protein(s) from trout thymocyte lysates that are similar in size (???57 kDa) to mammalian Lck. This antibody also reacted with permeabilized lymphocytes during FACS analysis, indicating its potential usage for cellular analyses of trout lymphocytes, thus representing an important tool for investigations of salmonid T-cell function.

  3. Proteomic shifts in embryonic stem cells with gene dose modifications suggest the presence of balancer proteins in protein regulatory networks.

    Directory of Open Access Journals (Sweden)

    Lei Mao

    Full Text Available Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of "balancer" proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the "elasticity" of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions.

  4. Human cell lines: A promising alternative for recombinant FIX production.

    Science.gov (United States)

    de Sousa Bomfim, Aline; Cristina Corrêa de Freitas, Marcela; Picanço-Castro, Virgínia; de Abreu Soares Neto, Mário; Swiech, Kamilla; Tadeu Covas, Dimas; Maria de Sousa Russo, Elisa

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins.

  5. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  6. Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application, we need to replace mouse embryonic fibroblastswith human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state. We successfully use human fibroblasts derived from aborted fetus and adult prepuce as feeder layer to maintain human embryonic stem cells growth. During the passage and growth on this feeder layer, the human embryonic stem cells can keep their undifferentiated state.

  7. Primitive cardiac cells from human embryonic stem cells.

    Science.gov (United States)

    Hudson, James; Titmarsh, Drew; Hidalgo, Alejandro; Wolvetang, Ernst; Cooper-White, Justin

    2012-06-10

    Pluripotent stem cell-derived cardiomyocytes are currently being investigated for in vitro human heart models and as potential therapeutics for heart failure. In this study, we have developed a differentiation protocol that minimizes the need for specific human embryonic stem cell (hESC) line optimization. We first reduced the heterogeneity that exists within the starting population of bulk cultured hESCs by using cells adapted to single-cell passaging in a 2-dimensional (2D) culture format. Compared with bulk cultures, single-cell cultures comprised larger fractions of TG30(hi)/OCT4(hi) cells, corresponding to an increased expression of pluripotency markers OCT4 and NANOG, and reduced expression of early lineage-specific markers. A 2D temporal differentiation protocol was then developed, aimed at reducing the inherent heterogeneity and variability of embryoid body-based protocols, with induction of primitive streak cells using bone morphogenetic protein 4 and activin A, followed by cardiogenesis via inhibition of Wnt signaling using the small molecules IWP-4 or IWR-1. IWP-4 treatment resulted in a large percentage of cells expressing low amounts of cardiac myosin heavy chain and expression of early cardiac progenitor markers ISL1 and NKX2-5, thus indicating the production of large numbers of immature cardiomyocytes (~65,000/cm(2) or ~1.5 per input hESC). This protocol was shown to be effective in HES3, H9, and, to a lesser, extent, MEL1 hESC lines. In addition, we observed that IWR-1 induced predominantly atrial myosin light chain (MLC2a) expression, whereas IWP-4 induced expression of both atrial (MLC2a) and ventricular (MLC2v) forms. The intrinsic flexibility and scalability of this 2D protocol mean that the output population of primitive cardiomyocytes will be particularly accessible and useful for the investigation of molecular mechanisms driving terminal cardiomyocyte differentiation, and potentially for the future treatment of heart failure.

  8. Cell shape regulates global histone acetylation in human mammaryepithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Le Beyec, Johanne; Xu, Ren; Lee, Sun-Young; Nelson, Celeste M.; Rizki, Aylin; Alcaraz, Jordi; Bissell, Mina J.

    2007-02-28

    Extracellular matrix (ECM) regulates cell morphology and gene expression in vivo; these relationships are maintained in three-dimensional (3D) cultures of mammary epithelial cells. In the presence of laminin-rich ECM (lrECM), mammary epithelial cells round up and undergo global histone deacetylation, a process critical for their functional differentiation. However, it remains unclear whether lrECM-dependent cell rounding and global histone deacetylation are indeed part of a common physical-biochemical pathway. Using 3D cultures as well as nonadhesive and micropatterned substrata, here we showed that the cell 'rounding' caused by lrECM was sufficient to induce deacetylation of histones H3 and H4 in the absence of biochemical cues. Microarray and confocal analysis demonstrated that this deacetylation in 3D culture is associated with a global increase in chromatin condensation and a reduction in gene expression. Whereas cells cultured on plastic substrata formed prominent stress fibers, cells grown in 3D lrECM or on micropatterns lacked these structures. Disruption of the actin cytoskeleton with cytochalasin D phenocopied the lrECM-induced cell rounding and histone deacetylation. These results reveal a novel link between ECM-controlled cell shape and chromatin structure, and suggest that this link is mediated by changes in the actin cytoskeleton.

  9. Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Te, E-mail: liute79@yahoo.com [School of Environmental Science and Engineering, Donghua University, Shanghai 201620 (China); Shanghai Geriatric Institute of Chinese Medicine, Shanghai 200031 (China); Cheng, Weiwei [International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai 200030 (China); Huang, Yongyi [Laboratoire PROTEE, Batiment R, Universite du Sud Toulon-Var, 83957 LA GARDE Cedex (France); Huang, Qin; Jiang, Lizhen [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Guo, Lihe, E-mail: liute79@yahoo.com [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

    2012-02-15

    Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal. Highlights: Black-Right-Pointing-Pointer microRNA-145 inhibits Sox2 expression in human iPS cells. Black-Right-Pointing-Pointer microRNA-145 suppresses the self-renewal and pluripotency of human iPS cells. Black-Right-Pointing-Pointer HuAECs regulate expression of microRNA-145 and Sox2 in human iPS cells. Black-Right-Pointing-Pointer HuAECs feeder

  10. Appearance of Human Plasma Cells Following Differentiation of Human B Cells in NOD/SCID Mouse Spleen

    OpenAIRE

    2003-01-01

    Relatively little is known for the differentiation and maturation process of human B cells to plasma cells. This is particularly important in reconstitution work involving transfer of autoantibodies. To address this issue, we transplanted human peripheral blood mononuclear cells (PBMC) directly into the spleen of irradiated NOD/SCID mice depleted of natural killer cell activity. Within 6 weeks, naïve B cells differentiated into memory B cells and, importantly, the numbers of human CD138+ plas...

  11. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins

    Directory of Open Access Journals (Sweden)

    Hayato Fukusumi

    2016-01-01

    Full Text Available Human neural progenitor cells (hNPCs have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi. Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes.

  12. Human microglial cells synthesize albumin in brain.

    Directory of Open Access Journals (Sweden)

    Sung-Min Ahn

    Full Text Available Albumin, an abundant plasma protein with multifunctional properties, is mainly synthesized in the liver. Albumin has been implicated in Alzheimer's disease (AD since it can bind to and transport amyloid beta (Abeta, the causative agent of AD; albumin is also a potent inhibitor of Abeta polymerization. Despite evidence of non-hepatic transcription of albumin in many tissues including kidney and pancreas, non-hepatic synthesis of albumin at the protein level has been rarely confirmed. In a pilot phase study of Human Brain Proteome Project, we found evidence that microglial cells in brain may synthesize albumin. Here we report, for the first time, the de novo synthesis of albumin in human microglial cells in brain. Furthermore, we demonstrate that the synthesis and secretion of albumin from microglial cells is enhanced upon microglial activation by Abeta(1-42- or lipopolysaccharide (LPS-treatment. These data indicate that microglial cells may play a beneficial role in AD by secreting albumin that not only inhibits Abeta polymerization but also increases its clearance.

  13. In vitro effects of trichothecenes on human dendritic cells.

    Science.gov (United States)

    Hymery, N; Sibiril, Y; Parent-Massin, D

    2006-09-01

    The aim of this work was to study the in vitro effects of trichothecenes on human dendritic cells. Trichothecenes are mycotoxins produced by fungi such as Fusarium, Myrothecium, and Stachybotrys. Two aspects have been explored in this work: the cytotoxicity of trichothecenes on immature dendritic cells to determine IC 50 (inhibition concentration), and the effects of trichothecenes on dendritic cell maturation process. Two mycotoxins (T-2 and DON) known to be immunotoxic have been tested on a model of monocyte-derived dendritic cells culture. Cytotoxic effects of T-2 toxin and DON on immature dendritic cells showed that DON is less potent than T-2 toxin. The exposure to trichothecenes during dendritic cell maturation upon addition of LPS or TNF-alpha markedly inhibited the up-regulation of maturation markers such as CD-86, HLA-DR and CCR7. Features of LPS or TNF-alpha -mediated maturation of dendritic cells, such as IL-10 and IL-12 secretions and endocytosis, were also impaired in response to trichothecenes treatment. These results suggest trichothecenes have adverse effects on dendritic cells and dendritic cell maturation process.

  14. Neocortical glial cell numbers in human brains

    DEFF Research Database (Denmark)

    Pelvig, D.P.; Pakkenberg, H.; Stark, A.K.

    2008-01-01

    and neurons and counting were done in each of the four lobes. The study showed that the different subpopulations of glial cells behave differently as a function of age; the number of oligodendrocytes showed a significant 27% decrease over adult life and a strong correlation to the total number of neurons...... while the total astrocyte number is constant through life; finally males have a 28% higher number of neocortical glial cells and a 19% higher neocortical neuron number than females. The overall total number of neocortical neurons and glial cells was 49.3 billion in females and 65.2 billion in males......, a difference of 24% with a high biological variance. These numbers can serve as reference values in quantitative studies of the human neocortex. (C) 2007 Elsevier Inc. All rights reserved Udgivelsesdato: 2008/11...

  15. Characterizing motility dynamics in human RPE cells

    Science.gov (United States)

    Liu, Zhuolin; Kurokawa, Kazuhiro; Zhang, Furu; Miller, Donald T.

    2017-02-01

    Retinal pigment epithelium (RPE) cells are vital to health of the outer retina, however, are often compromised in ageing and ocular diseases that lead to blindness. Early manifestation of RPE disruption occurs at the cellular level, but while in vivo biomarkers at this scale hold considerable promise, RPE cells have proven extremely challenging to image in the living human eye. Recently we addressed this problem by using organelle motility as a novel contrast agent to enhance the RPE cell in conjunction with 3D resolution of adaptive optics-optical coherence tomography (AO-OCT) to section the RPE layer. In this study, we expand on the central novelty of our method - organelle motility - by characterizing the dynamics of the motility in individual RPE cells, important because of its direct link to RPE physiology. To do this, AO-OCT videos of the same retinal patch were acquired at approximately 1 min intervals or less, time stamped, and registered in 3D with sub-cellular accuracy. Motility was quantified by an exponential decay time constant, the time for motility to decorrelate the speckle field across an RPE cell. In two normal subjects, we found the decay time constant to be just 3 seconds, thus indicating rapid motility in normal RPE cells.

  16. Human cell culture in a space bioreactor

    Science.gov (United States)

    Morrison, Dennis R.

    1988-01-01

    Microgravity offers new ways of handling fluids, gases, and growing mammalian cells in efficient suspension cultures. In 1976 bioreactor engineers designed a system using a cylindrical reactor vessel in which the cells and medium are slowly mixed. The reaction chamber is interchangeable and can be used for several types of cell cultures. NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first Space Bioreactor was designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small Bioreactor is being constructed for flight experiments in the Shuttle Middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption and control of low shear stress on cells.

  17. Human Adipose Derived Stem Cells Induced Cell Apoptosis and S Phase Arrest in Bladder Tumor

    Directory of Open Access Journals (Sweden)

    Xi Yu

    2015-01-01

    Full Text Available The aim of this study was to determine the effect of human adipose derived stem cells (ADSCs on the viability and apoptosis of human bladder cancer cells. EJ and T24 cells were cocultured with ADSCs or cultured with conditioned medium of ADSCs (ADSC-CM, respectively. The cell counting and colony formation assay showed ADSCs inhibited the proliferation of EJ and T24 cells. Cell viability assessment revealed that the secretions of ADSCs, in the form of conditioned medium, were able to decrease cancer cell viability. Wound-healing assay suggested ADSC-CM suppressed migration of T24 and EJ cells. Moreover, the results of the flow cytometry indicated that ADSC-CM was capable of inducing apoptosis of T24 cells and inducing S phase cell cycle arrest. Western blot revealed ADSC-CM increased the expression of cleaved caspase-3 and cleaved PARP, indicating that ADSC-CM induced apoptosis in a caspase-dependent way. PTEN/PI3K/Akt pathway and Bcl-2 family proteins were involved in the mechanism of this reaction. Our study indicated that ADSCs may provide a promising and practicable manner for bladder tumor therapy.

  18. In Vitro Brucella suis Infection Prevents the Programmed Cell Death of Human Monocytic Cells

    Science.gov (United States)

    Gross, Antoine; Terraza, Annie; Ouahrani-Bettache, Safia; Liautard, Jean-Pierre; Dornand, Jacques

    2000-01-01

    During the complex interaction between an infectious agent and a host organism, the pathogen can interfere with the host cell's programmed death to its own benefit. Induction or prevention of host cell apoptosis appears to be a critical step for determining the infection outcome. Members of the gram-negative bacterial genus Brucella are intracellular pathogens which preferentially invade monocytic cells and develop within these cells. We investigated the effect of Brucella suis infection on apoptosis of human monocytic phagocytes. The present study provides evidence that Brucella infection inhibited spontaneously occurring apoptosis in human monocytes. Prevention of monocyte apoptosis was not mediated by Brucella lipopolysaccharide and required bacterial survival within infected cells. Both invaded and noninvaded cells were protected, indicating that soluble mediators released during infection were involved in the phenomenon. Analysis of Brucella-infected monocytes revealed specific overexpression of the A1 gene, a member of the bcl-2 family implicated in the survival of hematopoietic cells. Brucella infection also rendered macrophage-like cells resistant to Fas ligand- or gamma interferon-induced apoptosis, suggesting that Brucella infection protected host cells from several cytotoxic processes occurring at different steps of the immune response. The present data clearly show that Brucella suis modulated the monocyte/macrophage's apoptotic response to the advantage of the pathogen, thus preventing host cell elimination. This might represent a strategy for Brucella development in infected hosts. PMID:10603407

  19. Mechanisms by Which Interleukin-6 Attenuates Cell Invasion and Tumorigenesis in Human Bladder Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Ke-Hung Tsui

    2013-01-01

    Full Text Available Interleukin-6, a multifunctional cytokine, contributes to tumor cell proliferation and differentiation. However, the biological mechanisms that are affected by the expression of interleukin-6 in bladder cancer cells remain unclear. We evaluated the effects of interleukin-6 expression in human bladder carcinoma cells in vitro and in vivo. The results of interleukin-6-knockdown experiments in T24 cells and interleukin-6-overexpression experiments in HT1376 cells revealed that interleukin-6 reduced cell proliferation, migration, and invasion in vitro. Xenograft animal studies indicated that the overexpression of interleukin-6 downregulated tumorigenesis of bladder cells and that interleukin-6 knockdown reversed this effect. The results of RT-PCR, immunoblotting, and reporter assays indicated that the overexpression of interleukin-6 upregulated the expression of the mammary serine protease inhibitor (MASPIN, N-myc downstream gene 1 (NDRG1, and KAI1 proteins in HT1376 cells and that interleukin-6 knockdown reduced the expression of these proteins in T24 cells. In addition, results of immunoblotting assays revealed that interleukin-6 modulated epithelial-mesenchymal transitions by upregulating the expression of the E-cadherin, while downregulation N-cadherin and vimentin proteins. Our results suggest that the effects of interleukin-6 on the regulation of epithelial-mesenchymal transitions and the expressions of the MASPIN, NDRG1, and KAI1 genes attribute to the modulation of tumorigenesis in human bladder carcinoma cells.

  20. Raman Spectroscopy of DNA Packaging in Individual Human Sperm Cells distinguishes Normal from Abnormal Cells

    Energy Technology Data Exchange (ETDEWEB)

    Huser, T; Orme, C; Hollars, C; Corzett, M; Balhorn, R

    2009-03-09

    Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

  1. Human dental pulp stem cells express many pluripotency regulators and differentiate into neuronal cells

    Institute of Scientific and Technical Information of China (English)

    Behnam Ebrahimi; Mohammad Mehdi Yaghoobi; Ali Mohammadi Kamal-abadi; Maryam Raoof

    2011-01-01

    Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR (RT-PCR), and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-1, jmjd1a, jmjd2c, and cyclin D1. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expres-sion significantly decreased, whereas expression of neuronal markers, such as microtubule asso-ciated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.

  2. Human umbilical vein endothelial cells synergize osteo/odontogenic differentiation of periodontal ligament stem cells in 3D cell sheets.

    Science.gov (United States)

    Pandula, P K C Prgeeth; Samaranayake, L P; Jin, L J; Zhang, C F

    2014-06-01

    To investigate the expression of osteo/odontogenic differentiation markers and vascular network formation in a 3D cell sheet with varying cell ratios of periodontal ligament stem cells (PDLSCs) and human umbilical vein endothelial cells (HUVECs). Human PDLSCs were isolated and characterized by flow cytometry, and co-cultured with HUVECs for the construction of cell sheets. Both types of cells were seeded on temperature-responsive culture dishes with PDLSCs alone, HUVECs alone and various ratios of the latter cells (1 : 1, 2 : 1, 5 : 1 and 1 : 5) to obtain confluent cell sheets. The expressions of osteo/odontogenic pathway markers, including alkaline phosphatase (ALP), bone sialoprotein (BSP) and runt-related transcription factor 2 (RUNX2), were analyzed at 3 and 7 d using RT-PCR. Further ALP protein quantification was performed at 7 and 14 d using ALP assay. The calcium nodule formation was assessed qualitatively and quantitatively by alizarin red assay. Histological evaluations of three cell sheet constructs treated with different combinations (PDLSC-PDLSC-PDLSC/PDLSC-HUVEC-PDLSC/co-culture-co-culture-co-culture) were performed with hematoxylin and eosin and immunofluorescence staining. Statistical analysis was performed using t-test (p culture groups compared with other groups (p cultures as compared with monoculture cell sheets (p cell sheet structure with endothelial cell islands within the constructed PDLSC-HUVEC-PDLSC and co-culture groups. Furthermore, HUVECs invaded the layered cell sheet, suggestive of rudimentary vascular network initiation. This study suggests that the PDLSC-HUVEC co-culture, cell sheet, model exhibits significantly high levels of osteo/odontogenic markers with signs of initial vascular formation. This novel 3D cell sheet-based approach may be potentially beneficial for periodontal regenerative therapy. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. GWAS of follicular lymphoma reveals allelic heterogeneity at 6p21.32 and suggests shared genetic susceptibility with diffuse large B-cell lymphoma.

    Science.gov (United States)

    Smedby, Karin E; Foo, Jia Nee; Skibola, Christine F; Darabi, Hatef; Conde, Lucia; Hjalgrim, Henrik; Kumar, Vikrant; Chang, Ellen T; Rothman, Nathaniel; Cerhan, James R; Brooks-Wilson, Angela R; Rehnberg, Emil; Irwan, Ishak D; Ryder, Lars P; Brown, Peter N; Bracci, Paige M; Agana, Luz; Riby, Jacques; Cozen, Wendy; Davis, Scott; Hartge, Patricia; Morton, Lindsay M; Severson, Richard K; Wang, Sophia S; Slager, Susan L; Fredericksen, Zachary S; Novak, Anne J; Kay, Neil E; Habermann, Thomas M; Armstrong, Bruce; Kricker, Anne; Milliken, Sam; Purdue, Mark P; Vajdic, Claire M; Boyle, Peter; Lan, Qing; Zahm, Shelia H; Zhang, Yawei; Zheng, Tongzhang; Leach, Stephen; Spinelli, John J; Smith, Martyn T; Chanock, Stephen J; Padyukov, Leonid; Alfredsson, Lars; Klareskog, Lars; Glimelius, Bengt; Melbye, Mads; Liu, Edison T; Adami, Hans-Olov; Humphreys, Keith; Liu, Jianjun

    2011-04-01

    Non-Hodgkin lymphoma (NHL) represents a diverse group of hematological malignancies, of which follicular lymphoma (FL) is a prevalent subtype. A previous genome-wide association study has established a marker, rs10484561 in the human leukocyte antigen (HLA) class II region on 6p21.32 associated with increased FL risk. Here, in a three-stage genome-wide association study, starting with a genome-wide scan of 379 FL cases and 791 controls followed by validation in 1,049 cases and 5,790 controls, we identified a second independent FL-associated locus on 6p21.32, rs2647012 (OR(combined)  = 0.64, P(combined)  = 2 × 10(-21)) located 962 bp away from rs10484561 (r(2)<0.1 in controls). After mutual adjustment, the associations at the two SNPs remained genome-wide significant (rs2647012:OR(adjusted)  = 0.70, P(adjusted)  =  4 × 10(-12); rs10484561:OR(adjusted)  = 1.64, P(adjusted)  = 5 × 10(-15)). Haplotype and coalescence analyses indicated that rs2647012 arose on an evolutionarily distinct haplotype from that of rs10484561 and tags a novel allele with an opposite (protective) effect on FL risk. Moreover, in a follow-up analysis of the top 6 FL-associated SNPs in 4,449 cases of other NHL subtypes, rs10484561 was associated with risk of diffuse large B-cell lymphoma (OR(combined)  = 1.36, P(combined)  =  1.4 × 10(-7)). Our results reveal the presence of allelic heterogeneity within the HLA class II region influencing FL susceptibility and indicate a possible shared genetic etiology with diffuse large B-cell lymphoma. These findings suggest that the HLA class II region plays a complex yet important role in NHL.

  4. GWAS of follicular lymphoma reveals allelic heterogeneity at 6p21.32 and suggests shared genetic susceptibility with diffuse large B-cell lymphoma.

    Directory of Open Access Journals (Sweden)

    Karin E Smedby

    2011-04-01

    Full Text Available Non-Hodgkin lymphoma (NHL represents a diverse group of hematological malignancies, of which follicular lymphoma (FL is a prevalent subtype. A previous genome-wide association study has established a marker, rs10484561 in the human leukocyte antigen (HLA class II region on 6p21.32 associated with increased FL risk. Here, in a three-stage genome-wide association study, starting with a genome-wide scan of 379 FL cases and 791 controls followed by validation in 1,049 cases and 5,790 controls, we identified a second independent FL-associated locus on 6p21.32, rs2647012 (OR(combined  = 0.64, P(combined  = 2 × 10(-21 located 962 bp away from rs10484561 (r(2<0.1 in controls. After mutual adjustment, the associations at the two SNPs remained genome-wide significant (rs2647012:OR(adjusted  = 0.70, P(adjusted  =  4 × 10(-12; rs10484561:OR(adjusted  = 1.64, P(adjusted  = 5 × 10(-15. Haplotype and coalescence analyses indicated that rs2647012 arose on an evolutionarily distinct haplotype from that of rs10484561 and tags a novel allele with an opposite (protective effect on FL risk. Moreover, in a follow-up analysis of the top 6 FL-associated SNPs in 4,449 cases of other NHL subtypes, rs10484561 was associated with risk of diffuse large B-cell lymphoma (OR(combined  = 1.36, P(combined  =  1.4 × 10(-7. Our results reveal the presence of allelic heterogeneity within the HLA class II region influencing FL susceptibility and indicate a possible shared genetic etiology with diffuse large B-cell lymphoma. These findings suggest that the HLA class II region plays a complex yet important role in NHL.

  5. Antiproliferative effect of rapamycin on human T-cell leukemia cell line Jurkat by cell cycle arrest and telomerase inhibition

    Institute of Scientific and Technical Information of China (English)

    Yan-min ZHAO; Qian ZHOU; Yun XU; Xiao-yu LAI; He HUANG

    2008-01-01

    Aim:To examine the ability of rapamycin to suppress growth and regulate telomerase activity in the human T-cell leukemia cell line Jurkat. Methods:Cell proliferation was assessed after exposure to rapamycin by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle progression and apoptosis were determined by flow cytometry. The proteins important for cell cycle progres-sion and Akt/mammalian target of rapamycin signaling cascade were assessed by Western blotting. Telomerase activity was quantified by telomeric repeat amplication protocol assay. The human telomerase reverse transcriptase (hTERT) mRNA levels were determined by semi-quantitative RT-PCR. Results:Rapamycin inhibited the proliferation of Jurkat, induced G1 phase arrest, unregulated the pro-tein level of p21 as well as p27, and downregulated cyclinD3, phospho-p70s6k, and phospho-s6, but had no effect on apoptosis. Treatment with rapamycin reduced telomerase activity, and reduced hTERT mRNA and protein expression. Conclusion:Rapamycin displayed a potent antileukemic effect in the human T-cell leukemia cell line by inhibition of cell proliferation through G1 cell cycle arrest and also through the suppression of telomerase activity, suggesting that rapamycin may have potential clinical implications in the treatment of some leukemias.

  6. Human autologous serum as a substitute for fetal bovine serum in human Schwann cell culture.

    Directory of Open Access Journals (Sweden)

    Parisa Goodarzi

    2014-04-01

    Full Text Available Nowadays, cell -based and tissue engineered products have opened new horizons in treatment of incurable nervous system disorders. The number of studies on the role of Schwann cells (SC in treating nervous disorders is higher than other cell types. Different protocols have been suggested for isolation and expansion of SC which most of them have used multiple growth factors, mitogens and fetal bovine sera (FBS in culture medium. Because of potential hazards of animal-derived reagents, this study was designed to evaluate the effect of replacing FBS with human autologous serum (HAS on SC's yield and culture parameters. Samples from 10 peripheral nerve biopsies were retrieved and processed under aseptic condition. The isolated cells cultured in FBS (1st group or autologous serum (2nd group. After primary culture the cells were seeded at 10000 cell/cm2 in a 12 wells cell culture plate for each group. At 100% confluency, the cell culture parameters (count, viability, purity and culture duration of 2 groups were compared using paired t-test. The average donors' age was 35.80 (SD=13.35 and except for 1 sample the others cultured successfully. In first group, the averages of cell purity, viability and culture duration were 97% (SD=1.32, 97/33% (SD=1.22 and 11.77 (SD=2.58 days respectively. This parameters were 97.33% (SD=1.00, 97.55% (SD=1.33 and 10.33 days (SD=1.65 in second group. The difference of cell count, purity and viability were not significant between 2 groups (P>0.05. The cells of second group reached to 100% confluency in shorter period of time (P=0.03. The results of this study showed that autologous serum can be a good substitute for FBS in human SC culture. This can reduce the costs and improve the safety of cell product for clinical application.

  7. TFF3 knockout in human pituitary adenoma cell HP75 facilitates cell apoptosis via mitochondrial pathway.

    Science.gov (United States)

    Gao, Feng; Pan, Suxia; Liu, Bing; Zhang, Huanzhi

    2015-01-01

    Trefoil factor 3 (TFF3), a regulatory protein composed of 59 amino acids, has been suggested to be involved in pathogenesis, proliferation, differentiation, invasion, migration and apoptosis in multiple malignant tumors. This study thus investigated the effect of TFF3 knockout in human pituitary adenoma cell line HP75 on cell apoptosis and related pathways. RNA interference approach was used to knock down the expression of TFF3 protein. The gene silencing was validated by RNA denaturing gel electrophoresis and Western blotting. The effect of TFF3 knockout on cell apoptosis was analyzed by Western blotting and flow cytometry. TFF3 protein level in pituitary adenoma was about 3.61 ± 0.48 folds of that in normal tissues (P TFF3, the apoptotic ration was significantly elevated (P TFF3 protein knockout can facilitate apoptosis of human pituitary adenoma HP75 cells via mitochondrial pathway.

  8. TFF3 knockout in human pituitary adenoma cell HP75 facilitates cell apoptosis via mitochondrial pathway

    Science.gov (United States)

    Gao, Feng; Pan, Suxia; Liu, Bing; Zhang, Huanzhi

    2015-01-01

    Trefoil factor 3 (TFF3), a regulatory protein composed of 59 amino acids, has been suggested to be involved in pathogenesis, proliferation, differentiation, invasion, migration and apoptosis in multiple malignant tumors. This study thus investigated the effect of TFF3 knockout in human pituitary adenoma cell line HP75 on cell apoptosis and related pathways. RNA interference approach was used to knock down the expression of TFF3 protein. The gene silencing was validated by RNA denaturing gel electrophoresis and Western blotting. The effect of TFF3 knockout on cell apoptosis was analyzed by Western blotting and flow cytometry. TFF3 protein level in pituitary adenoma was about 3.61 ± 0.48 folds of that in normal tissues (P TFF3, the apoptotic ration was significantly elevated (P TFF3 protein knockout can facilitate apoptosis of human pituitary adenoma HP75 cells via mitochondrial pathway. PMID:26823779

  9. Human somatic cell nuclear transfer is alive and well.

    Science.gov (United States)

    Cibelli, Jose B

    2014-06-05

    In this issue, Chung et al. (2014) generate human embryonic stem cells by fusing an adult somatic cell to a previously enucleated human oocyte, in agreement with recent reports by the Mitalipov and Egli groups. We can now safely say that human somatic cell nuclear transfer is alive and well.

  10. Effect of Melatonin on Human Dental Papilla Cells

    Directory of Open Access Journals (Sweden)

    Ryusuke Tachibana

    2014-09-01

    Full Text Available Melatonin regulates a variety of biological processes, which are the control of circadian rhythms, regulation of seasonal reproductive function and body temperature, free radical scavenging and so on. Our previous studies have shown that various cells exist in human and mouse tooth germs that express the melatonin 1a receptor (Mel1aR. However, little is known about the effects of melatonin on tooth development and growth. The present study was performed to examine the possibility that melatonin might exert its influence on tooth development. DP-805 cells, a human dental papilla cell line, were shown to express Mel1aR. Expression levels of mRNA for Mel1aR in DP-805 cells increased until 3 days after reaching confluence and decreased thereafter. Real-time reverse transcription-polymerase chain reaction showed that melatonin increased the expression of mRNAs for osteopontin (OPN, osteocalcin (OCN, bone sialoprotein (BSP, dentin matrix protein-1 (DMP-1 and dentin sialophosphoprotin (DSPP. Melatonin also enhanced the mineralized matrix formation in DP-805 cell cultures in a dose-dependent manner. These results strongly suggest that melatonin may play a physiological role in tooth development/growth by regulating the cellular function of odontogenic cells in tooth germs.

  11. Myeloid dendritic cells are potential players in human neurodegenerative diseases

    Directory of Open Access Journals (Sweden)

    Paola eBossù

    2015-12-01

    Full Text Available Alzheimer’s (AD and Parkinson’s (PD diseases are devastating neurodegenerative disturbances wherein neuroinflammation is a chronic pathogenic process with high therapeutic potential. Major mediators of AD/PD neuroimmune processes are resident immune cells, but immune cells derived from periphery may also participate and to some extent modify neuroinflammation. Specifically, blood borne myeloid cells emerge as crucial components of AD/PD progression and susceptibility. Among these, dendritic cells (DCs are key immune orchestrators and players of brain immune surveillance: we candidate them as potential mediators of both AD and PD and as relevant cell model for unraveling myeloid cell role in neurodegeneration. Hence, we recapitulate and discuss emerging data suggesting that blood-derived DCs play a role in experimental and human neurodegenerative diseases. In humans, in particular, DCs are modified by in vitro culture with neurodegeneration-associated pathogenic factors and dysregulated in AD patients, while the levels of DC precursors are decreased in AD and PD patients’ blood, possibly as an index of their recruitment to the brain. Overall, we emphasize the need to explore the impact of DCs on neurodegeneration to uncover peripheral immune mechanisms of pathogenic importance, recognize potential biomarkers and improve therapeutic approaches for neurodegenerative diseases.

  12. Pluripotent stem cell transcription factors during human odontogenesis.

    Science.gov (United States)

    da Cunha, Juliana Malta; da Costa-Neves, Adriana; Kerkis, Irina; da Silva, Marcelo Cavenaghi Pereira

    2013-09-01

    Stem cells are capable of generating various cell lines and can be obtained from adult or embryonic tissues for clinical therapies. Stem cells from deciduous dental pulp are among those that are easily obtainable from adult tissues and have been widely studied because of their ability to differentiate into a variety of cell lines in the presence of various chemical mediators. We have analyze the expression of several proteins related to the differentiation and proliferative potential of cell populations that compose the tooth germ of human fetuses. We evaluate 20 human fetuses of both genders. After being paraffin-embedded, cap and bell stages of tooth germ development were subjected to immunohistochemistry for the following markers: Oct-4, Nanog, Stat-3 and Sox-2. The studied antibodies showed nuclear or cytoplasmic immunnostaining within various anatomical structures and with various degrees of expression, indicating the action of these proteins during tooth development. We conclude that the interrelationship between these transcription factors is complex and associated with self-renewal and cell differentiation. Our results suggest that the expression of Oct-4, Nanog, Sox-2 and Stat-3 are related to differentiation in ameloblasts and odontoblasts.

  13. Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?

    Science.gov (United States)

    Roberts, R Michael; Loh, Kyle M; Amita, Mitsuyoshi; Bernardo, Andreia S; Adachi, Katsuyuki; Alexenko, Andrei P; Schust, Danny J; Schulz, Laura C; Telugu, Bhanu Prakash V L; Ezashi, Toshihiko; Pedersen, Roger A

    2014-05-01

    It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

  14. The association between human papillomavirus and oropharyngeal squamous cell Carcinoma

    DEFF Research Database (Denmark)

    Walvik, Lena; Svensson, Amanda Björk; Friborg, Jeppe

    2016-01-01

    There is emerging evidence of the association between human papillomavirus and a subset of head and neck cancers. However, the role of human papillomavirus as a causal factor is still debated. This review addresses the association between human papillomavirus and oropharyngeal squamous cell...... of well-defined premalignant lesions. However, a causal relationship between human papillomavirus infection and oropharyngeal squamous cell carcinoma seems evident....

  15. Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

    Science.gov (United States)

    Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

    2013-03-01

    Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

  16. Mutagenic effect of cadmium on tetranucleotide repeats in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Slebos, Robbert J.C. [Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States) and Department of Otolaryngology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States)]. E-mail: r.slebos@vanderbilt.edu; Li Ming [Department of Biostatistics, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Evjen, Amy N. [Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Coffa, Jordy [Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Shyr, Yu [Department of Biostatistics, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Yarbrough, Wendell G. [Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Department of Otolaryngology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States)

    2006-12-01

    Cadmium is a human carcinogen that affects cell proliferation, apoptosis and DNA repair processes that are all important to carcinogenesis. We previously demonstrated that cadmium inhibits DNA mismatch repair (MMR) in yeast cells and in human cell-free extracts (H.W. Jin, A.B. Clark, R.J.C. Slebos, H. Al-Refai, J.A. Taylor, T.A. Kunkel, M.A. Resnick, D.A. Gordenin, Cadmium is a mutagen that acts by inhibiting mismatch repair, Nat. Genet. 34 (3) (2003) 326-329), but cadmium also inhibits DNA excision repair. For this study, we selected a panel of three hypermutable tetranucleotide markers (MycL1, D7S1482 and DXS981) and studied their suitability as readout for the mutagenic effects of cadmium. We used a clonal derivative of the human fibrosarcoma cell line HT1080 to assess mutation levels in microsatellites after cadmium and/or N-methyl-N-nitro-N-nitrosoguanidine (MNNG) exposure to study effects of cadmium in the presence or absence of base damage. Mutations were measured in clonally expanded cells obtained by limiting dilution after exposure to zero dose, 0.5 {mu}M cadmium, 5 nM MNNG or a combination of 0.5 {mu}M cadmium and 5 nM MNNG. Exposure of HT1080-C1 to cadmium led to statistically significant increases in microsatellite mutations, either with or without concurrent exposure to MNNG. A majority of the observed mutant molecules involved 4-nucleotide shifts consistent with DNA slippage mutations that are normally repaired by MMR. These results provide evidence for the mutagenic effects of low, environmentally relevant levels of cadmium in intact human cells and suggest that inhibition of DNA repair is involved.

  17. Glycomics of human embryonic stem cells and human induced pluripotent stem cells.

    Science.gov (United States)

    Furukawa, Jun-Ichi; Okada, Kazue; Shinohara, Yasuro

    2016-10-01

    Most cells are coated by a dense glycocalyx composed of glycoconjugates such as glycosphingolipids, glycoproteins, and proteoglycans. The overall glycomic profile is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that regulate cell-cell adhesion, the immune response, microbial pathogenesis, and other cellular events. Many cell surface markers were discovered and identified as glycoconjugates such as stage-specific embryonic antigen, Tra-1-60/81 and various other cell surface molecules (e.g., cluster of differentiation). Recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The glycomic profiles of these cells are highly cell type-specific and reflect cellular alterations, such as development, differentiation and cancerous change. In this mini review, we briefly summarize the glycosylation spectra specific to hESCs and hiPSCs, which cover glycans of all major glycoconjugates (i.e., glycosphingolipids, N- and O-glycans of glycoproteins, and glycosaminoglycans) and free oligosaccharides.

  18. Isolation and characterization of human spermatogonial stem cells

    Directory of Open Access Journals (Sweden)

    Liu Shixue

    2011-10-01

    Full Text Available Abstract Background To isolate and characterization of human spermatogonial stem cells from stem spermatogonium. Methods The disassociation of spermatogonial stem cells (SSCs were performed using enzymatic digestion of type I collagenase and trypsin. The SSCs were isolated by using Percoll density gradient centrifugation, followed by differential surface-attachment method. Octamer-4(OCT4-positive SSC cells were further identified using immunofluorescence staining and flow cytometry technques. The purity of the human SSCs was also determined, and a co-culture system for SSCs and Sertoli cells was established. Results The cell viability was 91.07% for the suspension of human spermatogonial stem cells dissociated using a two-step enzymatic digestion process. The cells isolated from Percoll density gradient coupled with differential surface-attachement purification were OCT4 positive, indicating the cells were human spermatogonial stem cells. The purity of isolated human spermatogonial stem cells was 86.7% as assessed by flow cytometry. The isolated SSCs were shown to form stable human spermatogonial stem cell colonies on the feeder layer of the Sertoli cells. Conclusions The two-step enzyme digestion (by type I collagenase and trypsin process is an economical, simple and reproducible technique for isolating human spermatogonial stem cells. With little contamination and less cell damage, this method facilitates isolated human spermatogonial stem cells to form a stable cell colony on the supporting cell layer.

  19. Cultivation of Human Microvascular Endothelial Cells on Topographical Substrates to Mimic the Human Corneal Endothelium

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    Jie Shi Chua

    2013-03-01

    Full Text Available Human corneal endothelial cells have a limited ability to replicate in vivo and in vitro. Allograft transplantation becomes necessary when an accident or trauma results in excessive cell loss. The reconstruction of the cornea endothelium using autologous cell sources is a promising alternative option for therapeutic or in vitro drug testing applications. The native corneal endothelium rests on the Descemet’s membrane, which has nanotopographies of fibers and pores. The use of synthetic topographies mimics the native environment, and it is hypothesized that this can direct the behavior and growth of human microvascular endothelial cells (HMVECs to resemble the corneal endothelium. In this study, HMVECs are cultivated on substrates with micron and nano-scaled pillar and well topographies. Closely packed HMVEC monolayers with polygonal cells and well-developed tight junctions were formed on the topographical substrates. Sodium/potassium (Na+/K+ adenine triphosphatase (ATPase expression was enhanced on the microwells substrate, which also promotes microvilli formation, while more hexagonal-like cells are found on the micropillars samples. The data obtained suggests that the use of optimized surface patterning, in particular, the microtopographies, can induce HMVECs to adopt a more corneal endothelium-like morphology with similar barrier and pump functions. The mechanism involved in cell contact guidance by the specific topographical features will be of interest for future studies.

  20. Auditory function in the Tc1 mouse model of down syndrome suggests a limited region of human chromosome 21 involved in otitis media.

    Directory of Open Access Journals (Sweden)

    Stephanie Kuhn

    Full Text Available Down syndrome is one of the most common congenital disorders leading to a wide range of health problems in humans, including frequent otitis media. The Tc1 mouse carries a significant part of human chromosome 21 (Hsa21 in addition to the full set of mouse chromosomes and shares many phenotypes observed in humans affected by Down syndrome with trisomy of chromosome 21. However, it is unknown whether Tc1 mice exhibit a hearing phenotype and might thus represent a good model for understanding the hearing loss that is common in Down syndrome. In this study we carried out a structural and functional assessment of hearing in Tc1 mice. Auditory brainstem response (ABR measurements in Tc1 mice showed normal thresholds compared to littermate controls and ABR waveform latencies and amplitudes were equivalent to controls. The gross anatomy of the middle and inner ears was also similar between Tc1 and control mice. The physiological properties of cochlear sensory receptors (inner and outer hair cells: IHCs and OHCs were investigated using single-cell patch clamp recordings from the acutely dissected cochleae. Adult Tc1 IHCs exhibited normal resting membrane potentials and expressed all K(+ currents characteristic of control hair cells. However, the size of the large conductance (BK Ca(2+ activated K(+ current (I(K,f, which enables rapid voltage responses essential for accurate sound encoding, was increased in Tc1 IHCs. All physiological properties investigated in OHCs were indistinguishable between the two genotypes. The normal functional hearing and the gross structural anatomy of the middle and inner ears in the Tc1 mouse contrast to that observed in the Ts65Dn model of Down syndrome which shows otitis media. Genes that are trisomic in Ts65Dn but disomic in Tc1 may predispose to otitis media when an additional copy is active.

  1. The structure of the two amino-terminal domains of human intercellular adhesion molecule-1 suggests how it functions as a rhinovirus receptor.

    Science.gov (United States)

    Bella, J; Kolatkar, P R; Marlor, C W; Greve, J M; Rossmann, M G

    1999-08-01

    The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen (LFA-1) or macrophage-1 antigen (Mac-1). However, ICAM-1 is also utilized as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2 A resolution and fitted into a cryo-electron microscopy reconstruction of a rhinovirus-ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE and FG loops of the amino-terminal immunoglobulin-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1 which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is known to be mediated by a divalent cation bound to the I-(insertion) domain on the alpha chain of LFA-1 and the carboxy group of a conserved glutamic acid residue on ICAMs. Domain D1 has been docked with the known structure of the I-domain. The resultant model is consistent with mutational data and provides a structural framework for the adhesion between these molecules.

  2. The structure of the two amino-terminal domains of human ICAM-1 suggests how it functions as a rhinovirus receptor and as an LFA-1 integrin ligand.

    Science.gov (United States)

    Bella, J; Kolatkar, P R; Marlor, C W; Greve, J M; Rossmann, M G

    1998-04-14

    The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen (LFA-1) or macrophage-1 antigen (Mac-1). However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2-A resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus-ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is known to be mediated by a divalent cation bound to the insertion (I)-domain on the alpha chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs. Domain D1 has been docked with the known structure of the I-domain. The resultant model is consistent with mutational data and provides a structural framework for the adhesion between these molecules.

  3. Cell entry by human pathogenic arenaviruses.

    Science.gov (United States)

    Rojek, Jillian M; Kunz, Stefan

    2008-04-01

    The arenaviruses Lassa virus (LASV) in Africa and Machupo (MACV), Guanarito (GTOV) and Junin viruses (JUNV) in South America cause severe haemorrhagic fevers in humans with fatality rates of 15-35%. The present review focuses on the first steps of infection with human pathogenic arenaviruses, the interaction with their cellular receptor molecules and subsequent entry into the host cell. While similarities exist in genomic organization, structure and clinical disease caused by pathogenic Old World and New World arenaviruses these pathogens use different primary receptors. The Old World arenaviruses employ alpha-dystroglycan, a cellular receptor for proteins of the extracellular matrix, and the human pathogenic New World arenaviruses use the cellular cargo receptor transferrin receptor 1. While the New World arenavirus JUNV enters cells via clathrin-dependent endocytosis, evidence occurred for clathrin-independent entry of the prototypic Old World arenavirus lymphocytic choriomeningitis virus. Upon internalization, arenaviruses are delivered to the endosome, where pH-dependent membrane fusion is mediated by the envelope glycoprotein (GP). While arenavirus GPs share characteristics with class I fusion GPs of other enveloped viruses, unusual mechanistic features of GP-mediated membrane fusion have recently been discovered for arenaviruses with important implications for viral entry.

  4. Markers of T Cell Senescence in Humans

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    Weili Xu

    2017-08-01

    Full Text Available Many countries are facing the aging of their population, and many more will face a similar obstacle in the near future, which could be a burden to many healthcare systems. Increased susceptibility to infections, cardiovascular and neurodegenerative disease, cancer as well as reduced efficacy of vaccination are important matters for researchers in the field of aging. As older adults show higher prevalence for a variety of diseases, this also implies higher risk of complications, including nosocomial infections, slower recovery and sequels that may reduce the autonomy and overall quality of life of older adults. The age-related effects on the immune system termed as “immunosenescence” can be exemplified by the reported hypo-responsiveness to influenza vaccination of the elderly. T cells, which belong to the adaptive arm of the immune system, have been extensively studied and the knowledge gathered enables a better understanding of how the immune system may be affected after acute/chronic infections and how this matters in the long run. In this review, we will focus on T cells and discuss the surface and molecular markers that are associated with T cell senescence. We will also look at the implications that senescent T cells could have on human health and diseases. Finally, we will discuss the benefits of having these markers for investigators and the future work that is needed to advance the field of T cell senescence markers.

  5. Effects of active bufadienolide compounds on human cancer cells and CD4+CD25+Foxp3+ regulatory T cells in mitogen-activated human peripheral blood mononuclear cells.

    Science.gov (United States)

    Yuan, Bo; He, Jing; Kisoh, Keishi; Hayashi, Hideki; Tanaka, Sachiko; Si, Nan; Zhao, Hai-Yu; Hirano, Toshihiko; Bian, Baolin; Takagi, Norio

    2016-09-01

    The growth inhibitory effects of bufadienolide compounds were investigated in two intractable cancer cells, a human glioblastoma cell line U-87 and a pancreatic cancer cell line SW1990. Among four bufadienolide compounds, a dose-dependent cytotoxicity was observed in these cancer cells after treatment with gamabufotalin and arenobufagin. The IC50 values of the two compounds were 3-5 times higher in normal peripheral blood mononuclear cells (PBMCs) than these values for both cancer cell lines. However, similar phenomena were not observed for two other bufadienolide compounds, telocinobufagin and bufalin. These results thus suggest that gamabufotalin and arenobufagin possess selective cytotoxic activity against tumor cells rather than normal cells. Moreover, a clear dose-dependent lactate dehydrogenase (LDH) release, a well-known hallmark of necrosis, was observed in both cancer cells treated with gamabufotalin, suggesting that gamabufotalin-mediated cell death is predominantly associated with a necrosis-like phenotype. Of most importance, treatment with as little as 8 ng/ml of gamabufotalin, even an almost non-toxic concentration to PBMCs, efficiently downregulated the percentages of CD4+CD25+Foxp3+ regulator T (Treg) cells in mitogen-activated PBMCs. Given that Treg cells play a critical role in tumor immunotolerance by suppressing antitumor immunity, these results suggest that gamabufotalin may serve as a promising candidate, as an adjuvant therapeutic agent by manipulating Treg cells to enhance the efficacy of conventional anticancer drugs and lessen their side-effects. These findings provide insights into the clinical application of gamabufotalin for cancer patients with glioblastoma/pancreatic cancer based on its cytocidal effect against tumor cells as well as its depletion of Treg cells.

  6. Arecoline is cytotoxic for human endothelial cells.

    Science.gov (United States)

    Ullah, Mafaz; Cox, Stephen; Kelly, Elizabeth; Boadle, Ross; Zoellner, Hans

    2014-11-01

    Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333 μg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111 μg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4 h with arecoline at 333 μg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24 h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Immunity to pathogens taught by specialized human dendritic cell subsets.

    Directory of Open Access Journals (Sweden)

    Jens A. E. Geginat

    2015-10-01

    Full Text Available Dendritic cells (DC are specialized antigen-presenting cells (APC that have a key role in immune responses, because they bridge the innate and adaptive arms of the immune system. They mature upon recognition of pathogens and up-regulate MHC molecules and co-stimulatory receptors to activate antigen-specific CD4+ and CD8+ T-cells. It is now well established that DC are not a homogeneous population, but are composed of different subsets with specialized functions in immune responses to specific pathogens. Upon viral infections, plasmacytoid DC (pDC rapidly produce large amounts of IFN-α, which has potent anti-viral functions and activates several other immune cells. However, pDC are not particularly potent APC and induce the tolerogenic cytokine IL-10 in CD4+ T-cells. In contrast, myeloid DC (mDC are very potent APC and possess the unique capacity to prime naïve T-cells and consequently to initiate a primary adaptive immune response. Different subsets of myeloid DC with specialized functions have been identified. In mice, CD8α+ mDC capture antigenic material from necrotic cells, secrete high levels of IL-12, and prime Th1 and cytotoxic T cell responses to control intracellular pathogens. Conversely, CD8α- mDC preferentially prime CD4+ T-cells and promote Th2 or Th17 differentiation. BDCA-3+ mDC2 are the human homologue of CD8α+ mDC, since they share the expression of several key molecules, the capacity to cross-present antigens to CD8+ T-cells and to produce IFN-λ. However, although several features of the DC network are conserved between humans and mice, the expression of several relevant toll-like receptors as well as the production of cytokines that regulate T-cell differentiation are different. Intriguingly, recent data suggests specific roles for human DC subsets in immune responses against individual pathogens. The biology of human DC subsets holds the promise to be exploitable in translational medicine, in particular for the

  8. Dysregulation of gene expression in the artificial human trisomy cells of chromosome 8 associated with transformed cell phenotypes.

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    Hisakatsu Nawata

    Full Text Available A change in chromosome number, known as aneuploidy, is a common characteristic of cancer. Aneuploidy disrupts gene expression in human cancer cells and immortalized human epithelial cells, but not in normal human cells. However, the relationship between aneuploidy and cancer remains unclear. To study the effects of aneuploidy in normal human cells, we generated artificial cells of human primary fibroblast having three chromosome 8 (trisomy 8 cells by using microcell-mediated chromosome transfer technique. In addition to decreased proliferation, the trisomy 8 cells lost contact inhibition and reproliferated after exhibiting senescence-like characteristics that are typical of transformed cells. Furthermore, the trisomy 8 cells exhibited chromosome instability, and the overall gene expression profile based on microarray analyses was significantly different from that of diploid human primary fibroblasts. Our data suggest that aneuploidy, even a single chromosome gain, can be introduced into normal human cells and causes, in some cases, a partial cancer phenotype due to a disruption in overall gene expression.

  9. Secretome analysis of human oligodendrocytes derived from neural stem cells.

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    Woo Kyung Kim

    Full Text Available In this study, we investigated the secretome of human oligodendrocytes (F3.Olig2 cells generated from human neural stem cells by transduction with the gene encoding the Olig2 transcription factor. Using mRNA sequencing and protein cytokine arrays, we identified a number of biologically important secretory proteins whose expression has not been previously reported in oligodendrocytes. We found that F3.Olig2 cells secrete IL-6, PDGF-AA, GRO, GM-CSF, and M-CSF, and showed prominent expression of their corresponding receptors. Co-expression of ligands and receptors suggests that autocrine signaling loops may play important roles in both differentiation and maintenance of oligodendrocytes. We also found that F3.Olig2 cells secrete matrix metalloproteinases and matrix metalloproteinase-associated proteins associated with functional competence of oligodendrocytes. The results of our secretome analysis provide insights into the functional and molecular details of human oligodendrocytes. To the best of our knowledge, this is the first systematic analysis of the secretome of oligodendrocytes.

  10. Propionibacterium acnes inhibits FOXM1 and induces cell cycle alterations in human primary prostate cells.

    Science.gov (United States)

    Sayanjali, Behnam; Christensen, Gitte J M; Al-Zeer, Munir A; Mollenkopf, Hans-Joachim; Meyer, Thomas F; Brüggemann, Holger

    2016-11-01

    Propionibacterium acnes has been detected in diseased human prostate tissue, and cell culture experiments suggest that the bacterium can establish a low-grade inflammation. Here, we investigated its impact on human primary prostate epithelial cells. Microarray analysis confirmed the inflammation-inducing capability of P. acnes but also showed deregulation of genes involved in the cell cycle. qPCR experiments showed that viable P. acnes downregulates a master regulator of cell cycle progression, FOXM1. Flow cytometry experiments revealed that P. acnes increases the number of cells in S-phase. We tested the hypothesis that a P. acnes-produced berninamycin-like thiopeptide is responsible for this effect, since it is related to the FOXM1 inhibitor siomycin. The thiopeptide biosynthesis gene cluster was strongly expressed; it is present in subtype IB of P. acnes, but absent from type IA, which is most abundant on human skin. A knock-out mutant lacking the gene encoding the berninamycin-like peptide precursor was unable to downregulate FOXM1 and to halt the cell cycle. Our study reveals a novel host cell-interacting activity of P. acnes.

  11. Procoagulant properties of human MV3 melanoma cells

    Directory of Open Access Journals (Sweden)

    D.L.A. Geaquinto

    2008-02-01

    Full Text Available A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa, and ii direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.

  12. Functional heterogeneity of human effector CD8+ T cells.

    Science.gov (United States)

    Takata, Hiroshi; Naruto, Takuya; Takiguchi, Masafumi

    2012-02-09

    Effector CD8(+) T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF-γ and TNF-α. We investigated the difference between CXCR1(+) and CXCR1(-) subsets of human effector CD27(-)CD28(-)CD8(+) T cells. The subsets expressed cytolytic molecules similarly and exerted substantial cytolytic activity, whereas only the CXCR1(-) subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1(+) subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1(-) subset and that of pro-apoptotic death-associated protein kinase 1 (DAPK1) in the CXCR1(+) subset. The IL-2 producers were more frequently found in the IL-7R(+) subset of the CXCR1(-) effector CD8(+) T cells than in the IL-7R(-) subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1(-) subset. The present study has highlighted a novel subset of effector CD8(+) T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8(+) T cells.

  13. Salinomycin induces cell death with autophagy through activation of endoplasmic reticulum stress in human cancer cells.

    Science.gov (United States)

    Li, Tianliang; Su, Ling; Zhong, Ning; Hao, Xuexi; Zhong, Diansheng; Singhal, Sunil; Liu, Xiangguo

    2013-07-01

    Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. In this study, we found that salinomycin induced autophagy in human non-small cell lung cancer (NSCLC) cells. Furthermore, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. We also showed that salinomycin triggered more apoptosis and less autophagy in A549 cells in which CDH1 expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.

  14. Electron cryotomography of ESCRT assemblies and dividing Sulfolobus cells suggests that spiraling filaments are involved in membrane scission.

    Science.gov (United States)

    Dobro, Megan J; Samson, Rachel Y; Yu, Zhiheng; McCullough, John; Ding, H Jane; Chong, Parkson Lee-Gau; Bell, Stephen D; Jensen, Grant J

    2013-08-01

    The endosomal-sorting complex required for transport (ESCRT) is evolutionarily conserved from Archaea to eukaryotes. The complex drives membrane scission events in a range of processes, including cytokinesis in Metazoa and some Archaea. CdvA is the protein in Archaea that recruits ESCRT-III to the membrane. Using electron cryotomography (ECT), we find that CdvA polymerizes into helical filaments wrapped around liposomes. ESCRT-III proteins are responsible for the cinching of membranes and have been shown to assemble into helical tubes in vitro, but here we show that they also can form nested tubes and nested cones, which reveal surprisingly numerous and versatile contacts. To observe the ESCRT-CdvA complex in a physiological context, we used ECT to image the archaeon Sulfolobus acidocaldarius and observed a distinct protein belt at the leading edge of constriction furrows in dividing cells. The known dimensions of ESCRT-III proteins constrain their possible orientations within each of these structures and point to the involvement of spiraling filaments in membrane scission.

  15. Characteristics of Mitochondrial Transformation into Human Cells.

    Science.gov (United States)

    Kesner, E E; Saada-Reich, A; Lorberboum-Galski, H

    2016-01-01

    Mitochondria can be incorporated into mammalian cells by simple co-incubation of isolated mitochondria with cells, without the need of transfection reagents or any other type of intervention. This phenomenon was termed mitochondrial transformation, and although it was discovered in 1982, currently little is known regarding its mechanism(s). Here we demonstrate that mitochondria can be transformed into recipient cells very quickly, and co-localize with endogenous mitochondria. The isolated mitochondria interact directly with cells, which engulf the mitochondria with cellular extensions in a way, which may suggest the involvement of macropinocytosis or macropinocytosis-like mechanisms in mitochondrial transformation. Indeed, macropinocytosis inhibitors but not clathrin-mediated endocytosis inhibition-treatments, blocks mitochondria transformation. The integrity of the mitochondrial outer membrane and its proteins is essential for the transformation of the mitochondria into cells; cells can distinguish mitochondria from similar particles and transform only intact mitochondria. Mitochondrial transformation is blocked in the presence of the heparan sulfate molecules pentosan polysulfate and heparin, which indicate crucial involvement of cellular heparan sulfate proteoglycans in the mitochondrial transformation process.

  16. Characteristics of Mitochondrial Transformation into Human Cells

    Science.gov (United States)

    Kesner, E. E.; Saada-Reich, A.; Lorberboum-Galski, H.

    2016-01-01

    Mitochondria can be incorporated into mammalian cells by simple co-incubation of isolated mitochondria with cells, without the need of transfection reagents or any other type of intervention. This phenomenon was termed mitochondrial transformation, and although it was discovered in 1982, currently little is known regarding its mechanism(s). Here we demonstrate that mitochondria can be transformed into recipient cells very quickly, and co-localize with endogenous mitochondria. The isolated mitochondria interact directly with cells, which engulf the mitochondria with cellular extensions in a way, which may suggest the involvement of macropinocytosis or macropinocytosis-like mechanisms in mitochondrial transformation. Indeed, macropinocytosis inhibitors but not clathrin-mediated endocytosis inhibition-treatments, blocks mitochondria transformation. The integrity of the mitochondrial outer membrane and its proteins is essential for the transformation of the mitochondria into cells; cells can distinguish mitochondria from similar particles and transform only intact mitochondria. Mitochondrial transformation is blocked in the presence of the heparan sulfate molecules pentosan polysulfate and heparin, which indicate crucial involvement of cellular heparan sulfate proteoglycans in the mitochondrial transformation process. PMID:27184109

  17. Effects of small interfering RNAs targeting fascin on human esophageal squamous cell carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Garcia Jose

    2010-06-01

    Full Text Available Abstract Background Fascin induces membrane protrusions and cell motility. Fascin overexpression was associated with poor prognosis, and its downregulation reduces cell motility and invasiveness in esophageal squamous cell carcinoma (ESCC. Using a stable knockdown cell line, we revealed the effect of fascin on cell growth, cell adhesion and tumor formation. Methods We examined whether fascin is a potential target in ESCC using in vitro and in vivo studies utilizing a specific siRNA. We established a stable transfectant with downregulated fascin from KYSE170 cell line. Results The fascin downregulated cell lines showed a slower growth pattern by 40.3% (p In vivo, the tumor size was significantly smaller in the tumor with fascin knockdown cells than in mock cells by 95% at 30 days after inoculation. Conclusions These findings suggest that fascin overexpression plays a role in tumor growth and progression in ESCC and that cell death caused by its downregulation might be induced by cell adhesion loss. This indicates that targeting fascin pathway could be a novel therapeutic strategy for the human ESCC.

  18. Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses.

    Science.gov (United States)

    Sun, Xiangjie; Zeng, Hui; Kumar, Amrita; Belser, Jessica A; Maines, Taronna R; Tumpey, Terrence M

    2016-12-15

    A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage

  19. Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid- treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the differential recognition of MHC I molecules of xeno-endothelial cells by human NK cells could be the major reason for higher NK cytotoxicity to PAEC. KIR might be the primary molecule that transduced inhibitory signals when endothelial cells were injured by NK cells.

  20. Enhancement of Radiosensitivity by Roscovitine Pretreatment in Human Non-small Cell Lung Cancer A549 Cells

    OpenAIRE

    Feng, Zhang; Tao, Zhang; Zhong-Ping, GU; Yong-An, Zhou; yong, Han; Xiao-Fei, LI; Xiao-Ping, WANG; Qing-Shu, CHENG; Qi-Bing, MEI; Department of Thoracic Surgery, Tangdu Hospital, The Fourth Military Medical University; Department of Pharmacology, The Fourth Military Medical University

    2008-01-01

    Roscovitine has been reported to have anti-proliferative properties and is in process of undergoing clinical trials. In addition to its intrinsic anticancer properties, it has recently been suggested that roscovitine may also enhance the activity of traditional chemo- and radio-therapies in certain cancer cell lines. The purpose of this study was to define the activity of roscovitine in increasing radiosensitivity of human non-small cell lung cancer (NSCLC) cell line A549 cells in vitro. A549...

  1. Sulforaphane induces DNA single strand breaks in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sestili, Piero, E-mail: piero.sestili@uniurb.it [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Paolillo, Marco [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Lenzi, Monia [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy); Colombo, Evelin; Vallorani, Luciana; Casadei, Lucia; Martinelli, Chiara [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Fimognari, Carmela [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy)

    2010-07-07

    Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human umbilical vein endothelial cells (HUVEC). 1-3 h treatments with 10-30 {mu}M SFR elicited intracellular ROS formation (as assayed with dihydrorhodamine, DHR, oxidation) as well as DNA breakage (as assessed with fast halo assay, FHA). These effects lacked cell-type specificity, since could be observed in both Jurkat and HUVEC. Differential-pH FHA analysis of damaged DNA showed that SFR causes frank DNA single strand breaks (SSBs); no DNA double strand breaks (DSBs) were found within the considered treatment times (up to 3 h). SFR-derived ROS were formed at the mitochondrial respiratory chain (MRC) level: indeed rotenone or myxothiazol (MRC Complex I and III inhibitors, respectively) abrogated ROS formation. Furthermore ROS were not formed in Jurkat cells pharmacologically depleted of respiring mitochondria (MRC-/Jurkat). Formation of ROS was causally linked to the induction of SSBs: indeed all the experimental conditions capable of preventing ROS formation also prevented the damage of nuclear DNA from SFR-intoxicated cells. As to the toxicological relevance of SSBs, we found that their prevention slightly but significantly attenuated SFR cytotoxicity, suggesting that high-dose SFR toxicity is the result of a complex series of events among which GSH depletion seems to play a pivotal role. In conclusion, the present study identifies a novel mechanism contributing to SFR toxicity which - since DNA damage is a prominent mechanism underlying the cytotoxic activity of established antineoplastic agents - might help to exploit the therapeutic value

  2. Serum Levels of Human MIC-1/GDF15 Vary in a Diurnal Pattern, Do Not Display a Profile Suggestive of a Satiety Factor and Are Related to BMI.

    Directory of Open Access Journals (Sweden)

    Vicky Wang-Wei Tsai

    Full Text Available The TGF-b superfamily cytokine MIC-1/GDF15 circulates in the blood of healthy humans. Its levels rise substantially in cancer and other diseases and this may sometimes lead to development of an anorexia/cachexia syndrome. This is mediated by a direct action of MIC-1/GDF15 on feeding centres in the hypothalamus and brainstem. More recent studies in germline gene deleted mice also suggest that this cytokine may play a role in physiological regulation of energy homeostasis. To further characterize the role of MIC-1/GDF15 in physiological regulation of energy homeostasis in man, we have examined diurnal and food associated variation in serum levels and whether variation in circulating levels relate to BMI in human monozygotic twin pairs. We found that the within twin pair differences in serum MIC-1/GDF15 levels were significantly correlated with within twin pair differences in BMI, suggesting a role for MIC-1/GDF15 in the regulation of energy balance in man. MIC-1/GDF15 serum levels altered slightly in response to a meal, but comparison with variation its serum levels over a 24 hour period suggested that these changes are likely to be due to bimodal diurnal variation which can alter serum MIC-1/GDF15 levels by about plus or minus 10% from the mesor. The lack of a rapid and substantial postprandial increase in MIC-1/GDF15 serum levels suggests that MIC1/GDF15 is unlikely to act as a satiety factor. Taken together, our findings suggest that MIC-1/GDF15 may be a physiological regulator of energy homeostasis in man, most probably due to actions on long-term regulation of energy homeostasis.

  3. Beneficial or harmful influence of phytosterols on human cells?

    Science.gov (United States)

    Rubis, Blazej; Paszel, Anna; Kaczmarek, Mariusz; Rudzinska, Magdalena; Jelen, Henryk; Rybczynska, Maria

    2008-12-01

    So far, a protective influence of phytosterols on the human organism and atherogenesis has been suggested. Most studies have concentrated on the cytotoxic efficacy of phytosterols on cancer cells. However, there are only a few reports showing their influence on normal cells. The aim of the present study was to determine whether dietary plant sterols and their thermal processing products could influence the viability of normal, abdominal endothelial cells that play a crucial role in atherogenesis. Thus, we studied the effect of rapeseed oil-extract components, beta-sitosterol, cholesterol and their epoxy-derivatives, 5 alpha,6 alpha-epoxy-beta-sitosterol and 5 alpha,6 alpha-epoxycholesterol, on the proliferation and viability of human abdominal aorta endothelial cells HAAE-2 in vitro. We showed strong cytotoxic properties of beta-sitosterol in HAAE-2 cells (half maximal inhibitory concentration (IC50) = 1.99 (SEM 0.56) microm) and, interestingly, a weaker cytotoxic effect of 5 alpha,6 alpha-epoxy-beta-sitosterol (IC50>200 microm). Moreover, we observed a significantly stronger cytotoxic activity of beta-sitosterol than cholesterol (IC50 = 8.99 (SEM 2.74) microm). We also revealed that beta-sitosterol as well as cholesterol caused apoptosis, inducing caspase-3 activity in the cells (60 % increase compared with control cells) that corresponded to the DNA fragmentation analysis in a terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) study. Although absorption of plant sterols is low compared with cholesterol, they can still influence other physiological functions. Since they effectively reduce serum LDL-cholesterol and atherosclerotic risk but also decrease the viability of cancer cells as well as normal cells in a time- and dose-dependent manner in vitro, their influence on other metabolic processes remains to be elucidated.

  4. Differential cytotoxicity of copper ferrite nanoparticles in different human cells.

    Science.gov (United States)

    Ahmad, Javed; Alhadlaq, Hisham A; Alshamsan, Aws; Siddiqui, Maqsood A; Saquib, Quaiser; Khan, Shams T; Wahab, Rizwan; Al-Khedhairy, Abdulaziz A; Musarrat, Javed; Akhtar, Mohd Javed; Ahamed, Maqusood

    2016-10-01

    Copper ferrite nanoparticles (NPs) have the potential to be applied in biomedical fields such as cell labeling and hyperthermia. However, there is a lack of information concerning the toxicity of copper ferrite NPs. We explored the cytotoxic potential of copper ferrite NPs in human lung (A549) and liver (HepG2) cells. Copper ferrite NPs were crystalline and almost spherically shaped with an average diameter of 35 nm. Copper ferrite NPs induced dose-dependent cytotoxicity in both types of cells, evident by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide and neutral red uptake assays. However, we observed a quite different susceptibility in the two kinds of cells regarding toxicity of copper ferrite NPs. Particularly, A549 cells showed higher susceptibility against copper ferrite NP exposure than those of HepG2 cells. Loss of mitochondrial membrane potential due to copper ferrite NP exposure was observed. The mRNA level as well as activity of caspase-3 enzyme was higher in cells exposed to copper ferrite NPs. Cellular redox status was disturbed as indicated by induction of reactive oxygen species (oxidant) generation and depletion of the glutathione (antioxidant) level. Moreover, cytotoxicity induced by copper ferrite NPs was efficiently prevented by N-acetylcysteine treatment, which suggests that reactive oxygen species generation might be one of the possible mechanisms of cytotoxicity caused by copper ferrite NPs. To the best of our knowledge, this is the first report showing the cytotoxic potential of copper ferrite NPs in human cells. This study warrants further investigation to explore the mechanisms of differential toxicity of copper ferrite NPs in different types of cells. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Theracurmin® efficiently inhibits the growth of human prostate and bladder cancer cells via induction of apoptotic cell death and cell cycle arrest.

    Science.gov (United States)

    Kang, Minyong; Ho, Jin-Nyoung; Kook, Ha Rim; Lee, Sangchul; Oh, Jong Jin; Hong, Sung Kyu; Lee, Sang Eun; Byun, Seok-Soo

    2016-03-01

    In the present study, we aimed to investigate the anticancer properties of Theracurmin®, a novel form of the yellow curry pigment curcumin, as well as explore the molecular mechanisms of the potential anticancer effects of Theracurmin® on human prostate cancer and bladder cancer cells in vitro. The proliferation of cancer cells was examined by using the Cell Counting Kit-8. The clonogenic growth potential was determined by clonogenic assay. Cell cycle distribution was evaluated by flow cytometry using propidium iodide staining. Western blot analysis was applied to explore the expression patterns of molecules associated with apoptotic cell death and cell cycle checkpoint. We noted that Theracurmin® and curcumin exhibited similar anticancer effects in both androgen-dependent and -independent human prostate cancer cells in a dose- and time-dependent manner. These agents reduced cell viability and clonogenic growth potential by inducing apoptosis and cell cycle disturbance in human prostate cancer cells. Theracurmin® and curcumin also exerted marked anticancer effects on human bladder cancer cells, even in cisplatin-resistant T24R2 cells, in a dose- and time-dependent manner. Moreover, Theracurmin® and curcumin treatment decreased cell viability and clonogenicity via induction of apoptotic cell death and cell cycle dysregulation in human bladder cancer cells. In conclusion, our study suggests that Theracurmin® has potential as an anticancer agent in complementary and alternative medicine for these urological cancers.

  6. Study on workloads of human care worker with the introduction of IT system - the characteristics of work loads by observational research and the suggestions for KAIZEN.

    Science.gov (United States)

    Mizuno, Yuki; Yoshikawa, Toru; Matsuda, Fumiko; Takeuchi, Yuriko; Motegi, Nobuyuki; Ikegami, Thor; Sakai, Kazuhiro

    2012-01-01

    The purpose of this study was to clarify the characteristic of workloads on human care worker with the introduction of IT system, and suggested the support measures for KAIZEN in Japan. The investigation method is workflow line and hearing with a focus on work observation. The objects were 8 human care workers of the acute hospital that introduced an electronic system. By the introduction of the electronic chart, the nurse station sojourn time decreased, sickroom sojourn time increased, and time about direct nursing care to a patient increased. In addition, access to patient information became easy, and the offer of the health care service based on correct information came to be possible in real time. By The point of workflow line, it was effect that moving lengths decreased in order to install the electronic chart in patients' rooms. Though, it was a problem that it hasn't formed where to place the instruments such as sphygmomanometer, clinical thermometer and others.

  7. The targeted inactivation of TRβ gene in thyroid follicular cells suggests a new mechanism of regulation of thyroid hormone production.

    Science.gov (United States)

    Selmi-Ruby, Samia; Bouazza, Lamia; Obregon, Maria-Jesus; Conscience, Aude; Flamant, Frédéric; Samarut, Jacques; Borson-Chazot, Françoise; Rousset, Bernard

    2014-02-01

    Thyroid epithelial cells, or thyrocytes, express functional thyroid hormone receptors but no precise role has yet been assigned to either TRα or TRβ in the thyroid gland. In this study, we analyzed the impact of inactivating the TRβ gene in the thyroid of mice. First, we generated a mouse line named Thyr-Cre, expressing the Cre recombinase under the control of the thyroglobulin gene promoter, which led to a complete recombination of floxed genes in thyrocytes. Thyr-Cre mice were then crossed with TRβ floxed mice (TRβ(flox/flox)) to obtain a thyrocyte-selective deletion of TRβ. Thyr-TRβ(-/-) mice were characterized by a decrease in the size and functional activity of the thyroid gland. These alterations were associated with a decrease in plasma TSH concentration. Surprisingly, Thyr-TRβ(-/-) displayed elevated serum T(4) and rT(3) concentrations with no significant change in serum T(3) levels. Their intrathyroidal free T(4) and rT(3) contents were also elevated, whereas the ratio of serum T(4) to thyroid free T(4) was decreased by comparison with wild-type littermates. Also, within the thyroid, deiodinases D1 and D2 were reduced as well as the expression levels of genes encoding monocarboxylate transporters (Mct8 and Mct10). Such a decrease in intrathyroidal deiodination of T(4) and in the expression of genes encoding thyroid hormone transporters may contribute to the primary overproduction of T(4) observed in Thyr-TRβ(-/-) mice. In conclusion, these data show that the control of thyroid hormone production involves not only TRβ-dependent mechanisms acting at the level of hypothalamus and pituitary but also TRβ-dependent mechanisms acting at the thyroid level.

  8. Identification of a candidate stem cell in human gallbladder

    Directory of Open Access Journals (Sweden)

    Rohan Manohar

    2015-05-01

    In conclusion, we have isolated a distinct clonogenic population of epithelial cells from primary human fetal gallbladder with stem cell characteristics and found it to be unique compared to IHBD cells.

  9. Brugia malayi infective larvae fail to activate Langerhans cells and dermal dendritic cells in human skin.

    Science.gov (United States)

    Cotton, R N; McDonald-Fleming, R; Boyd, A; Spates, K; Nutman, T B; Tolouei Semnani, R

    2015-02-01

    Filarial infection in humans is initiated when a mosquito deposits third-stage parasite larvae (L3) in the skin. Langerhans cells (LCs) and dermal dendritic cells (DDCs) are the first cells that the parasite encounters, and L3s must evade these highly effective antigen-presenting cells to establish infection. To assess LC and DDC responses to L3 in human skin, we employed three models of increasing physiologic relevance: in vitro-generated LCs, epidermal blister explants and full-thickness human skin sections. In vitro-generated LCs expressed TLR1-10 and robustly produced IL-6 and TNF-α in response to PolyI:C, but pre-exposure to L3s did not alter inflammatory cytokine production or TLR expression. L3s did not modulate expression of LC markers CDH1, CD207, or CD1a, or the regulatory products TSLP or IDO in epidermal explants or in vitro-generated LC. LC, CD14+ DDC, CD1c+ DC and CD141+ DC from human skin sections were analysed by flow cytometry. While PolyI:C potently induced CCL22 production in LC, CD1c+ DC, and CD141+ DC, and IL-10 production in LC, L3s did not modulate the numbers of or cytokine production by any skin DC subset. L3s broadly failed to activate or modulate LCs or DDCs, suggesting filarial larvae expertly evade APC detection in human skin.

  10. Genetic engineering of human ES and iPS cells using TALE nucleases

    OpenAIRE

    Hockemeyer, Dirk; Wang, Haoyi; Kiani, Samira; Lai, Christine S.; Gao, Qing; Cassady, John P.; Cost, Gregory J.; Zhang, Lei; Santiago, Yolanda; Miller, Jeffrey C; Zeitler, Bryan; Cherone, Jennifer M.; Meng, Xiangdong; Hinkley, Sarah J; Rebar, Edward J.

    2011-01-01

    Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator–like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that T...

  11. Evaluation of the cytotoxicity of geosmin and 2-methylisoborneol using cultured human, monkey, and dog cells.

    Science.gov (United States)

    Mochida, Kyo

    2009-03-01

    The cytotoxicity of musty odor-emitting substances, geosmin (GM) and 2-methylisoborneol, at a concentration of 10 ng/L - 300 mg/L was investigated using cultured mammalian cells. These two compounds exhibited no cytotoxicity in either the colony-formation of human KB cells or WST-1 assays of human-, monkey-, and dog-derived cells. These results suggest that the maximum concentration (700 ng/L) of GM found in the water of Lake Shinji is not toxic.

  12. Human embryonic stem cells and patent protection

    Directory of Open Access Journals (Sweden)

    Radovanović Sanja M.

    2015-01-01

    Full Text Available Given the importance of biotechnological research in modern diagnostics and therapeutics, on the one hand, and stimulative function of a patent, on the other hand, this work deals with the question of the possibility of pa-tent protection of human embryonic stem cells. Taking into account that this is a biotechnological invention, the key question that this paper highlights is the interpretation of the provisions of their patentability. Namely, thanks to the advanced methods of isolation, purification and preparation for implementation, modern patent systems do not exclude a priori living organisms from patent protection. Therefore, the analysis of representative administrative decisions or court rulings sought to define the criteria that would be applied in order to give patent protection to a certain biotechnological invention (stem cells while others do not.

  13. Generation of human induced pluripotent stem cells from dermal fibroblasts

    OpenAIRE

    2008-01-01

    The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. Technologies including somatic cell nuclear transfer and cell fusion might generate such cells but are hindered by issues that might prevent them from being used clinically. Here, we describe methods to use dermal fibroblasts easily obtained from an individual human to generate human induced pluripotent stem (iPS) cells by ...

  14. DNA damage responses in human induced pluripotent stem cells and embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Olga Momcilovic

    Full Text Available BACKGROUND: Induced pluripotent stem (iPS cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation, iPS cells activate checkpoint signaling, evidenced by phosphorylation of ATM, NBS1, CHEK2, and TP53, localization of ATM to the double strand breaks (DSB, and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2 phase of the cell cycle, displaying a lack of the G(1/S cell cycle arrest similar to human embryonic stem (ES cells. Furthermore, both cell types remove DSB within six hours of γ-irradiation, form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally, we report elevated expression of genes involved in DNA damage signaling, checkpoint function, and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts. CONCLUSIONS/SIGNIFICANCE: High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage, dramatic changes in cell cycle structure, including a high percentage of cells in the S phase, increased radiosensitivity and loss of DNA damage-induced G(1/S cell cycle arrest, were

  15. Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β1

    Institute of Scientific and Technical Information of China (English)

    Guan-Bin Song; Jian Qin; Qing Luo; Xiao-Dong Shen; Run-Bin Yan; Shao-Xi Cai

    2005-01-01

    AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721)to human umbilical vein endothelial cells (ECV-304),expression of adhesive molecule integrinβ1 in SMMC-7721cells and its contribution to this adhesive course.METHODS: Adhesive force of SMMC-7721 cells to endothelialcells was measured using micropipette aspiration technique.Synchronous G1 and S phase SMMC-7721 cells wereachieved by thymine-2-deoxyriboside and colchicinessequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronousrates of SMMC-7721 cells and expression of integrinβ1 inSMMC-7721 cells were detected by flow cytometer.RESULTS: The percentage of cell cycle phases of generalSMMC-7721 cells was 11.01% in G2/M phases, 53.51% inG0/G1 phase, and 35.48% in S phase. The synchronous ratesof G1 and S phase SMMC-7721 cells amounted to 74.09%and 98.29%, respectively. The adhesive force of SMMC-7721cells to endothelial cells changed with the variations ofadhesive time and presented behavior characteristics ofadhesion and de-adhesion. S phase SMMC-7721 cells had higheradhesive forces than G1 phase cells [(307.65±92.10)× 10-10Nvs (195.42±60.72)×10-10N, P<0.01]. The expressivefluorescent intensity of integrinβ1 in G1 phase SMMC-7721cells was depressed more significantly than the values ofS phase and general SMMC-7721cells. The contribution ofadhesive integrinβ1 was about 53% in this adhesive course.CONCLUSION: SMMC-7721 cells can be synchronizedpreferably in G1 and S phases with thymine-2-deoxyribosideand colchicines. The adhesive molecule integrinβ1 expressesa high level in SMMC-7721 cells and shows differences invarious cell cycles, suggesting integrin β1 plays an importantrole in adhesion to endothelial cells. The change of adhesiveforces in different cell cycle SMMC-7721 cells indicatesthat S phase cells play predominant roles possibly whilethey interact with endothelial cells.

  16. Telomerase RNA accumulates in Cajal bodies in human cancer cells.

    Science.gov (United States)

    Zhu, Yusheng; Tomlinson, Rebecca L; Lukowiak, Andrew A; Terns, Rebecca M; Terns, Michael P

    2004-01-01

    Telomerase synthesizes telomeric DNA repeats at the ends of eukaryotic chromosomes. The RNA component of the enzyme (hTR) provides the template for telomere synthesis, which is catalyzed by telomerase reverse transcriptase (hTERT). Little is known regarding the subcellular localization of hTR and hTERT and the pathway by which telomerase is assembled. Here we report the first glimpse of the detailed subcellular localization of endogenous hTR in human cells, which we obtained by fluorescence in situ hybridization (FISH). Our studies have revealed a distinctive hTR localization pattern in cancer cells. We have found that hTR accumulates within intranuclear foci called Cajal bodies in all typical tumor-derived cell lines examined (in which telomerase is active), but not in primary or ALT cells (where little or no hTERT is present). Accumulation of hTR in the Cajal bodies of primary cells is induced when hTERT is ectopically expressed. Moreover, we report that hTERT is also found in Cajal bodies. Our data suggest that Cajal bodies are involved in the assembly and/or function of human telomerase.

  17. Human leptospirosis in Seychelles: A prospective study confirms the heavy burden of the disease but suggests that rats are not the main reservoir.

    Directory of Open Access Journals (Sweden)

    Leon Biscornet

    2017-08-01

    Full Text Available Leptospirosis is a bacterial zoonosis caused by pathogenic Leptospira for which rats are considered as the main reservoir. Disease incidence is higher in tropical countries, especially in insular ecosystems. Our objectives were to determine the current burden of leptospirosis in Seychelles, a country ranking first worldwide according to historical data, to establish epidemiological links between animal reservoirs and human disease, and to identify drivers of transmission.A total of 223 patients with acute febrile symptoms of unknown origin were enrolled in a 12-months prospective study and tested for leptospirosis through real-time PCR, IgM ELISA and MAT. In addition, 739 rats trapped throughout the main island were investigated for Leptospira renal carriage. All molecularly confirmed positive samples were further genotyped.A total of 51 patients fulfilled the biological criteria of acute leptospirosis, corresponding to an annual incidence of 54.6 (95% CI 40.7-71.8 per 100,000 inhabitants. Leptospira carriage in Rattus spp. was overall low (7.7% but dramatically higher in Rattus norvegicus (52.9% than in Rattus rattus (4.4%. Leptospira interrogans was the only detected species in both humans and rats, and was represented by three distinct Sequence Types (STs. Two were novel STs identified in two thirds of acute human cases while noteworthily absent from rats.This study shows that human leptospirosis still represents a heavy disease burden in Seychelles. Genotype data suggests that rats are actually not the main reservoir for human disease. We highlight a rather limited efficacy of preventive measures so far implemented in Seychelles. This could result from ineffective control measures of excreting animal populations, possibly due to a misidentification of the main contaminating reservoir(s. Altogether, presented data stimulate the exploration of alternative reservoir animal hosts.

  18. Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.

    Science.gov (United States)

    Alshabibi, Manal A; Al Huqail, Al Joharah; Khatlani, Tanvir; Abomaray, Fawaz M; Alaskar, Ahmed S; Alawad, Abdullah O; Kalionis, Bill; Abumaree, Mohamed Hassan

    2017-09-15

    Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by

  19. Why Right is Might: How the Social Science on Radicalisation suggests that International Human Rights Norms actually help frame Effective Counterterrorism Policies

    Directory of Open Access Journals (Sweden)

    Tom Parker

    2012-08-01

    Full Text Available Many states appear to turn instinctively to hard power resources when confronted with a terrorist threat. Yet existing research on violent extremism and radicalisation leading to terrorism suggests that such responses might well exacerbate the problem. Terrorist groups actively seek to exploit the push-pull dynamic that drives radicalisation and violent extremism, while one case study after the other indicates that states thereby appear to play actively into their hands. Social science research suggests that international human rights norms assist compliant states to moderate responses, build legitimacy, and ultimately craft effective counterterrorism strategies. A close reading of the literature on radicalisation and terrorist group formation offers qualitative evidence to support this conclusion. 

  20. In vivo liver regeneration potential of human induced pluripotent stem cells from diverse origins.

    Science.gov (United States)

    Liu, Hua; Kim, Yonghak; Sharkis, Saul; Marchionni, Luigi; Jang, Yoon-Young

    2011-05-11

    Human induced pluripotent stem cells (iPSCs) are a potential source of hepatocytes for liver transplantation to treat end-stage liver disease. In vitro differentiation of human iPSCs into hepatic cells has been achieved using a multistage differentiation protocol, but whether these cells are functional and capable of engrafting and regenerating diseased liver tissue is not clear. We show that human iPSC-derived hepatic cells at various differentiation stages can engraft the liver in a mouse transplantation model. Using the same differentiation and transplantation protocols, we also assessed the ability of human iPSCs derived from each of the three developmental germ layer tissues (that is, ectoderm, mesoderm, and endoderm) to regenerate mouse liver. These iPSC lines, with similar but distinct global DNA methylation patterns, differentiated into multistage hepatic cells with an efficiency similar to that of human embryonic stem cells. Human hepatic cells at various differentiation stages derived from iPSC lines of different origins successfully repopulated the liver tissue of mice with liver cirrhosis. They also secreted human-specific liver proteins into mouse blood at concentrations comparable to that of proteins secreted by human primary hepatocytes. Our results demonstrate the engraftment and liver regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo and suggest that human iPSCs of distinct origins and regardless of their parental epigenetic memory can efficiently differentiate along the hepatic lineage.

  1. Roles of CDX2 and EOMES in human induced trophoblast progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying, E-mail: ying.chen@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Michigan State University, Grand Rapids, MI 49503 (United States); Wang, Kai [Department of Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Michigan State University, Grand Rapids, MI 49503 (United States); Gong, Yun Guo; Khoo, Sok Kean [Genomic Microarray Core Facility, Van Andel Research Institute, Grand Rapids, MI 49503 (United States); Leach, Richard, E-mail: Richard.Leach@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Michigan State University, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group, Grand Rapids, MI 49503 (United States)

    2013-02-08

    Highlights: ► CDX2 and EOMES play critical roles in human induced trophoblast progenitors (iTP). ► iTP cells directly transformed from fibroblasts. ► Differentiation of iTP cells into extravillous trophoblasts and syncytiotrophoblasts. -- Abstract: Abnormal trophoblast lineage proliferation and differentiation in early pregnancy have been associated with the pathogenesis of placenta diseases of pregnancy. However, there is still a gap in understanding the molecular mechanisms of early placental development due to the limited primary trophoblast cultures and fidelity of immortalized trophoblast lines. Trophoblasts stem (TS) cells, an in vitro model of trophectoderm that can differentiate into syncytiotrophoblasts and extravillous trophoblasts, can be an attractive tool for early pregnancy research. TS cells are well established in mouse but not in humans due to insufficient knowledge of which trophoblast lineage-specific transcription factors are involved in human trophectoderm (TE) proliferation and differentiation. Here, we applied induced pluripotent stem cell technique to investigate the human trophoblast lineage-specific transcription factors. We established human induced trophoblast progenitor (iTP) cells by direct reprogramming the fibroblasts with a pool of mouse trophoblast lineage-specific transcription factors consisting of CDX2, EOMES, and ELF5. The human iTP cells exhibit epithelial morphology and can be maintained in vitro for more than 2 months. Gene expression profile of these cells was tightly clustered with human trophectoderm but not with human neuron progenitor cells, mesenchymal stem cells, or endoderm cells. These cells are capable of differentiating into cells with an invasive capacity, suggesting extravillous trophoblasts. They also form multi-nucleated cells which secrete human chorionic gonadotropin and estradiol, consistent with a syncytiotrophoblast phenotype. Our results provide the evidence that transcription factors CDX2 and

  2. Human Serum Albumin (HSA) Suppresses the Effects of Glycerol Monolaurate (GML) on Human T Cell Activation and Function

    Science.gov (United States)

    Zhang, Michael S.; Houtman, Jon C. D.

    2016-01-01

    Glycerol monolaurate (GML) is a monoglyceride with well characterized anti-microbial properties. Because of these properties, GML is widely used in food, cosmetics, and personal care products and currently being tested as a therapeutic for menstrual associated toxic shock syndrome, superficial wound infections, and HIV transmission. Recently, we have described that GML potently suppresses select T cell receptor (TCR)-induced signaling events, leading to reduced human T cell effector functions. However, how soluble host factors present in the blood and at sites of infection affect GML-mediated human T cell suppression is unknown. In this study, we have characterized how human serum albumin (HSA) affects GML-induced inhibition of human T cells. We found that HSA and other serum albumins bind to 12 carbon acyl side chain of GML at low micromolar affinities and restores the TCR-induced formation of LAT, PLC-γ1, and AKT microclusters at the plasma membrane. Additionally, HSA reverses GML mediated inhibition of AKT phosphorylation and partially restores cytokine production in GML treated cells. Our data reveal that HSA, one of the most abundant proteins in the human serum and at sites of infections, potently reverses the suppression of human T cells by GML. This suggests that GML-driven human T cell suppression depends upon the local tissue environment, with albumin concentration being a major determinant of GML function. PMID:27764189

  3. Expression of cyclooxygenase-2 in human esophageal squamous cell carcinomas

    Institute of Scientific and Technical Information of China (English)

    Jian-Gang Jiang; Dao-Wen Wang; Jiang-Bo Tang; Chun-Lian Chen; Bao-Xing Liu; Xiang-Ning Fu; Zhi-Hui Zhu; Wei Qu; Katherine Cianflone; Michael P. Waalkes

    2004-01-01

    AIM: To determine whether cyclooxygenase-2 (COX-2) was expressed in human esophageal squamous cell carcinoma.METHODS: Quantitative reverse transcription-polymerase chain reaction (RT-PCR), western blotting, immunohistochemistry and immunofluorescence were used to assess the expression level of COX-2 in esophageal tissue.RESULTS: COX-2 mRNA levels were increased by >80-fold in esophageal squamous cell carcinoma when compared to adjacent noncancerous tissue. COX-2 protein was present in 21 of 30 cases of esophageal squamous cell carcinoma tissues, but was undetectable in noncancerous tissue. Immunohistochemistry was performed to directly show expression of COX-2 in tumor tissue.CONCLUSION: These results suggest that COX-2 may be an important factor for esophageal cancer and inhibition of COX-2 may be helpful for prevention and possibly treatment of this cancer.

  4. Manipulation on human red blood cells with femtosecond optical tweezers

    Institute of Scientific and Technical Information of China (English)

    Ming Zhou; Haifeng Yang; Jianke Di; Enlan Zhao

    2008-01-01

    Different types of femtosecond optical tweezers have become a powerful tool in the modern biological field. However, how to control the irregular targets, including biological cells, using femtosecond optical tweezers remains to be explored. In this study, human red blood cells (hRBCs) are manipulated with femtosecond optical tweezers, and their states under different laser powers are investigated. The results indicate that optical potential traps only can capture the edge of hRBCs under the laser power from 1.4 to 2.8 mW, while it can make hRBCs turn over with the laser power more than 2.8 roW. It is suggested that femtosecond optical tweezers could not only manipulate biological cells, but also subtly control its states by adjusting the laser power.

  5. Derivation of human embryonic stem cells in defined conditions.

    Science.gov (United States)

    Ludwig, Tenneille E; Levenstein, Mark E; Jones, Jeffrey M; Berggren, W Travis; Mitchen, Erika R; Frane, Jennifer L; Crandall, Leann J; Daigh, Christine A; Conard, Kevin R; Piekarczyk, Marian S; Llanas, Rachel A; Thomson, James A

    2006-02-01

    We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

  6. Shielding of the Geomagnetic Field Alters Actin Assembly and Inhibits Cell Motility in Human Neuroblastoma Cells.

    Science.gov (United States)

    Mo, Wei-Chuan; Zhang, Zi-Jian; Wang, Dong-Liang; Liu, Ying; Bartlett, Perry F; He, Rong-Qiao

    2016-03-31

    Accumulating evidence has shown that absence of the geomagnetic field (GMF), the so-called hypomagnetic field (HMF) environment, alters the biological functions in seemingly non-magnetosensitive cells and organisms, which indicates that the GMF could be sensed by non-iron-rich and non-photo-sensing cells. The underlying mechanisms of the HMF effects on those cells are closely related to their GMF sensation but remain poorly understood so far. Previously, we found that the HMF represses expressions of genes associated with cell migration and cytoskeleton assembly in human neuroblastoma cells (SH-SY5Y cell line). Here, we measured the HMF-induced changes on cell morphology, adhesion, motility and actin cytoskeleton in SH-SY5Y cells. The HMF inhibited cell adhesion and migration accompanied with a reduction in cellular F-actin amount. Moreover, following exposure to the HMF, the number of cell processes was reduced and cells were smaller in size and more round in shape. Furthermore, disordered kinetics of actin assembly in vitro were observed during exposure to the HMF, as evidenced by the presence of granule and meshed products. These results indicate that elimination of the GMF affects assembly of the motility-related actin cytoskeleton, and suggest that F-actin is a target of HMF exposure and probably a mediator of GMF sensation.

  7. Trehalose improves cell proliferation and dehydration tolerance of human HaCaT cells

    Directory of Open Access Journals (Sweden)

    Lee Kyung Eun

    2015-01-01

    Full Text Available Trehalose is a disaccharide molecule that serves as a natural osmotic regulator in halophilic microorganisms and plants but not in mammals. We observed that human HaCaT cells supplied with trehalose improved cell proliferation and extended viability under dehydration. In HaCaT cells, in response to increasing concentrations of exogenous trehalose, the levels of heat shock protein (HSP 70 increased and matrix metalloproteinase (MMP 1 decreased. Proteome analysis of trehalose-treated HaCaT cells revealed remarkable increases in the levels of proteins involved in cell signaling and the cell cycle, including p21 activated kinase I, Sec I family domain protein and elongation factor G. Moreover, the proteins for cell stress resistance, tryptophan hydroxylase, serine/cysteine proteinase inhibitors and vitamin D receptors were also increased. In addition, the proteins responsible for the maintenance of the cytoskeleton and cellular structures including procollagen-lysine dioxygenase, vinculin and ezrin were increased. Proteomic data revealed that trehalose affected HaCaT cells by inducing the proteins involved in cell proliferation. These results suggest that trehalose improves the proliferation and dehydration tolerance of HaCaT cells by inducing proteins involved in cell growth and dehydration protection.

  8. Entry of Oncolytic Herpes Simplex Virus into Human Squamous Cell Carcinoma Cells by Ultrasound

    Directory of Open Access Journals (Sweden)

    Shusuke Okunaga

    2015-10-01

    Full Text Available Low-intensity ultrasound is a useful method to introduce materials into cells due to the transient formation of micropores, called sonoporations, on the cell membrane. Whether oncolytic herpes simplex virus type 1 (HSV-1 can be introduced into oral squamous cell carcinoma (SCC cells through membrane pores remains undetermined. Human SCC cell line SAS and oncolytic HSV-1 RH2, which was deficient in the 134.5 gene and fusogenic, were used. Cells were exposed to ultrasound in the presence or absence of microbubbles. The increase of virus entry was estimated by plaque numbers. Viral infection was hardly established without the adsorption step, but plaque number was increased by the exposure of HSV-1-inoculated cells to ultrasound. Plaque number was also increased even if SAS cells were exposed to ultrasound and inoculated with RH2 without the adsorption step. This effect was abolished when the interval from ultrasound exposure to virus inoculation was prolonged. Scanning electron microscopy revealed depressed spots on the cell surface after exposure to ultrasound. These results suggest that oncolytic HSV-1 RH2 can be introduced into SAS cells through ultrasound-mediated pores of the cell membrane that are resealed after an interval.

  9. Abnormal mitosis triggers p53-dependent cell cycle arrest in human tetraploid cells.

    Science.gov (United States)

    Kuffer, Christian; Kuznetsova, Anastasia Yurievna; Storchová, Zuzana

    2013-08-01

    Erroneously arising tetraploid mammalian cells are chromosomally instable and may facilitate cell transformation. An increasing body of evidence shows that the propagation of mammalian tetraploid cells is limited by a p53-dependent arrest. The trigger of this arrest has not been identified so far. Here we show by live cell imaging of tetraploid cells generated by an induced cytokinesis failure that most tetraploids arrest and die in a p53-dependent manner after the first tetraploid mitosis. Furthermore, we found that the main trigger is a mitotic defect, in particular, chromosome missegregation during bipolar mitosis or spindle multipolarity. Both a transient multipolar spindle followed by efficient clustering in anaphase as well as a multipolar spindle followed by multipolar mitosis inhibited subsequent proliferation to a similar degree. We found that the tetraploid cells did not accumulate double-strand breaks that could cause the cell cycle arrest after tetraploid mitosis. In contrast, tetraploid cells showed increased levels of oxidative DNA damage coinciding with the p53 activation. To further elucidate the pathways involved in the proliferation control of tetraploid cells, we knocked down specific kinases that had been previously linked to the cell cycle arrest and p53 phosphorylation. Our results suggest that the checkpoint kinase ATM phosphorylates p53 in tetraploid cells after abnormal mitosis and thus contributes to proliferation control of human aberrantly arising tetraploids.

  10. Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells.

    Science.gov (United States)

    Brzeszczynska, J; Samuel, K; Greenhough, S; Ramaesh, K; Dhillon, B; Hay, D C; Ross, J A

    2014-06-01

    It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular, the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory, the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel, provides a robust model of human development and in the future, may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally, we demonstrate that following continued cell culture, stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model of disease.

  11. Expression and alternative splicing pattern of human telomerase reverse transcriptase in human lung cancer cells.

    Science.gov (United States)

    Fujiwara, Masachika; Kamma, Hiroshi; Wu, Wenwen; Hamasaki, Makoto; Kaneko, Setsuko; Horiguchi, Hisashi; Matsui-Horiguchi, Miwa; Satoh, Hiroaki

    2004-04-01

    Telomerase activity is generally considered to be necessary for cancer cells to avoid senescence. The expression of human telomerase reverse transcriptase (hTERT) is believed to be a rate-limiting step in telomerase activation. Recently, it has been proposed that the alternative splicing of hTERT is also involved in regulation of telomerase activity. However, the regulatory mechanism of telomerase in cancer cells has not been thoroughly investigated. To clarify it in lung cancer cells, we measured the expression of the hTERT transcript, analyzed its alternative splicing by RT-PCR, and compared it with telomerase activity and telomere length. The expression of the hTERT transcript was positively correlated with telomerase activity in lung cancer cells. Cancer cells with high telomerase activity contained 4 splicing variants of hTERT, and the full-length variant was 31.3-54.2% of the total transcripts. Cells of the TKB-20 cell line, which has extremely low telomerase activity, showed a different splicing pattern of hTERT in addition to low expression. The functional full-length variant was scarcely detected in TKB-20 cells, suggesting that the telomerase activity was repressed by alternative splicing of hTERT. Telomere length was not necessarily correlated with telomerase activity or hTERT expression in lung cancer cells. Cells of the TKB-4 cell line that also showed relatively low telomerase activity (as TKB-20 cells) had long telomeres. In conclusion, hTERT expression is regulated at both the transcriptional and post-transcriptional levels in lung cancer cells, and the alternative splicing of hTERT is involved in the control of telomerase activity.

  12. Risedronate inhibits human osteosarcoma cell invasion

    Directory of Open Access Journals (Sweden)

    Jung Sung

    2009-07-01

    Full Text Available Abstract Background Osteosarcoma is a highly malignant bone tumor and is the most commonly encountered malignant bone tumor in children and adolescents. Furthermore, significant numbers of patients eventually develop pulmonary metastases and succumb to the disease even after conventional multi-agent chemotherapy and surgical excision. Several solid tumors display enhanced expression of matrix metalloproteinases (MMPs, and recently clinical trials have been initiated on MMP-inhibitors. On the other hand, bisphosphonates (BPs, which have a profound effect on bone resorption, are widely used to treat osteoclast-mediated bone diseases. BPs are also known to inhibit tumor growths and metastases in some tumors such as breast cancer, renal cell carcinoma, and prostate cancer. Methods Two osteosarcoma cell lines (SaOS-2 and U2OS were treated with risedronate (0, 0.1, 1, 10 μM for 48 hours. Cell viabilities were determined using MTT assay, the mRNA levels of MMP-2 and MMP-9 were analyzed by reverse-transcription polymerase chain reaction, the amount of MMP-2 and MMP-9 protein were analyzed by Westernblot, the activities of MMP-2 and MMP-9 were observed by Gelatin zymography, and Matrigel invasion assays were used to investigate the invasive potential of osteosarcoma cell lines before and after risedronate treatment. Results The invasiveness of osteosarcoma cell lines (SaOS-2, U2OS were reduced in a dose dependent manner follow 48 hour treatment of up to 10 μM of the risedronate at which concentration no cytotoxicity occurred. Furthermore, the gelatinolytic activities and protein and mRNA levels of MMP-2 and MMP-9 were also suppressed by increasing risedronate concentrations. Conclusion Given that MMP-2 and MMP-9 are instrumental in tumor cell invasion, our results suggest the risedronate could reduce osteosarcoma cell invasion.

  13. Human germ cell formation in xenotransplants of induced pluripotent stem cells carrying X chromosome aneuploidies.

    Science.gov (United States)

    Dominguez, Antonia A; Chiang, H Rosaria; Sukhwani, Meena; Orwig, Kyle E; Reijo Pera, Renee A

    2014-09-22

    Turner syndrome is caused by complete or partial loss of the second sex chromosome and is characterized by spontaneous fetal loss in >90% of conceptions. Survivors possess an array of somatic and germline clinical characteristics. Induced pluripotent stem cells (iPSCs) offer an opportunity for insight into genetic requirements of the X chromosome linked to Turner syndrome. We derived iPSCs from Turner syndrome and control individuals and examined germ cell development as a function of X chromosome composition. We demonstrate that two X chromosomes are not necessary for reprogramming or maintenance of pluripotency and that there are minimal differences in gene expression, at the single cell level, linked to X chromosome aneuploidies. Formation of germ cells, as assessed in vivo through a murine xenotransplantation model, indicated that undifferentiated iPSCs, independent of X chromosome composition, are capable of forming germ-cell-like cells (GCLCs) in vivo. In combination with clinical data regarding infertility in women with X chromosome aneuploidies, results suggest that two intact X chromosomes are not required for human germ cell formation, qualitatively or quantitatively, but rather are likely to be required for maintenance of human germ cells to adulthood.

  14. Mycobacterium tuberculosis infection induces non-apoptotic cell death of human dendritic cells

    LENUS (Irish Health Repository)

    Ryan, Ruth CM

    2011-10-24

    Abstract Background Dendritic cells (DCs) connect innate and adaptive immunity, and are necessary for an efficient CD4+ and CD8+ T cell response after infection with Mycobacterium tuberculosis (Mtb). We previously described the macrophage cell death response to Mtb infection. To investigate the effect of Mtb infection on human DC viability, we infected these phagocytes with different strains of Mtb and assessed viability, as well as DNA fragmentation and caspase activity. In parallel studies, we assessed the impact of infection on DC maturation, cytokine production and bacillary survival. Results Infection of DCs with live Mtb (H37Ra or H37Rv) led to cell death. This cell death proceeded in a caspase-independent manner, and without nuclear fragmentation. In fact, substrate assays demonstrated that Mtb H37Ra-induced cell death progressed without the activation of the executioner caspases, 3\\/7. Although the death pathway was triggered after infection, the DCs successfully underwent maturation and produced a host-protective cytokine profile. Finally, dying infected DCs were permissive for Mtb H37Ra growth. Conclusions Human DCs undergo cell death after infection with live Mtb, in a manner that does not involve executioner caspases, and results in no mycobactericidal effect. Nonetheless, the DC maturation and cytokine profile observed suggests that the infected cells can still contribute to TB immunity.

  15. Sustained CD8+ T-cell responses induced after acute parvovirus B19 infection in humans

    DEFF Research Database (Denmark)

    Norbeck, Oscar; Isa, Adiba; Pöhlmann, Christoph

    2005-01-01

    Murine models have suggested that CD8+ T-cell responses peak early in acute viral infections and are not sustained, but no evidence for humans has been available. To address this, we longitudinally analyzed the CD8+ T-cell response to human parvovirus B19 in acutely infected individuals. We...... observed striking CD8+ T-cell responses, which were sustained or even increased over many months after the resolution of acute disease, indicating that CD8+ T cells may play a prominent role in the control of parvovirus B19 and other acute viral infections of humans, including potentially those generated...

  16. Human embryonic stem cells and metastatic colorectal cancer cells shared the common endogenous human microRNA-26b.

    Science.gov (United States)

    Ma, Yan-Lei; Zhang, Peng; Wang, Feng; Moyer, Mary Pat; Yang, Jian-Jun; Liu, Zhi-Hua; Peng, Jia-Yuan; Chen, Hong-Qi; Zhou, Yu-Kun; Liu, Wei-Jie; Qin, Huan-Long

    2011-09-01

    The increase in proliferation and the lack of differentiation of cancer cells resemble what occur in the embryonic stem cells during physiological process of embryogenesis. There are also striking similarities in the behaviour between the invasive placental cells and invasive cancer cells. In the present study, microarrays were used to analyse the global expression of microRNAs in a human embryonic stem cell line (i.e. HUES-17) and four colorectal cancer (CRC) cell lines (i.e. LoVo, SW480, HT29 and Caco-2) with different metastatic potentialities. Only the expression of miR-26b was significant decreased in HUES-17s and LoVo cells, compared with other three cell lines (P cell growth and the induction of apoptosis in LoVo cells in vitro, and the inhibition of tumour growth in vivo. Moreover, the potential targets of miR-26b was predicted by using bioinformatics, and then the predicted target genes were further validated by comparing gene expression profiles between LoVo and NCM460 cell lines. Four genes (TAF12, PTP4A1, CHFR and ALS2CR2) with intersection were found to be the targets of miR-26b. MetaCore network analysis further showed that the regulatory pathways of miR-26b were significantly associated with the invasiveness and metastasis of CRC cells. These data suggest that miR-26b might serve as a novel prognostic factor and a potential therapeutic target for CRC.

  17. Induction of human pancreatic beta cell replication by inhibitors of dual specificity tyrosine regulated kinase

    Science.gov (United States)

    Wang, Peng; Alvarez-Perez, Juan-Carlos; Felsenfeld, Dan P.; Liu, Hongtao; Sivendran, Sharmila; Bender, Aaron; Kumar, Anil; Sanchez, Roberto; Scott, Donald K.; Garcia-Ocaña, Adolfo; Stewart, Andrew F.

    2015-01-01

    Types 1 and 2 diabetes affect some 380 million people worldwide. Both result ultimately from a deficiency of functional pancreatic insulin-producing beta cells. Beta cells proliferate in humans during a brief temporal window beginning around the time of birth, with peak beta cell labeling indices achieving approximately 2% in first year of life1-4. In embryonic life and after early childhood, beta cell replication rates are very low. While beta cell expansion seems an obvious therapeutic approach to beta cell deficiency, adult human beta cells have proven recalcitrant to such efforts1-8. Hence, there remains an urgent need for diabetes therapeutic agents that can induce regeneration and expansion of adult human beta cells in vivo or ex vivo. Here, we report the results of a high-throughput small molecule screen (HTS) revealing a novel class of human beta cell mitogenic compounds, analogues of the small molecule, harmine. We also define dual specificity tyrosine-regulated kinase-1a (DYRK1A) as the likely target of harmine, and the Nuclear Factors of activated T-cells (NFAT) family of transcription factors as likely mediators of human beta cell proliferation as well as beta cell differentiation. These observations suggest that harmine analogues (“harmalogs”) may have unique therapeutic promise for human diabetes therapy. Enhancing potency and beta cell specificity are important future challenges. PMID:25751815

  18. Human iPSC-Derived Endothelial Cell Sprouting Assay in ...

    Science.gov (United States)

    Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by a lack of definition to the substratum and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). Thiol-ene photopolymerization was used to rapidly encapsulate iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres and subsequently to rapidly encapsulate iPSC-EC-containing hydrogel spheres in a cell-free over-layer. The hydrogel sprouting array here maintained pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. The sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors, which suggests the functional role of vascular endothelial growth factor, NF-κB, matrix metalloproteinase-2/9, protein kinase activity, and β-tubulin in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds (pVDCs) from the US Environmental Protection Agency’s ToxCast library identified five compounds th

  19. A gene expression signature classifying telomerase and ALT immortalization reveals an hTERT regulatory network and suggests a mesenchymal stem cell origin for ALT

    DEFF Research Database (Denmark)

    Lafferty-Whyte, K; Cairney, C J; Will, M B

    2009-01-01

    Telomere length is maintained by two known mechanisms, the activation of telomerase or alternative lengthening of telomeres (ALT). The molecular mechanisms regulating the ALT phenotype are poorly understood and it is unknown how the decision of which pathway to activate is made at the cellular le......TERT in different tumour types and normal tissues. We also show evidence to suggest a novel mesenchymal stem cell origin for ALT immortalization in cell lines and mesenchymal tissues....

  20. Cell-to-cell contact of human monocytes with infected arterial smooth-muscle cells enhances growth of Chlamydia pneumoniae.

    Science.gov (United States)

    Puolakkainen, Mirja; Campbell, Lee Ann; Lin, Tsun-Mei; Richards, Theresa; Patton, Dorothy L; Kuo, Cho-Chou

    2003-02-01

    Chlamydia pneumoniae can infect arterial cells. It has been shown that coculture of human monocytes (U937) and endothelial cells promotes infection of C. pneumoniae in endothelial cells and that the enhancement was mediated by a soluble factor (insulin-like growth factor 2) secreted by monocytes. In this study, it is shown that coculture of monocytes with C. pneumoniae enhances infection of C. pneumoniae in arterial smooth-muscle cells 5.3-fold at a monocyte-to-smooth-muscle cell ratio of 5. However, unlike endothelial cells, no enhancement was observed if monocytes were placed in cell culture inserts or if conditioned medium from monocyte cultures was used, which suggests that cell-to-cell contact is critical. The addition of mannose 6-phosphate or octyl glucoside, a nonionic detergent containing a sugar group, to cocultures inhibited the enhancement. These findings suggest that the monocyte-smooth-muscle cell interaction may be mediated by mannose 6-phosphate receptors present on monocytes.

  1. Vesicular uptake of macromolecules by human placental amniotic epithelial cells.

    Science.gov (United States)

    Sharshiner, Rita; Brace, Robert A; Cheung, Cecilia Y

    2017-09-01

    Studies in animal models have shown that unidirectional vesicular transport of amniotic fluid across the amnion plays a primary role in regulating amniotic fluid volume. Our objective was to explore vesicle type, vesicular uptake and intracellular distribution of vesicles in human amnion cells using high- and super-resolution fluorescence microscopy. Placental amnion was obtained at cesarean section and amnion cells were prepared and cultured. At 20%-50% confluence, the cells were incubated with fluorophore conjugated macromolecules for 1-30 min at 22 °C or 37 °C. Fluorophore labeled macromolecules were selected as markers of receptor-mediated caveolar and clathrin-coated vesicular uptake as well as non-specific endocytosis. After fluorophore treatment, the cells were fixed, imaged and vesicles counted using Imaris(®) software. Vesicular uptake displayed first order saturation kinetics with half saturation times averaging 1.3 min at 37 °C compared to 4.9 min at 22 °C, with non-specific endocytotic uptake being more rapid at both temperatures. There was extensive cell-to-cell variability in uptake rate. Under super-resolution microscopy, the pattern of intracellular spatial distribution was distinct for each macromolecule. Co-localization of fluorescently labeled macromolecules was very low at vesicular dimensions. In human placental amnion cells, 1) vesicular uptake of macromolecules is rapid, consistent with the concept that vesicular transcytosis across the amnion plays a role in the regulation of amniotic fluid volume; 2) uptake is temperature dependent and variable among individual cells; 3) the unique intracellular distributions suggest distinct functions for each vesicle type; 4) non-receptor mediated vesicular uptake may be a primary vesicular uptake mechanism. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. First insight into the kinome of human regulatory T cells.

    Directory of Open Access Journals (Sweden)

    Sebastian König

    Full Text Available Regulatory T cells (Tregs are essential for controlling peripheral tolerance by the active suppression of various immune cells including conventional T effector cells (Teffs. Downstream of the T cell receptor (TCR, more than 500 protein kinases encoded by the human genome have to be considered in signaling cascades regulating the activation of Tregs and Teffs, respectively. Following TCR engagement, Tregs posses a number of unique attributes, such as constitutive expression of Foxp3, hyporesponsiveness and poor cytokine production. Furthermore, recent studies showed that altered regulation of protein kinases is important for Treg function. These data indicate that signaling pathways in Tregs are distinctly organized and alterations at the level of protein kinases contribute to the unique Treg phenotype. However, kinase-based signaling networks in Tregs are poorly understood and necessitate further systematic characterization. In this study, we analyzed the differential expression of kinases in Tregs and Teffs by using a kinase-selective proteome strategy. In total, we revealed quantitative information on 185 kinases expressed in the human CD4(+ T cell subsets. The majority of kinases was equally abundant in both T cell subsets, but 11 kinases were differentially expressed in Tregs. Most strikingly, Tregs showed an altered expression of cell cycle kinases including CDK6. Quantitative proteomics generates first comparative insight into the kinase complements of the CD4(+ Teff and Treg subset. Treg-specific expression pattern of 11 protein kinases substantiate the current opinion that TCR-mediated signaling cascades are altered in Tregs and further suggests that Tregs exhibit significant specificities in cell-cycle control and progression.

  3. Specific binding of benzodiazepines to human breast cancer cell lines.

    Science.gov (United States)

    Beinlich, A; Strohmeier, R; Kaufmann, M; Kuhl, H

    1999-01-01

    Binding of [3H]Ro5-4864, a peripheral benzodiazepine receptor (PBR) agonist, to BT-20 human, estrogen- (ER) and progesterone- (PR) receptor negative breast cancer cells was characterized. It was found to be specific, dose-dependent and saturable with a single population of binding sites. Dissociation constant (K(D)) was 8.5 nM, maximal binding capacity (Bmax) 339 fM/10(6) cells. Ro5-4864 (IC50 17.3 nM) and PK 11195 (IC50 12.3 nM) were able to compete with [3H]Ro5-4864 for binding, indicating specificity of interaction with PBR. Diazepam was able to displace [3H]Ro5-4864 from binding only at high concentrations (>1 microM), while ODN did not compete for PBR binding. Thymidine-uptake assay showed a biphasic response of cell proliferation. While low concentrations (100 nM) of Ro5-4864, PK 11195 and diazepam increased cell growth by 10 to 20%, higher concentrations (10-100 microM) significantly inhibited cell proliferation. PK 11195, a potent PBR ligand, was able to attenuate growth of BT-20 cells stimulated by 100 nM Ro5-4864 and to reverse growth reduction caused by 1 and 10 microM Ro5-4864, but not by 50 microM and 100 microM. This indicates that the antimitotic activity of higher concentrations of Ro5-4864 is independent of PBR binding. It is suggested, that PBR are involved in growth regulation of certain human breast cancer cell lines, possibly by supplying proliferating cells with energy, as their endogenous ligand is a polypeptide transporting Acyl-CoA.

  4. Allergen-induced migration of human cells in allergic severe combined immunodeficiency mice.

    Science.gov (United States)

    Duez, C; Akoum, H; Marquillies, P; Cesbron, J Y; Tonnel, A B; Pestel, J

    1998-02-01

    Recently, we have shown that severe combined immunodeficiency (SCID) mice, intraperitoneally reconstituted with peripheral blood mononuclear cells (PBMC) from Dermatophagoides pteronyssinus (Dpt)-sensitive patients, produced human IgE and developed a pulmonary inflammatory-type reaction after exposure to allergen aerosol. In order to understand the potential mechanisms involved in the human cell migration in SCID mice, we analysed their phenotypic profile in the lungs, spleen and thymus, 2 months after Dpt inhalation. The human cell recruitment in these organs was found to be allergen-dependent as CD45+ human cells were only detected in hu-SCID mice after Dpt exposure. The composition of the pulmonary human T-cell infiltrate, preferentially memory (CD45RO), activated (human leucocyte antigen (HLA)-DR) and CD4+ cells, was similar to that described in asthmatic patients. However, CD20+ B cells were predominately recruited in the spleen and thymus and may be IgE-producing cells in the spleen. In the lungs, the percentage of human leucocytes expressing the alpha-chain of the lymphocyte function-associated antigen-1 (LFA-1) (CD11a) was higher than those of CD49d+ or CD54+ cells, in contrast to the spleen and thymus, suggesting a potential role of LFA-1 in the human cell migration towards SCID mice lung. In conclusion, this model could be useful in the study of factors implicated in the cellular migration towards the lymphoid organs during an allergic reaction.

  5. Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis

    Institute of Scientific and Technical Information of China (English)

    Lintao Wang; Yanyan Peng; Kaikai Shi; Haixiao Wang; Jianlei Lu; Yanli Li; Changyan Ma

    2015-01-01

    Recent studies have revealed that osthole,an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson,a traditional Chinese medicine,possesses anticancer activity.However,its effect on breast cancer cells so far has not been elucidated clearly.In the present study,we evaluated the effects of osthole on the proliferation,cell cycle and apoptosis of human breast cancer cells MDA-MB 435.We demonstrated that osthole is effective in inhibiting the proliferation of MDA-MB 435 cells,The mitochondrion-mediated apoptotic pathway was involved in apoptosis induced by osthole,as indicated by activation of caspase-9 and caspase-3 followed by PARP degradation.The mechanism underlying its effect on the induction of G1 phase arrest was due to the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression.Were observed taken together,these findings suggest that the anticancer efficacy of osthole is mediated via induction of cell cycle arrest and apoptosis in human breast cancer cells and osthole may be a potential chemotherapeutic agent against human breast cancer.

  6. Lung endothelial cells strengthen, but brain endothelial cells weaken barrier properties of a human alveolar epithelium cell culture model.

    Science.gov (United States)

    Neuhaus, Winfried; Samwer, Fabian; Kunzmann, Steffen; Muellenbach, Ralf M; Wirth, Michael; Speer, Christian P; Roewer, Norbert; Förster, Carola Y

    2012-11-01

    The blood-air barrier in the lung consists of the alveolar epithelium, the underlying capillary endothelium, their basement membranes and the interstitial space between the cell layers. Little is known about the interactions between the alveolar and the blood compartment. The aim of the present study was to gain first insights into the possible interplay between these two neighbored cell layers. We established an in vitro Transwell model of the alveolar epithelium based on human cell line H441 and investigated the influence of conditioned medium obtained from human lung endothelial cell line HPMEC-ST1.6R on the barrier properties of the H441 layers. As control for tissue specificity H441 layers were exposed to conditioned medium from human brain endothelial cell line hCMEC/D3. Addition of dexamethasone was necessary to obtain stable H441 cell layers. Moreover, dexamethasone increased expression of cell type I markers (caveolin-1, RAGE) and cell type II marker SP-B, whereas decreased the transepithelial electrical resistance (TEER) in a concentration dependent manner. Soluble factors obtained from the lung endothelial cell line increased the barrier significantly proven by TEER values and fluorescein permeability on the functional level and by the differential expression of tight junctional proteins on the molecular level. In contrast to this, soluble factors derived from brain endothelial cells weakened the barrier significantly. In conclusion, soluble factors from lung endothelial cells can strengthen the alveolar epithelium barrier in vitro, which suggests communication between endothelial and epithelial cells regulating the integrity of the blood-air barrier.

  7. Introduction: characterization and functions of human T regulatory cells.

    Science.gov (United States)

    Romagnani, Sergio

    2005-06-01

    The field of human T regulatory (Treg) cells is a rapidly progressing, but still confused field of immunology. The effects of dendritic cell (DC) manipulation in Treg generation and the main features of human "natural" Treg cells, as well as of different populations of adaptive Treg subsets, are still partially unclear. However, it is clear that Treg cells play an important role in human diseases, such as autoimmune disorders, allergy, HIV infection, tumors and graft-versus-host disease.

  8. Human pancreatic cell autotransplantation following total pancreatectomy.

    Science.gov (United States)

    Traverso, L W; Abou-Zamzam, A M; Longmire, W P

    1981-01-01

    During total pancreaticoduodenectomy for chronic pancreatitis, four patients received an intraportal pancreatic mixed-cell autograft prepared by collagenase digestion. The technique of this autotransplantation procedure was successfully developed using a normal canine pancreas, but has proved difficult to apply in the human chronic pancreatitis model. Our four patients became insulin-dependent, with proof of intrahepatic insulin production in only one patient. Three factors have contributed to the lack of graft success: 1) the preoperative endocrine status, 2) systemic hypotension and portal hypertension secondary to graft infusion, and 3) difficulty applying the successful technique in a normal dog pancreas to an extensively scarred human pancreas. The preoperative insulin response during a glucose tolerance test was blunted or delayed in the three patients tested. An immediate decrease in blood pressure and rise in portal pressure occurred in every patient and prevented infusion of the entire graft (30-50%) in three patients. Unfortunately, the patient with the most compromised insulin status was the only patient able to receive the entire graft. Our experience would indicate that further refinements in technique are necessary to prevent the vascular reaction and allow infusion of the entire graft. Furthermore, normal islet cell function is necessary before a successful graft can be expected. PMID:6781424

  9. Flowers of Camellia nitidissima cause growth inhibition, cell-cycle dysregulation and apoptosis in a human esophageal squamous cell carcinoma cell line

    Science.gov (United States)

    Dai, Lu; Li, Ji-Lin; Liang, Xin-Qiang; Li, Lin; Feng, Yan; Liu, Hai-Zhou; Wei, Wen-Er; Ning, Shu-Fang; Zhang, Li-Tu

    2016-01-01

    The present study aimed to investigate the chemo-preventive effect of Camellia nitidissima flowers water extract (CNFE) on the Eca109 human esophageal squamous cell carcinoma (ESCC) cell line. The antiproliferative effect on Eca109 cells was determined using the trypan blue exclusion assay. The effects of CNFE on apoptosis and cell cycle arrest were investigated by flow cytometry. CNFE inhibited cell growth in both a dose- and time-dependent manner in Eca109 cells. CNFE also caused dose- and time-dependent apoptosis of these cells. Treatment of cells with CNFE resulted in dose-dependent G0/G1 phase arrest of the cell cycle. The data demonstrated that CNFE serves antiproliferative effects against human ESCC Eca109 cells by inducing apoptosis and interrupting the cell cycle. These results suggested that CNFE has the potential to be a chemoprotective agent for ESCC. PMID:27314447

  10. Human embryonic stem cell-derived mesenchymal stem cells as cellular delivery vehicles for prodrug gene therapy of glioblastoma.

    Science.gov (United States)

    Bak, Xiao Ying; Lam, Dang Hoang; Yang, Jingye; Ye, Kai; Wei, Esther Lee Xing; Lim, Sai Kiang; Wang, Shu

    2011-11-01

    Mesenchymal stem cells (MSCs) possess tumor-tropic properties and consequently have been used to deliver therapeutic agents for cancer treatment. Their potential in cancer therapy highlights the need for a consistent and renewable source for the production of uniform human MSCs suitable for clinical applications. In this study, we seek to investigate whether human embryonic stem cells can be used as a cell source to fulfill this goal. We generated MSC-like cells from two human embryonic stem cell lines, HuES9 and H1, and observed that MSC-like cells derived from human embryonic stem cells were able to migrate into human glioma intracranial xenografts after being injected into the cerebral hemisphere contralateral to the tumor inoculation site. We engineered these cells with baculoviral and lentiviral vectors, respectively, for transient and stable expression of the herpes simplex virus thymidine kinase gene. In tumor-bearing mice the engineered MSC-like cells were capable of inhibiting tumor growth and prolonging survival in the presence of ganciclovir after they were injected either directly into the xenografts or into the opposite hemisphere. Our findings suggest that human embryonic stem cell-derived MSCs may be a viable and attractive alternative for large-scale derivation of targeting vehicles for cancer therapy.

  11. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    OpenAIRE

    Mehta, Rajvi H.

    2014-01-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF...

  12. Iodide uptake in human anaplastic thyroid carcinoma cells after transfer of the human thyroid peroxidase gene

    Energy Technology Data Exchange (ETDEWEB)

    Haberkom, U. [Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center, Heidelberg (Germany); Dept. of Nuclear Medicine, Univ. of Heidelberg (Germany); Altmann, A.; Jiang, S.; Morr, I.; Mahmut, M. [Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center, Heidelberg (Germany); Eisenhut, M. [Dept. of Nuclear Medicine, Univ. of Heidelberg (Germany)

    2001-05-01

    Human thyroperoxidase (hTPO) is critical for the accumulation of iodide in thyroid tissues. Poorly differentiated and anaplastic thyroid tumours which lack thyroid-specific gene expression fail to accumulate iodide and, therefore, do not respond to iodine-131 therapy. We consequently investigated whether transfer of the hTPO gene is sufficient to restore the iodide-trapping capacity in undifferentiated thyroid and non-thyroid tumour cells. The human anaplastic thyroid carcinoma cell lines C643 and SW1736, the rat Morris hepatoma cell line MH3924A and the rat papillary thyroid carcinoma cell line L2 were used as in vitro model systems. Employing a bicistronic retroviral vector based on the myeloproliferative sarcoma virus for the transfer of the hTPO and the neomycin resistance gene, the C643 cells and SW1736 cells were transfected while the L2 cells and MH3924A cells were infected with retroviral particles. Seven recombinant C643 and seven SW1736 cell lines as well as four recombinant L2 and four MH3924A cell lines were established by neomycin selection. They were studied for hTPO expression using an antibody-based luminescence kit, followed by determination of the enzyme activity in the guaiacol assay and of the iodide uptake capacity in the presence of Na{sup 125}I. Genetically modified cell lines expressed up to 1,800 times more hTPO as compared to wild type tumour cells. The level of hTPO expression varied significantly between individual neomycin-resistant cell lines, suggesting that the recombinant retroviral DNA was integrated at different sites of the cellular genome. The accumulation of iodide, however, was not significantly enhanced in individual recombinant cell lines, irrespective of low or high hTPO expression. Moreover, there was no correlation between hTPO expression and enzyme activity in individual cell lines. The transduction of the hTPO gene per se is not sufficient to restore iodide trapping in non-iodide-concentrating tumour cells. Future

  13. Efficient Induction and Isolation of Human Primordial Germ Cell-Like Cells from Competent Human Pluripotent Stem Cells.

    Science.gov (United States)

    Irie, Naoko; Surani, M Azim

    2017-01-01

    We recently reported a robust and defined culture system for the specification of human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs), both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in vitro (Irie et al. Cell 160: 253-268, 2015). Similar attempts previously produced hPGCLCs from hPSCs at a very low efficiency, and the resulting cells were not fully characterized. A key step, which facilitated efficient hPGCLC specification from hPSCs, was the induction of a "competent" state for PGC fate via the medium containing a cocktail of four inhibitors. The competency of hPSCs can be maintained indefinitely and interchangeably with the conventional/low-competent hPSCs. Specification of hPGCLC occurs following sequential expression of key germ cell fate regulators, notably SOX17 and BLIMP1, as well as initiation of epigenetic resetting over 5 days. The hPGCLCs can be isolated using specific cell surface markers without the need for generating germ cell-specific reporter hPSC lines. This powerful method for the induction and isolation of hPGCLCs can be applied to both hESCs and iPSCs, which can be used for advances in human germ line biology.

  14. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells

    Institute of Scientific and Technical Information of China (English)

    Li-Wei Zheng; Logan Linthicum; Pamela K DenBesten; Yan Zhang

    2013-01-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCI) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which ,vas also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.

  15. Sequestration of human cytomegalovirus by human renal and mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Twite, Nicolas [Institute for Medical Immunology, Université Libre de Bruxelles, Rue A. Bolland 8, B-6041 Charleroi (Belgium); Andrei, Graciela [Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven (Belgium); Kummert, Caroline [ImmuneHealth, Rue A. Bolland 8, B-6041 Charleroi (Belgium); Donner, Catherine [Department of Obstetrics and Gynecology, Erasme Hospital, Route de Lennik 808, 1070 Brussels (Belgium); Perez-Morga, David [Laboratory of Molecular Parasitology, Institut de Biologie et Médecine Moléculaires, Université Libre de Bruxelles, Gosselies (Belgium); De Vos, Rita [Pathology Department, U.Z. Leuven, Minderbroedersstraat 12, Leuven (Belgium); Snoeck, Robert, E-mail: Robert.Snoeck@Rega.kuleuven.be [Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven (Belgium); Marchant, Arnaud, E-mail: arnaud.marchant@ulb.ac.be [Institute for Medical Immunology, Université Libre de Bruxelles, Rue A. Bolland 8, B-6041 Charleroi (Belgium); ImmuneHealth, Rue A. Bolland 8, B-6041 Charleroi (Belgium)

    2014-07-15

    Urine and breast milk represent the main routes of human cytomegalovirus (HCMV) transmission but the contribution of renal and mammary epithelial cells to viral excretion remains unclear. We observed that kidney and mammary epithelial cells were permissive to HCMV infection and expressed immediate early, early and late antigens within 72 h of infection. During the first 24 h after infection, high titers of infectious virus were measured associated to the cells and in culture supernatants, independently of de novo synthesis of virus progeny. This phenomenon was not observed in HCMV-infected fibroblasts and suggested the sequestration and the release of HCMV by epithelial cells. This hypothesis was supported by confocal and electron microscopy analyses. The sequestration and progressive release of HCMV by kidney and mammary epithelial cells may play an important role in the excretion of the virus in urine and breast milk and may thereby contribute to HCMV transmission. - Highlights: • Primary renal and mammary epithelial cells are permissive to HCMV infection. • HCMV is sequestered by epithelial cells and this phenomenon does not require viral replication. • HCMV sequestration by epithelial cells is reduced by antibodies and IFN-γ.

  16. Human embryonic stem cells as models for aneuploid chromosomal syndromes.

    Science.gov (United States)

    Biancotti, Juan-Carlos; Narwani, Kavita; Buehler, Nicole; Mandefro, Berhan; Golan-Lev, Tamar; Yanuka, Ofra; Clark, Amander; Hill, David; Benvenisty, Nissim; Lavon, Neta

    2010-09-01

    Syndromes caused by chromosomal aneuploidies are widely recognized genetic disorders in humans and often lead to spontaneous miscarriage. Preimplantation genetic screening is used to detect chromosomal aneuploidies in early embryos. Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may serve as models for human syndromes caused by aneuploidies. We have established 25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro development. The hESC lines exhibited morphology and expressed markers typical of hESCs. They demonstrated long-term proliferation capacity and pluripotent differentiation. Karyotype analysis revealed that two-third of the cell lines carry a normal euploid karyotype, while one-third remained aneuploid throughout the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X (Turner syndrome). On the basis of the level of single nucleotide polymorphism heterozygosity in the aneuploid chromosomes, we determined whether the aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene expression profiles of the trisomic cell lines suggested that all three chromosomes are actively transcribed. Our analysis allowed us to determine which tissues are most affected by the presence of a third copy of either chromosome 13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic development. The results presented here suggest that aneuploid embryos can serve as an alternative source for either normal euploid or aneuploid hESC lines, which represent an invaluable tool to study developmental aspects of chromosomal abnormalities in humans.

  17. Radiosensitivity of Human Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Bergoc, R. M.; Medina, V.; Cricco, G.; Mohamed, N.; Martin, G.; Nunez, M.; Croci, M.; Crescenti, E. J.; Rivera, E. S.

    2004-07-01

    Cutaneous melanoma is a skin cancer resulting from the malign transformation of skin-pigment cells, the melanocytes. The radiotherapy, alone or in combination with other treatment, is an important therapy for this disease. the objective of this paper was to determine in vitro the radiosensitivity of two human melanoma cell lines with different metastatic capability: WM35 and MI/15, and to study the effect of drugs on radiobiological parameters. The Survival Curves were adjusted to the mathematical Linear-quadratic model using GrapsPad Prism software. Cells were seeded in RPMI medium (3000-3500 cells/flask), in triplicate and irradiated 24 h later. The irradiation was performed using an IBL 437C H Type equipment (189 TBq, 7.7 Gy/min) calibrated with a TLD 700 dosimeter. The range of Doses covered from 0 to 10 Gy and the colonies formed were counted at day 7th post-irradiation. Results obtained were: for WM35, {alpha}=0.37{+-}0.07 Gy''-1 and {beta}=0.06{+-}0.02 Gy''-2, for M1/15m {alpha}=0.47{+-}0.03 Gy''-1 and {beta}=0.06{+-}0.01 Gy''-2. The {alpha}/{beta} values WM35: {alpha}/{beta} values WM35: {alpha}/{beta}=6.07 Gy and M1/15: {alpha}/{beta}{sub 7}.33 Gy were similar, independently of their metastatic capabillity and indicate that both lines exhibit high radioresistance. Microscopic observation of irradiated cells showed multinuclear cells with few morphologic changes non-compatible with apoptosis. By means of specific fluorescent dyes and flow cytometry analysis we determined the intracellular levels of the radicals superoxide and hydrogen peroxide and their modulation in response to ionizing radiation. The results showed a marked decreased in H{sub 2}O{sub 2} intracellular levels with a simultaneous increase in superoxide that will be part of a mechanism responsible for induction of cell radioresistance. This response triggered by irradiated cells could not be abrogated by different treatments like histamine or the

  18. Necdin modulates proliferative cell survival of human cells in response to radiation-induced genotoxic stress

    Directory of Open Access Journals (Sweden)

    Lafontaine Julie

    2012-06-01

    Full Text Available Abstract Background The finite replicative lifespan of cells, termed cellular senescence, has been proposed as a protective mechanism against the proliferation of oncogenically damaged cells, that fuel cancer. This concept is further supported by the induction of premature senescence, a process which is activated when an oncogene is expressed in normal primary cells as well as following intense genotoxic stresses. Thus, deregulation of genes that control this process, like the tumor suppressor p53, may contribute to promoting cancer by allowing cells to bypass senescence. A better understanding of the genes that contribute to the establishment of senescence is therefore warranted. Necdin interacts with p53 and is also a p53 target gene, although the importance of Necdin in the p53 response is not clearly understood. Methods In this study, we first investigated Necdin protein expression during replicative senescence and premature senescence induced by gamma irradiation and by the overexpression of oncogenic RasV12. Gain and loss of function experiments were used to evaluate the contribution of Necdin during the senescence process. Results Necdin expression declined during replicative aging of IMR90 primary human fibroblasts or following induction of premature senescence. Decrease in Necdin expression seemed to be a consequence of the establishment of senescence since the depletion of Necdin in human cells did not induce a senescence-like growth arrest nor a flat morphology or SA-β-galactosidase activity normally associated with senescence. Similarly, overexpression of Necdin did not affect the life span of IMR90 cells. However, we demonstrate that in normal human cells, Necdin expression mimicked the effect of p53 inactivation by increasing radioresistance. Conclusion This result suggests that Necdin potentially attenuate p53 signaling in response to genotoxic stress in human cells and supports similar results describing an inhibitory function

  19. Arsenic trioxide inhibits cell proliferation and human papillomavirus oncogene expression in cervical cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hongtao [Department of Pathology, School of Medicine, Southeast University, Nanjing 210009 (China); Gao, Peng [Department of Internal Medicine, University of Iowa, Iowa City, IA 52242 (United States); Zheng, Jie, E-mail: jiezheng54@126.com [Department of Pathology, School of Medicine, Southeast University, Nanjing 210009 (China)

    2014-09-05

    Highlights: • As{sub 2}O{sub 3} inhibits growth of cervical cancer cells and expression of HPV oncogenes in these cells. • HPV-negative cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-positive cervical cancer cells. • HPV-18 positive cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-16 positive cancer cells. • Down-regulation of HPV oncogenes by As{sub 2}O{sub 3} is partially due to the diminished AP-1 binding. - Abstract: Arsenic trioxide (As{sub 2}O{sub 3}) has shown therapeutic effects in some leukemias and solid cancers. However, the molecular mechanisms of its anticancer efficacy have not been clearly elucidated, particularly in solid cancers. Our previous data showed that As{sub 2}O{sub 3} induced apoptosis of human papillomavirus (HPV) 16 DNA-immortalized human cervical epithelial cells and cervical cancer cells and inhibited the expression of HPV oncogenes in these cells. In the present study, we systemically examined the effects of As{sub 2}O{sub 3} on five human cervical cancer cell lines and explored the possible molecular mechanisms. MTT assay showed that HPV-negative C33A cells were more sensitive to growth inhibition induced by As{sub 2}O{sub 3} than HPV-positive cervical cancer cells, and HPV 18-positive HeLa and C4-I cells were more sensitive to As{sub 2}O{sub 3} than HPV 16-positive CaSki and SiHa cells. After As{sub 2}O{sub 3} treatment, both mRNA and protein levels of HPV E6 and E7 obviously decreased in all HPV positive cell lines. In contrast, p53 and Rb protein levels increased in all tested cell lines. Transcription factor AP-1 protein expression decreased significantly in HeLa, CaSki and C33A cells with ELISA method. These results suggest that As{sub 2}O{sub 3} is a potential anticancer drug for cervical cancer.

  20. Generation of human induced pluripotent stem cells from dermal fibroblasts.

    Science.gov (United States)

    Lowry, W E; Richter, L; Yachechko, R; Pyle, A D; Tchieu, J; Sridharan, R; Clark, A T; Plath, K

    2008-02-26

    The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. Technologies including somatic cell nuclear transfer and cell fusion might generate such cells but are hindered by issues that might prevent them from being used clinically. Here, we describe methods to use dermal fibroblasts easily obtained from an individual human to generate human induced pluripotent stem (iPS) cells by ectopic expression of the defined transcription factors KLF4, OCT4, SOX2, and C-MYC. The resultant cell lines are morphologically indistinguishable from human embryonic stem cells (HESC) generated from the inner cell mass of a human preimplantation embryo. Consistent with these observations, human iPS cells share a nearly identical gene-expression profile with two established HESC lines. Importantly, DNA fingerprinting indicates that the human iPS cells were derived