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Sample records for human cells harboring

  1. Human Lymphoid Tissues Harbor a Distinct CD69+CXCR6+ NK Cell Population.

    Science.gov (United States)

    Lugthart, Gertjan; Melsen, Janine E; Vervat, Carly; van Ostaijen-Ten Dam, Monique M; Corver, Willem E; Roelen, Dave L; van Bergen, Jeroen; van Tol, Maarten J D; Lankester, Arjan C; Schilham, Marco W

    2016-07-01

    Knowledge of human NK cells is based primarily on conventional CD56(bright) and CD56(dim) NK cells from blood. However, most cellular immune interactions occur in lymphoid organs. Based on the coexpression of CD69 and CXCR6, we identified a third major NK cell subset in lymphoid tissues. This population represents 30-60% of NK cells in marrow, spleen, and lymph node but is absent from blood. CD69(+)CXCR6(+) lymphoid tissue NK cells have an intermediate expression of CD56 and high expression of NKp46 and ICAM-1. In contrast to circulating NK cells, they have a bimodal expression of the activating receptor DNAX accessory molecule 1. CD69(+)CXCR6(+) NK cells do not express the early markers c-kit and IL-7Rα, nor killer cell Ig-like receptors or other late-differentiation markers. After cytokine stimulation, CD69(+)CXCR6(+) NK cells produce IFN-γ at levels comparable to CD56(dim) NK cells. They constitutively express perforin but require preactivation to express granzyme B and exert cytotoxicity. After hematopoietic stem cell transplantation, CD69(+)CXCR6(+) lymphoid tissue NK cells do not exhibit the hyperexpansion observed for both conventional NK cell populations. CD69(+)CXCR6(+) NK cells constitute a separate NK cell population with a distinct phenotype and function. The identification of this NK cell population in lymphoid tissues provides tools to further evaluate the cellular interactions and role of NK cells in human immunity.

  2. Generation of GFP Reporter Human Induced Pluripotent Stem Cells Using AAVS1 Safe Harbor Transcription Activator-Like Effector Nuclease.

    Science.gov (United States)

    Luo, Yongquan; Rao, Mahendra; Zou, Jizhong

    2014-05-16

    Generation of a fluorescent GFP reporter line in human induced pluripotent stem cells (hiPSCs) provides enormous potentials in both basic stem cell research and regenerative medicine. A protocol for efficiently generating such an engineered reporter line by gene targeting is highly desired. Transcription activator-like effector nucleases (TALENs) are a new class of artificial restriction enzymes that have been shown to significantly promote homologous recombination by >1000-fold. The AAVS1 (adeno-associated virus integration site 1) locus is a "safe harbor" and has an open chromatin structure that allows insertion and stable expression of transgene. Here, we describe a step-by-step protocol from determination of TALENs activity, hiPSC culture, and delivery of a donor into AAVS1 targeting site, to validation of targeted integration by PCR and Southern blot analysis using hiPSC line, and a pair of open-source AAVS1 TALENs.

  3. Human bone marrow harbors cells with neural crest-associated characteristics like human adipose and dermis tissues.

    Science.gov (United States)

    Coste, Cécile; Neirinckx, Virginie; Sharma, Anil; Agirman, Gulistan; Rogister, Bernard; Foguenne, Jacques; Lallemend, François; Gothot, André; Wislet, Sabine

    2017-01-01

    Adult neural crest stem-derived cells (NCSC) are of extraordinary high plasticity and promising candidates for use in regenerative medicine. Several locations such as skin, adipose tissue, dental pulp or bone marrow have been described in rodent, as sources of NCSC. However, very little information is available concerning their correspondence in human tissues, and more precisely for human bone marrow. The main objective of this study was therefore to characterize NCSC from adult human bone marrow. In this purpose, we compared human bone marrow stromal cells to human adipose tissue and dermis, already described for containing NCSC. We performed comparative analyses in terms of gene and protein expression as well as functional characterizations. It appeared that human bone marrow, similarly to adipose tissue and dermis, contains NESTIN+ / SOX9+ / TWIST+ / SLUG+ / P75NTR+ / BRN3A+/ MSI1+/ SNAIL1+ cells and were able to differentiate into melanocytes, Schwann cells and neurons. Moreover, when injected into chicken embryos, all those cells were able to migrate and follow endogenous neural crest migration pathways. Altogether, the phenotypic characterization and migration abilities strongly suggest the presence of neural crest-derived cells in human adult bone marrow.

  4. Perivascular Mesenchymal Stem Cells From the Adult Human Brain Harbor No Instrinsic Neuroectodermal but High Mesodermal Differentiation Potential.

    Science.gov (United States)

    Lojewski, Xenia; Srimasorn, Sumitra; Rauh, Juliane; Francke, Silvan; Wobus, Manja; Taylor, Verdon; Araúzo-Bravo, Marcos J; Hallmeyer-Elgner, Susanne; Kirsch, Matthias; Schwarz, Sigrid; Schwarz, Johannes; Storch, Alexander; Hermann, Andreas

    2015-10-01

    Brain perivascular cells have recently been identified as a novel mesodermal cell type in the human brain. These cells reside in the perivascular niche and were shown to have mesodermal and, to a lesser extent, tissue-specific differentiation potential. Mesenchymal stem cells (MSCs) are widely proposed for use in cell therapy in many neurological disorders; therefore, it is of importance to better understand the "intrinsic" MSC population of the human brain. We systematically characterized adult human brain-derived pericytes during in vitro expansion and differentiation and compared these cells with fetal and adult human brain-derived neural stem cells (NSCs) and adult human bone marrow-derived MSCs. We found that adult human brain pericytes, which can be isolated from the hippocampus and from subcortical white matter, are-in contrast to adult human NSCs-easily expandable in monolayer cultures and show many similarities to human bone marrow-derived MSCs both regarding both surface marker expression and after whole transcriptome profile. Human brain pericytes showed a negligible propensity for neuroectodermal differentiation under various differentiation conditions but efficiently generated mesodermal progeny. Consequently, human brain pericytes resemble bone marrow-derived MSCs and might be very interesting for possible autologous and endogenous stem cell-based treatment strategies and cell therapeutic approaches for treating neurological diseases. Perivascular mesenchymal stem cells (MSCs) recently gained significant interest because of their appearance in many tissues including the human brain. MSCs were often reported as being beneficial after transplantation in the central nervous system in different neurological diseases; therefore, adult brain perivascular cells derived from human neural tissue were systematically characterized concerning neural stem cell and MSC marker expression, transcriptomics, and mesodermal and inherent neuroectodermal differentiation

  5. The human vascular endothelial cell line HUV-EC-C harbors the integrated HHV-6B genome which remains stable in long term culture.

    Science.gov (United States)

    Shioda, Setsuko; Kasai, Fumio; Ozawa, Midori; Hirayama, Noriko; Satoh, Motonobu; Kameoka, Yousuke; Watanabe, Ken; Shimizu, Norio; Tang, Huamin; Mori, Yasuko; Kohara, Arihiro

    2017-07-28

    Human herpes virus 6 (HHV-6) is a common human pathogen that is most often detected in hematopoietic cells. Although human cells harboring chromosomally integrated HHV-6 can be generated in vitro, the availability of such cell lines originating from in vivo tissues is limited. In this study, chromosomally integrated HHV-6B has been identified in a human vascular endothelial cell line, HUV-EC-C (IFO50271), derived from normal umbilical cord tissue. Sequence analysis revealed that the viral genome was similar to the HHV-6B HST strain. FISH analysis using a HHV-6 DNA probe showed one signal in each cell, detected at the distal end of the long arm of chromosome 9. This was consistent with a digital PCR assay, validating one copy of the viral DNA. Because exposure of HUV-EC-C to chemicals did not cause viral reactivation, long term cell culture of HUV-EC-C was carried out to assess the stability of viral integration. The growth rate was altered depending on passage numbers, and morphology also changed during culture. SNP microarray profiles showed some differences between low and high passages, implying that the HUV-EC-C genome had changed during culture. However, no detectable change was observed in chromosome 9, where HHV-6B integration and the viral copy number remained unchanged. Our results suggest that integrated HHV-6B is stable in HUV-EC-C despite genome instability.

  6. Annexin A2 promotes the migration and invasion of human hepatocellular carcinoma cells in vitro by regulating the shedding of CD147-harboring microvesicles from tumor cells.

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    Full Text Available It has been reported that Annexin A2 (ANXA2 is up-regulated in hepatocellular carcinoma (HCC, but the roles of ANXA2 in the migration and invasion of HCC cells have not been determined. In this study, we found that ANXA2-specific siRNA (si-ANXA2 significantly inhibited the migration and invasion of HCC cells co-cultured with fibroblasts in vitro. In addition, the production of MMP-2 by fibroblasts cultured in supernatant collected from si-ANXA2-transfected HCC cells was notably down-regulated. ANXA2 was also found to be co-localized and co-immunoprecipitated with CD147. Further investigation revealed that the expression of ANXA2 in HCC cells affected the shedding of CD147-harboring membrane microvesicles, acting as a vehicle for CD147 in tumor-stromal interactions and thereby regulating the production of MMP-2 by fibroblasts. Together, these results suggest that ANXA2 enhances the migration and invasion potential of HCC cells in vitro by regulating the trafficking of CD147-harboring membrane microvesicles.

  7. Annexin A2 Promotes the Migration and Invasion of Human Hepatocellular Carcinoma Cells In Vitro by Regulating the Shedding of CD147-Harboring Microvesicles from Tumor Cells

    Science.gov (United States)

    Cai, Lei; Song, Zhen-Shun; Cao, Da-Yong; Tao, Kai-Shan; Zhou, Wen-Ping; Chen, Zhi-Nan; Dou, Ke-Feng

    2013-01-01

    It has been reported that Annexin A2 (ANXA2) is up-regulated in hepatocellular carcinoma (HCC), but the roles of ANXA2 in the migration and invasion of HCC cells have not been determined. In this study, we found that ANXA2-specific siRNA (si-ANXA2) significantly inhibited the migration and invasion of HCC cells co-cultured with fibroblasts in vitro. In addition, the production of MMP-2 by fibroblasts cultured in supernatant collected from si-ANXA2-transfected HCC cells was notably down-regulated. ANXA2 was also found to be co-localized and co-immunoprecipitated with CD147. Further investigation revealed that the expression of ANXA2 in HCC cells affected the shedding of CD147-harboring membrane microvesicles, acting as a vehicle for CD147 in tumor-stromal interactions and thereby regulating the production of MMP-2 by fibroblasts. Together, these results suggest that ANXA2 enhances the migration and invasion potential of HCC cells in vitro by regulating the trafficking of CD147-harboring membrane microvesicles. PMID:23950866

  8. Identification of hepatic niche harboring human acute lymphoblastic leukemic cells via the SDF-1/CXCR4 axis.

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    Itaru Kato

    Full Text Available In acute lymphoblastic leukemia (ALL patients, the bone marrow niche is widely known to be an important element of treatment response and relapse. Furthermore, a characteristic liver pathology observed in ALL patients implies that the hepatic microenvironment provides an extramedullary niche for leukemic cells. However, it remains unclear whether the liver actually provides a specific niche. The mechanism underlying this pathology is also poorly understood. Here, to answer these questions, we reconstituted the histopathology of leukemic liver by using patients-derived primary ALL cells into NOD/SCID/Yc (null mice. The liver pathology in this model was similar to that observed in the patients. By using this model, we clearly demonstrated that bile duct epithelial cells form a hepatic niche that supports infiltration and proliferation of ALL cells in the liver. Furthermore, we showed that functions of the niche are maintained by the SDF-1/CXCR4 axis, proposing a novel therapeutic approach targeting the extramedullary niche by inhibition of the SDF-1/CXCR4 axis. In conclusion, we demonstrated that the liver dissemination of leukemia is not due to nonselective infiltration, but rather systematic invasion and proliferation of leukemic cells in hepatic niche. Although the contribution of SDF-1/CXCR4 axis is reported in some cancer cells or leukemic niches such as bone marrow, we demonstrated that this axis works even in the extramedullary niche of leukemic cells. Our findings form the basis for therapeutic approaches that target the extramedullary niche by inhibiting the SDF-1/CXCR4 axis.

  9. Orange County Littoral Cell CRSMP Harbor Receiver Sites 2012

    Data.gov (United States)

    California Department of Resources — Harbor reciever sites from Everest (2009) 'Harbor Area Management Plan, In-Harbor Beach Replenishment Strategy', Technical Report. Prepared for Harbor Resources...

  10. A regimen combining the Wee1 inhibitor AZD1775 with HDAC inhibitors targets human acute myeloid leukemia cells harboring various genetic mutations.

    Science.gov (United States)

    Zhou, L; Zhang, Y; Chen, S; Kmieciak, M; Leng, Y; Lin, H; Rizzo, K A; Dumur, C I; Ferreira-Gonzalez, A; Dai, Y; Grant, S

    2015-04-01

    AZD1775 targets the cell cycle checkpoint kinase Wee1 and potentiates genotoxic agent cytotoxicity through p53-dependent or -independent mechanisms. Here, we report that AZD1775 interacted synergistically with histone deacetylase inhibitors (HDACIs, for example, Vorinostat), which interrupt the DNA damage response, to kill p53-wild type (wt) or -deficient as well as FLT3-ITD leukemia cells in association with pronounced Wee1 inhibition and diminished cdc2/Cdk1 Y15 phosphorylation. Similarly, Wee1 shRNA knockdown significantly sensitized cells to HDACIs. Although AZD1775 induced Chk1 activation, reflected by markedly increased Chk1 S296/S317/S345 phosphorylation leading to inhibitory T14 phosphorylation of cdc2/Cdk1, these compensatory responses were sharply abrogated by HDACIs. This was accompanied by premature mitotic entry, multiple mitotic abnormalities and accumulation of early S-phase cells displaying increased newly replicated DNA, culminating in robust DNA damage and apoptosis. The regimen was active against patient-derived acute myelogenous leukemia (AML) cells harboring either wt or mutant p53 and various next-generation sequencing-defined mutations. Primitive CD34(+)/CD123(+)/CD38(-) populations enriched for leukemia-initiating progenitors, but not normal CD34(+) hematopoietic cells, were highly susceptible to this regimen. Finally, combining AZD1775 with Vorinostat in AML murine xenografts significantly reduced tumor burden and prolonged animal survival. A strategy combining Wee1 with HDACI inhibition warrants further investigation in AML with poor prognostic genetic aberrations.

  11. Codon optimization of the human papillomavirus E7 oncogene induces a CD8+ T cell response to a cryptic epitope not harbored by wild-type E7.

    Directory of Open Access Journals (Sweden)

    Felix K M Lorenz

    Full Text Available Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine.

  12. Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

    Science.gov (United States)

    Fouladi, B.; Waldren, C. A.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (Lobrich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.

  13. Rat full term amniotic fluid harbors highly potent stem cells.

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    Mun-Fun, Hoo; Ferdaos, Nurfarhana; Hamzah, Siti Nurusaadah; Ridzuan, Noridzzaida; Hisham, Nurul Afiqah; Abdullah, Syahril; Ramasamy, Rajesh; Cheah, Pike See; Thilakavathy, Karrupiah; Yazid, Mohd Nazri; Nordin, Norshariza

    2015-10-01

    Amniotic fluid stem cells (AFSCs) are commonly isolated from mid-term amniotic fluid (AF) of animals and human collected via an invasive technique, amniocentesis. Alternatively, AFSCs could be collected at full-term. However, it is unclear whether AFSCs are present in the AF at full term. Here, we aimed to isolate and characterize stem cells isolated from AF of full term pregnant rats. Three stem cell lines have been established following immuno-selection against the stem cell marker, c-kit. Two of the new lines expressed multiple markers of pluripotency until more than passage 90. Further, they spontaneously differentiated into derivatives of the three primary germ layers through the formation of good quality embryoid bodies (EBs), and can be directly differentiated into neural lineage. Their strong stemness and potent neurogenic properties highlight the presence of highly potent stem cells in AF of full-term pregnancies, which could serve as a potential source of stem cells for regenerative medicine.

  14. Adaptive responses to dasatinib-treated lung squamous cell cancer cells harboring DDR2 mutations.

    Science.gov (United States)

    Bai, Yun; Kim, Jae-Young; Watters, January M; Fang, Bin; Kinose, Fumi; Song, Lanxi; Koomen, John M; Teer, Jamie K; Fisher, Kate; Chen, Yian Ann; Rix, Uwe; Haura, Eric B

    2014-12-15

    DDR2 mutations occur in approximately 4% of lung squamous cell cancer (SCC) where the tyrosine kinase inhibitor dasatinib has emerged as a new therapeutic option. We found that ERK and AKT phosphorylation was weakly inhibited by dasatinib in DDR2-mutant lung SCC cells, suggesting that dasatinib inhibits survival signals distinct from other oncogenic receptor tyrosine kinases (RTK) and/or compensatory signals exist that dampen dasatinib activity. To gain better insight into dasatinib's action in these cells, we assessed altered global tyrosine phosphorylation (pY) after dasatinib exposure using a mass spectrometry-based quantitative phosphoproteomics approach. Overlaying protein-protein interaction relationships upon this dasatinib-regulated pY network revealed decreased phosphorylation of Src family kinases and their targets. Conversely, dasatinib enhanced tyrosine phosphorylation in a panel of RTK and their signaling adaptor complexes, including EGFR, MET/GAB1, and IGF1R/IRS2, implicating a RTK-driven adaptive response associated with dasatinib. To address the significance of this observation, these results were further integrated with results from a small-molecule chemical library screen. We found that dasatinib combined with MET and insulin-like growth factor receptor (IGF1R) inhibitors had a synergistic effect, and ligand stimulation of EGFR and MET rescued DDR2-mutant lung SCC cells from dasatinib-induced loss of cell viability. Importantly, we observed high levels of tyrosine-phosphorylated EGFR and MET in a panel of human lung SCC tissues harboring DDR2 mutations. Our results highlight potential RTK-driven adaptive-resistant mechanisms upon DDR2 targeting, and they suggest new, rationale cotargeting strategies for DDR2-mutant lung SCC.

  15. Centrosomal and mitotic abnormalities in cell lines derived from papillary thyroid cancer harboring specific gene alterations

    National Research Council Canada - National Science Library

    Maric, Irena; Viaggi, Silvia; Caria, Paola; Frau, Daniela V; Degan, Paolo; Vanni, Roberta

    2011-01-01

    .... We investigated the centrosome status and mitotic abnormalities in three thyroid carcinoma-derived cell lines, each maintaining the specific, biologically relevant gene alteration harbored by the parental tumors...

  16. Non-Human Primates Harbor Diverse Mammalian and Avian Astroviruses Including Those Associated with Human Infections.

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    Erik A Karlsson

    Full Text Available Astroviruses (AstVs are positive sense, single-stranded RNA viruses transmitted to a wide range of hosts via the fecal-oral route. The number of AstV-infected animal hosts has rapidly expanded in recent years with many more likely to be discovered because of the advances in viral surveillance and next generation sequencing. Yet no study to date has identified human AstV genotypes in animals, although diverse AstV genotypes similar to animal-origin viruses have been found in children with diarrhea and in one instance of encephalitis. Here we provide important new evidence that non-human primates (NHP can harbor a wide variety of mammalian and avian AstV genotypes, including those only associated with human infection. Serological analyses confirmed that >25% of the NHP tested had antibodies to human AstVs. Further, we identified a recombinant AstV with parental relationships to known human AstVs. Phylogenetic analysis suggests AstVs in NHP are on average evolutionarily much closer to AstVs from other animals than are AstVs from bats, a frequently proposed reservoir. Our studies not only demonstrate that human astroviruses can be detected in NHP but also suggest that NHP are unique in their ability to support diverse AstV genotypes, further challenging the paradigm that astrovirus infection is species-specific.

  17. DINOFLAGELLATE CYST RECORD AND HUMAN DISTURBANCE IN NEW BEDFORD HARBOR, MA AND NARRAGANSETT BAY ESTUARIES

    Science.gov (United States)

    We studied the dinoflagellate cyst records in sediments from New Bedford Harbor and Apponagansett Bay over the last 350 yr provides to determine if cysts are sensitive to environmental change caused by human activity in the watershed. Changes in the total number, and absolute and...

  18. Pressure-natriuresis and -diuresis in transgenic rats harboring both human renin and human angiotensinogen genes.

    Science.gov (United States)

    Dehmel, B; Mervaala, E; Lippoldt, A; Gross, V; Bohlender, J; Ganten, D; Luft, F C

    1998-12-01

    The hypertensive double transgenic rat harboring both the human renin and human angiotensinogen genes (dTGR) offers a unique opportunity to study the human renin-angiotensin system in an experimental animal model. Since nothing is known about the control of sodium and water excretion in these rats, this study was performed to compare pressure-natriuresis relationships in hypertensive dTGR and normotensive control rats harboring only the human renin gene (hREN), in order to determine how the pressure-natriuresis relationship is reset in hypertensive dTGR. To differentiate between extrinsic and intrinsic renal mechanisms, experiments were performed with and without renal denervation, and with and without infusions of vasopressin, norepinephrine, 17-OH-corticosterone, and aldosterone. Human and rat angiotensinogen and renin mRNA expression were also determined. In hREN without controlled renal function, urine flow and sodium excretion increased from 13 to 169 microl/min per g kidney wet weight (kwt) and from 1 to 30 micromol/min per g kwt, respectively, as renal perfusion pressure was increased from 67 to 135 mmHg. Renal blood flow (RBF) and GFR ranged between 3 to 7 and 0.9 to 1.5 ml/min per g kwt. In dTGR, pressure-natriuresis-diuresis relationships were shifted approximately 40 mmHg rightward. RBF was lower in dTGR than in hREN; GFR was not different. In dTGR with neurohormonal factors controlled, RBF was decreased and pressure-natriuresis-diuresis curves were not different compared to dTGR curves without these interventions. By light microscopy, the kidneys of these 6-wk-old dTGR and hREN rats were normal and indistinguishable. Both human and rat renin and angiotensinogen mRNA were expressed in the kidneys of dTGR. The two renin mRNA were decreased in dTGR, indicating a physiologic downregulation of renin gene expression by high BP. It is concluded that the renal pressure-natriuresis mechanism is reset toward higher pressure levels in dTGR and participates in the

  19. Human case of bacteremia caused by Streptococcus canis sequence type 9 harboring the scm gene.

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    Taniyama, Daisuke; Abe, Yoshihiko; Sakai, Tetsuya; Kikuchi, Takahide; Takahashi, Takashi

    2017-01-01

    Streptococcus canis (Sc) is a zoonotic pathogen that is transferred mainly from companion animals to humans. One of the major virulence factors in Sc is the M-like protein encoded by the scm gene, which is involved in anti-phagocytic activities, as well as the recruitment of plasminogen to the bacterial surface in cooperation with enolase, and the consequent enhancement of bacterial transmigration and survival. This is the first reported human case of uncomplicated bacteremia following a dog bite, caused by Streptococcus canis harboring the scm gene. The similarity of the 16S rRNA from the infecting species to that of the Sc type strain, as well as the amplification of the species-specific cfg gene, encoding a co-hemolysin, was used to confirm the species identity. Furthermore, the isolate was confirmed as sequence type 9. The partial scm gene sequence harbored by the isolate was closely related to those of other two Sc strains. While this isolate did not possess the erm(A), erm(B), or mef(A), macrolide/lincosamide resistance genes, it was not susceptible to azithromycin: its susceptibility was intermediate. Even though human Sc bacteremia is rare, clinicians should be aware of this microorganism, as well as Pasteurella sp., Prevotella sp., and Capnocytophaga sp., when examining and treating patients with fever who maintain close contact with companion animals.

  20. Brown adipose tissue harbors a distinct sub-population of regulatory T cells.

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    Dasa Medrikova

    Full Text Available Regulatory T (Treg cells are critical determinants of both immune responses and metabolic control. Here we show that systemic ablation of Treg cells compromised the adaptation of whole-body energy expenditure to cold exposure, correlating with impairment in thermogenic marker gene expression and massive invasion of pro-inflammatory macrophages in brown adipose tissue (BAT. Indeed, BAT harbored a unique sub-set of Treg cells characterized by a unique gene signature. As these Treg cells respond to BAT activation upon cold exposure, this study defines a BAT-specific Treg sub-set with direct implications for the regulation of energy homeostasis in response to environmental stress.

  1. Transcriptome profiling identifies genes and pathways deregulated upon floxuridine treatment in colorectal cancer cells harboring GOF mutant p53

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    Arindam Datta

    2016-06-01

    Full Text Available Mutation in TP53 is a common genetic alteration in human cancers. Certain tumor associated p53 missense mutants acquire gain-of-function (GOF properties and confer oncogenic phenotypes including enhanced chemoresistance. The colorectal cancers (CRC harboring mutant p53 are generally aggressive in nature and difficult to treat. To identify a potential gene expression signature of GOF mutant p53-driven acquired chemoresistance in CRC, we performed transcriptome profiling of floxuridine (FUdR treated SW480 cells expressing mutant p53R273H (GEO#: GSE77533. We obtained several genes differentially regulated between FUdR treated and untreated cells. Further, functional characterization and pathway analysis revealed significant enrichment of crucial biological processes and pathways upon FUdR treatment in SW480 cells. Our data suggest that in response to chemotherapeutics treatment, cancer cells with GOF mutant p53 can modulate key cellular pathways to withstand the cytotoxic effect of the drugs. The genes and pathways identified in the present study can be further validated and targeted for better chemotherapy response in colorectal cancer patients harboring mutant p53.

  2. Oral keratinocytes support non-replicative infection and transfer of harbored HIV-1 to permissive cells

    Directory of Open Access Journals (Sweden)

    Giacaman Rodrigo A

    2008-07-01

    Full Text Available Abstract Background Oral keratinocytes on the mucosal surface are frequently exposed to HIV-1 through contact with infected sexual partners or nursing mothers. To determine the plausibility that oral keratinocytes are primary targets of HIV-1, we tested the hypothesis that HIV-1 infects oral keratinocytes in a restricted manner. Results To study the fate of HIV-1, immortalized oral keratinocytes (OKF6/TERT-2; TERT-2 cells were characterized for the fate of HIV-specific RNA and DNA. At 6 h post inoculation with X4 or R5-tropic HIV-1, HIV-1gag RNA was detected maximally within TERT-2 cells. Reverse transcriptase activity in TERT-2 cells was confirmed by VSV-G-mediated infection with HIV-NL4-3Δenv-EGFP. AZT inhibited EGFP expression in a dose-dependent manner, suggesting that viral replication can be supported if receptors are bypassed. Within 3 h post inoculation, integrated HIV-1 DNA was detected in TERT-2 cell nuclei and persisted after subculture. Multiply spliced and unspliced HIV-1 mRNAs were not detectable up to 72 h post inoculation, suggesting that HIV replication may abort and that infection is non-productive. Within 48 h post inoculation, however, virus harbored by CD4 negative TERT-2 cells trans infected co-cultured peripheral blood mononuclear cells (PBMCs or MOLT4 cells (CD4+ CCR5+ by direct cell-to-cell transfer or by releasing low levels of infectious virions. Primary tonsil epithelial cells also trans infected HIV-1 to permissive cells in a donor-specific manner. Conclusion Oral keratinocytes appear, therefore, to support stable non-replicative integration, while harboring and transmitting infectious X4- or R5-tropic HIV-1 to permissive cells for up to 48 h.

  3. DINOFLAGELLATE CYST RECORDS AND HUMAN DISTURBANCE IN TWO NEIGHBORING ESTUARIES, NEW BEDFORD HARBOR AND APPONAGANSETT BAY, MASSACHUSETTS, USA

    Science.gov (United States)

    The dinoflagellate cyst records in sediments from New Bedford Harbor and Apponagansett Bay demonstrate sensitivity to environmental change caused by human activity in the watersheds over the last 500 years. Changes in the species richness, as well as absolute and relative abundan...

  4. Antitumor effect of afatinib, as a human epidermal growth factor receptor 2-targeted therapy, in lung cancers harboring HER2 oncogene alterations.

    Science.gov (United States)

    Suzawa, Ken; Toyooka, Shinichi; Sakaguchi, Masakiyo; Morita, Mizuki; Yamamoto, Hiromasa; Tomida, Shuta; Ohtsuka, Tomoaki; Watanabe, Mototsugu; Hashida, Shinsuke; Maki, Yuho; Soh, Junichi; Asano, Hiroaki; Tsukuda, Kazunori; Miyoshi, Shinichiro

    2016-01-01

    Human epidermal growth factor receptor 2 (HER2) is a member of the HER family of proteins containing four receptor tyrosine kinases. It plays an important role in the pathogenesis of certain human cancers. In non-small-cell lung cancer (NSCLC), HER2 amplification or mutations have been reported. However, little is known about the benefit of HER2-targeted therapy for NSCLCs harboring HER2 alterations. In this study, we investigated the antitumor effect of afatinib, an irreversible epidermal growth factor receptor (EGFR)-HER2 dual inhibitor, in lung cancers harboring HER2 oncogene alterations, including novel HER2 mutations in the transmembrane domain, which we recently identified. Normal bronchial epithelial cells, BEAS-2B, ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, and G660D) showed constitutive autophosphorylation of HER2 and activation of downstream signaling. They were sensitive to afatinib, but insensitive to gefitinib. Furthermore, we examined the antitumor activity of afatinib and gefitinib in several NSCLC cell lines, and investigated the association between their genetic alterations and sensitivity to afatinib treatment. In HER2-altered NSCLC cells (H2170, Calu-3, and H1781), afatinib downregulated the phosphorylation of HER2 and EGFR as well as their downstream signaling, and induced an antiproliferative effect through G1 arrest and apoptotic cell death. In contrast, HER2- or EGFR-non-dependent NSCLC cells were insensitive to afatinib. In addition, these effects were confirmed in vivo by using a xenograft mouse model of HER2-altered lung cancer cells. Our results suggest that afatinib is a therapeutic option as a HER2-targeted therapy for NSCLC harboring HER2 amplification or mutations.

  5. Antitumor Efficacy of the Dual PI3K/mTOR Inhibitor PF-04691502 in a Human Xenograft Tumor Model Derived from Colorectal Cancer Stem Cells Harboring a PIK3CA Mutation.

    Directory of Open Access Journals (Sweden)

    Douglas D Fang

    Full Text Available PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide mutations can help predict the antitumor activity of phosphatidylinositol-3-kinase (PI3K/mammalian target of rapamycin (mTOR pathway inhibitors in both preclinical and clinical settings. In light of the recent discovery of tumor-initiating cancer stem cells (CSCs in various tumor types, we developed an in vitro CSC model from xenograft tumors established in mice from a colorectal cancer patient tumor in which the CD133+/EpCAM+ population represented tumor-initiating cells. CD133+/EpCAM+ CSCs were enriched under stem cell culture conditions and formed 3-dimensional tumor spheroids. Tumor spheroid cells exhibited CSC properties, including the capability for differentiation and self-renewal, higher tumorigenic potential and chemo-resistance. Genetic analysis using an OncoCarta™ panel revealed a PIK3CA (H1047R mutation in these cells. Using a dual PI3K/mTOR inhibitor, PF-04691502, we then showed that blockage of the PI3K/mTOR pathway inhibited the in vitro proliferation of CSCs and in vivo xenograft tumor growth with manageable toxicity. Tumor growth inhibition in mice was accompanied by a significant reduction of phosphorylated Akt (pAKT (S473, a well-established surrogate biomarker of PI3K/mTOR signaling pathway inhibition. Collectively, our data suggest that PF-04691502 exhibits potent anticancer activity in colorectal cancer by targeting both PIK3CA (H1047R mutant CSCs and their derivatives. These results may assist in the clinical development of PF-04691502 for the treatment of a subpopulation of colorectal cancer patients with poor outcomes.

  6. Lithium chloride decreases proliferation and migration of C6 glioma cells harboring isocitrate dehydrogenase 2 mutant via GSK-3β.

    Science.gov (United States)

    Fu, Yuejun; Zheng, Yali; Chan, Kok-Gan; Liang, Aihua; Hu, Fengyun

    2014-06-01

    The gene encoding isocitrate dehydrogenase (IDH) is somatically mutated predominantly in secondary glioblastoma multiforme. Mutations of IDH1 and IDH2 lead to simultaneous loss and gain of activities in the production of α-ketoglutarate and 2-hydroxyglutarate, respectively. Lithium chloride was recently proved efficient in inhibiting glioma cell migration. The mechanism of lithium chloride on C6 glioma cells harboring IDH2 mutation has not been studied. Here, we found lithium chloride induced inhibitive effects on cell proliferation of both C6 glioma cells with and without IDH2 mutation, although IDH2 mutation increased the stability of HIF-1α. GSK-3β could be phosphorylated at Ser9 and its activity was inhibited when C6 glioma cells were treated by lithium chloride. The degree of phosphorylation in IDH2(R172G) treatment group was lower than that as compared to the control and IDH2 treatment groups. At the same time, the accumulation of β-catenin in C6 cell nucleus was decreased. Moreover, although the β-catenin and HIF-1α increased the secretion of metalloproteinase-2,-9 in C6 glioma cells harboring IDH2 mutation, the migration potential of lithium chloride-treated C6 glioma cells harboring the IDH2 and its mutant was uniform. These results indicated lithium chloride could decrease the proliferation and migration potential of C6 glioma cells harboring IDH2 mutation.

  7. MENINGES HARBOR CELLS EXPRESSING NEURAL PRECURSOR MARKERS DURING DEVELOPMENT AND ADULTHOOD

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    Francesco eBifari

    2015-10-01

    Full Text Available Brain and skull developments are tightly synchronized, allowing the cranial bones to dynamically adapt to the brain shape. At the brain-skull interface, meninges produce the trophic signals necessary for normal corticogenesis and bone development. Meninges harbor different cell populations, including cells forming the endosteum of the cranial vault. Recently, we and other groups have described the presence in meninges of a cell population endowed with neural differentiation potential in vitro and, after transplantation, in vivo. However, whether meninges may be a niche for neural progenitor cells during embryonic development and in adulthood remains to be determined.In this work we provide the first description of the distribution of neural precursor markers in rat meninges during development up to adulthood. We conclude that meninges share common properties with the classical neural stem cell niche, as they: i are a highly proliferating tissue; ii host cells expressing neural precursor markers such as nestin, vimentin, Sox2 and doublecortin; and iii are enriched in extracellular matrix components (e.g. fractones known to bind and concentrate growth factors. This study underlines the importance of meninges as a potential niche for endogenous precursor cells during development and in adulthood.

  8. Matrine Activates PTEN to Induce Growth Inhibition and Apoptosis in V600EBRAF Harboring Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Shuiying Wang

    2013-07-01

    Full Text Available Here, we report a natural chemical Matrine, which exhibits anti-melanoma potential with its PTEN activation mechanism. Matrine effectively inhibited proliferation of several carcinoma cell lines, including melanoma V600EBRAF harboring M21 cells. Flow cytometry analysis showed Matrine induced G0/G1 cell cycle arrest in M21 cells dose-dependently. Apoptosis in M21 cells induced by Matrine was identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL analysis and Annexin-V/FITC staining. Molecular mechanistic study suggested that Matrine upregulated both mRNA level and protein expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN, leading to inhibition of the PI3K/Akt pathway. Downregulation of phosphor-Aktser473 by Matrine activated p21 and Bax, which contributed to G0/G1 cell cycle and apoptosis. Besides, Matrine enhanced the PI3K/Akt inhibition effects to inhibit the cell proliferation with PI3K inhibitor, LY2940002. In summary, our findings suggest Matrine is a promising antitumor drug candidate with its possible PTEN activation mechanisms for treating cancer diseases, such as melanomas.

  9. Meninges harbor cells expressing neural precursor markers during development and adulthood.

    Science.gov (United States)

    Bifari, Francesco; Berton, Valeria; Pino, Annachiara; Kusalo, Marijana; Malpeli, Giorgio; Di Chio, Marzia; Bersan, Emanuela; Amato, Eliana; Scarpa, Aldo; Krampera, Mauro; Fumagalli, Guido; Decimo, Ilaria

    2015-01-01

    Brain and skull developments are tightly synchronized, allowing the cranial bones to dynamically adapt to the brain shape. At the brain-skull interface, meninges produce the trophic signals necessary for normal corticogenesis and bone development. Meninges harbor different cell populations, including cells forming the endosteum of the cranial vault. Recently, we and other groups have described the presence in meninges of a cell population endowed with neural differentiation potential in vitro and, after transplantation, in vivo. However, whether meninges may be a niche for neural progenitor cells during embryonic development and in adulthood remains to be determined. In this work we provide the first description of the distribution of neural precursor markers in rat meninges during development up to adulthood. We conclude that meninges share common properties with the classical neural stem cell niche, as they: (i) are a highly proliferating tissue; (ii) host cells expressing neural precursor markers such as nestin, vimentin, Sox2 and doublecortin; and (iii) are enriched in extracellular matrix components (e.g., fractones) known to bind and concentrate growth factors. This study underlines the importance of meninges as a potential niche for endogenous precursor cells during development and in adulthood.

  10. Redesigning Recombinase Specificity for Safe Harbor Sites in the Human Genome.

    Directory of Open Access Journals (Sweden)

    Mark C Wallen

    Full Text Available Site-specific recombinases (SSRs are valuable tools for genetic engineering due to their ability to manipulate DNA in a highly specific manner. Engineered zinc-finger and TAL effector recombinases, in particular, are two classes of SSRs composed of custom-designed DNA-binding domains fused to a catalytic domain derived from the resolvase/invertase family of serine recombinases. While TAL effector and zinc-finger proteins can be assembled to recognize a wide range of possible DNA sequences, recombinase catalytic specificity has been constrained by inherent base requirements present within each enzyme. In order to further expand the targeted recombinase repertoire, we used a genetic screen to isolate enhanced mutants of the Bin and Tn21 recombinases that recognize target sites outside the scope of other engineered recombinases. We determined the specific base requirements for recombination by these enzymes and demonstrate their potential for genome engineering by selecting for variants capable of specifically recombining target sites present in the human CCR5 gene and the AAVS1 safe harbor locus. Taken together, these findings demonstrate that complementing functional characterization with protein engineering is a potentially powerful approach for generating recombinases with expanded targeting capabilities.

  11. Mitochondrial-associated cell death mechanisms are reset to an embryonic-like state in aged donor-derived iPS cells harboring chromosomal aberrations.

    Science.gov (United States)

    Prigione, Alessandro; Hossini, Amir M; Lichtner, Björn; Serin, Akdes; Fauler, Beatrix; Megges, Matthias; Lurz, Rudi; Lehrach, Hans; Makrantonaki, Eugenia; Zouboulis, Christos C; Adjaye, James

    2011-01-01

    Somatic cells reprogrammed into induced pluripotent stem cells (iPSCs) acquire features of human embryonic stem cells (hESCs) and thus represent a promising source for cellular therapy of debilitating diseases, such as age-related disorders. However, reprogrammed cell lines have been found to harbor various genomic alterations. In addition, we recently discovered that the mitochondrial DNA of human fibroblasts also undergoes random mutational events upon reprogramming. Aged somatic cells might possess high susceptibility to nuclear and mitochondrial genome instability. Hence, concerns over the oncogenic potential of reprogrammed cells due to the lack of genomic integrity may hinder the applicability of iPSC-based therapies for age-associated conditions. Here, we investigated whether aged reprogrammed cells harboring chromosomal abnormalities show resistance to apoptotic cell death or mitochondrial-associated oxidative stress, both hallmarks of cancer transformation. Four iPSC lines were generated from dermal fibroblasts derived from an 84-year-old woman, representing the oldest human donor so far reprogrammed to pluripotency. Despite the presence of karyotype aberrations, all aged-iPSCs were able to differentiate into neurons, re-establish telomerase activity, and reconfigure mitochondrial ultra-structure and functionality to a hESC-like state. Importantly, aged-iPSCs exhibited high sensitivity to drug-induced apoptosis and low levels of oxidative stress and DNA damage, in a similar fashion as iPSCs derived from young donors and hESCs. Thus, the occurrence of chromosomal abnormalities within aged reprogrammed cells might not be sufficient to over-ride the cellular surveillance machinery and induce malignant transformation through the alteration of mitochondrial-associated cell death. Taken together, we unveiled that cellular reprogramming is capable of reversing aging-related features in somatic cells from a very old subject, despite the presence of genomic

  12. Mitochondrial-associated cell death mechanisms are reset to an embryonic-like state in aged donor-derived iPS cells harboring chromosomal aberrations.

    Directory of Open Access Journals (Sweden)

    Alessandro Prigione

    Full Text Available Somatic cells reprogrammed into induced pluripotent stem cells (iPSCs acquire features of human embryonic stem cells (hESCs and thus represent a promising source for cellular therapy of debilitating diseases, such as age-related disorders. However, reprogrammed cell lines have been found to harbor various genomic alterations. In addition, we recently discovered that the mitochondrial DNA of human fibroblasts also undergoes random mutational events upon reprogramming. Aged somatic cells might possess high susceptibility to nuclear and mitochondrial genome instability. Hence, concerns over the oncogenic potential of reprogrammed cells due to the lack of genomic integrity may hinder the applicability of iPSC-based therapies for age-associated conditions. Here, we investigated whether aged reprogrammed cells harboring chromosomal abnormalities show resistance to apoptotic cell death or mitochondrial-associated oxidative stress, both hallmarks of cancer transformation. Four iPSC lines were generated from dermal fibroblasts derived from an 84-year-old woman, representing the oldest human donor so far reprogrammed to pluripotency. Despite the presence of karyotype aberrations, all aged-iPSCs were able to differentiate into neurons, re-establish telomerase activity, and reconfigure mitochondrial ultra-structure and functionality to a hESC-like state. Importantly, aged-iPSCs exhibited high sensitivity to drug-induced apoptosis and low levels of oxidative stress and DNA damage, in a similar fashion as iPSCs derived from young donors and hESCs. Thus, the occurrence of chromosomal abnormalities within aged reprogrammed cells might not be sufficient to over-ride the cellular surveillance machinery and induce malignant transformation through the alteration of mitochondrial-associated cell death. Taken together, we unveiled that cellular reprogramming is capable of reversing aging-related features in somatic cells from a very old subject, despite the presence

  13. Antitumor effects and molecular mechanisms of ponatinib on endometrial cancer cells harboring activating FGFR2 mutations.

    Science.gov (United States)

    Kim, Do-Hee; Kwak, Yeonui; Kim, Nam Doo; Sim, Taebo

    2016-01-01

    Aberrant mutational activation of FGFR2 is associated with endometrial cancers (ECs). AP24534 (ponatinib) currently undergoing clinical trials has been known to be an orally available multi-targeted tyrosine kinase inhibitor. Our biochemical kinase assay showed that AP24534 is potent against wild-type FGFR1-4 and 5 mutant FGFRs (V561M-FGFR1, N549H-FGFR2, K650E-FGFR3, G697C-FGFR3, N535K-FGFR4) and possesses the strongest kinase-inhibitory activity on N549H-FGFR2 (IC50 of 0.5 nM) among all FGFRs tested. We therefore investigated the effects of AP24534 on endometrial cancer cells harboring activating FGFR2 mutations and explored the underlying molecular mechanisms. AP24534 significantly inhibited the proliferation of endometrial cancer cells bearing activating FGFR2 mutations (N549K, K310R/N549K, S252W) and mainly induced G1/S cell cycle arrest leading to apoptosis. AP24534 also diminished the kinase activity of immunoprecipitated FGFR2 derived from MFE-296 and MFE-280 cells and reduced the phosphorylation of FGFR2 and FRS2 on MFE-296 and AN3CA cells. AP24534 caused substantial reductions in ERK phosphorylation, PLCγ signaling and STAT5 signal transduction on ECs bearing FGFR2 activating mutations. Akt signaling pathway was also deactivated by AP24534. AP24534 causes the chemotherapeutic effect through mainly the blockade of ERK, PLCγ and STAT5 signal transduction on ECs. Moreover, AP24534 inhibited migration and invasion of endometrial cancer cells with FGFR2 mutations. In addition, AP24534 significantly blocked anchorage-independent growth of endometrial cancer cells. We, for the first time, report the molecular mechanisms by which AP24534 exerts antitumor effects on ECs with FGFR2 activating mutations, which would provide mechanistic insight into ongoing clinical investigations of AP24534 for ECs.

  14. Geochemical evaluation of the land use and human activities at a Medieval harbor site, Masuda city, Shimane Prefecture, Japan

    Science.gov (United States)

    Dalai, Banzragch; Ishiga, Hiroaki

    2014-05-01

    Large-scale harbor and settlement sites from the latter half of the eleventh through sixteenth centuries have recently been discovered in the northern part of Masuda City, Shimane Prefecture, Japan. The sites were constructed at the river mouth delta of the Takatsu and Masuda rivers, facing the Sea of Japan. In former time, the mouths of the two rivers are thought to have formed a shallow lagoon connecting with the Sea of Japan. The harbor was thus well located for ships sailing along the sea coast, especially for conducting trade with the China mainland and the Korean peninsula. Archaeological investigations have identified over 800 construction pits, blacksmith hearths, harbor structures and numerous fragments of ceramic porcelain originating both from within Japan and from Asia (China, Korea, Vietnam and Thailand). It seems that the maritime trade network operated from this Medieval harbor site by the Masuda Clan was on an East Asian scale. Consequently, the harbor site can be expected to have received a considerable amount of ancient anthropogenic matter. Concentrations of 22 elements in 66 soil samples from the Nakazu Higashihara site were determined by X-ray fluorescence spectroscopy, in order to identify the land use and human impacts on soil chemistry at the harbor site. The results show that significant differences in geochemical compositional exist between the northern and southern parts of the site due to differences in lithology and land use practice. The south area was a production area of this harbor site. Three different activity areas were recognized within this area (fire pit and charcoal area, building pillars, and a blacksmith furnace area), based on geochemical and archaeological information. Cluster analysis shows a strong relationship exists between As, Pb, Cu, Br, TS, MnO and P2O5 in the fire pit and charcoal area. These charcoal materials were likely derived from fuel used in firing and heating. Close relationships occur between Cr, Sr, Sc

  15. Human brain harbors single nucleotide somatic variations in functionally relevant genes possibly mediated by oxidative stress [version 3; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Anchal Sharma

    2017-01-01

    Full Text Available Somatic variation in DNA can cause cells to deviate from the preordained genomic path in both disease and healthy conditions. Here, using exome sequencing of paired tissue samples, we show that the normal human brain harbors somatic single base variations measuring up to 0.48% of the total variations. Interestingly, about 64% of these somatic variations in the brain are expected to lead to non-synonymous changes, and as much as 87% of these represent G:C>T:A transversion events. Further, the transversion events in the brain were mostly found in the frontal cortex, whereas the corpus callosum from the same individuals harbors the reference genotype. We found a significantly higher amount of 8-OHdG (oxidative stress marker in the frontal cortex compared to the corpus callosum of the same subjects (pT:A transversions in the cortex. We found significant enrichment for axon guidance and related pathways for genes harbouring somatic variations. This could represent either a directed selection of genetic variations in these pathways or increased susceptibility of some loci towards oxidative stress. This study highlights that oxidative stress possibly influence single nucleotide somatic variations in normal human brain.

  16. A subset of prostatic basal cell carcinomas harbor the MYB rearrangement of adenoid cystic carcinoma.

    Science.gov (United States)

    Bishop, Justin A; Yonescu, Raluca; Epstein, Jonathan I; Westra, William H

    2015-08-01

    Adenoid cystic carcinoma (ACC) is a basaloid tumor consisting of myoepithelial and ductal cells typically arranged in a cribriform pattern. Adenoid cystic carcinoma is generally regarded as a form of salivary gland carcinoma, but it can arise from sites unassociated with salivary tissue. A rare form of prostate carcinoma exhibits ACC-like features; it is no longer regarded as a true ACC but rather as prostatic basal cell carcinoma (PBCC) and within the spectrum of basaloid prostatic proliferations. True ACCs often harbor MYB translocations resulting in the MYB-NFIB fusion protein. MYB analysis could clarify the true nature of prostatic carcinomas that exhibit ACC features and thus help refine the classification of prostatic basaloid proliferations. Twelve PBCCs were identified from the pathology consultation files of Johns Hopkins Hospital. The histopathologic features were reviewed, and break-apart fluorescence in situ hybridization for MYB was performed. All 12 cases exhibited prominent basaloid histology. Four were purely solid, 7 exhibited a cribriform pattern reminiscent of salivary ACC, and 1 had a mixed pattern. The MYB rearrangement was detected in 2 (29%) of 7 ACC-like carcinomas but in none (0%) of the 5 PBCCs with a prominent solid pattern. True ACCs can arise in the prostate as is evidenced by the presence of the characteristic MYB rearrangement. When dealing with malignant basaloid proliferations in the prostate, recommendations to consolidate ACCs with other tumor types may need to be reassessed, particularly in light of the rapidly advancing field of biologic therapy where the identification of tumor-specific genetic alterations presents novel therapeutic targets.

  17. Outcome of allogeneic hematopoietic stem cell transplantation in adult patients with acute myeloid leukemia harboring trisomy 8.

    Science.gov (United States)

    Konuma, Takaaki; Kondo, Tadakazu; Yamashita, Takuya; Uchida, Naoyuki; Fukuda, Takahiro; Ozawa, Yukiyasu; Ohashi, Kazuteru; Ogawa, Hiroyasu; Kato, Chiaki; Takahashi, Satoshi; Kanamori, Heiwa; Eto, Tetsuya; Nakaseko, Chiaki; Kohno, Akio; Ichinohe, Tatsuo; Atsuta, Yoshiko; Takami, Akiyoshi; Yano, Shingo

    2017-03-01

    Trisomy 8 (+8) is one of the most common cytogenetic abnormalities in adult patients with acute myeloid leukemia (AML). However, the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) in adult patients with AML harboring +8 remains unclear. To evaluate, the outcome and prognostic factors in patients with AML harboring +8 as the only chromosomal abnormality or in association with other abnormalities, we retrospectively analyzed the Japanese registration data of 631 adult patients with AML harboring +8 treated with allogeneic HSCT between 1990 and 2013. In total, 388 (61%) patients were not in remission at the time of HSCT. With a median follow-up of 38.5 months, the probability of overall survival and the cumulative incidence of relapse at 3 years were 40 and 34%, respectively. In the multivariate analysis, two or more additional cytogenetic abnormalities and not being in remission at the time of HSCT were significantly associated with a higher overall mortality and relapse. Nevertheless, no significant impact on the outcome was observed in cases with one cytogenetic abnormality in addition to +8. Although more than 60% of the patients received HSCT when not in remission, allogeneic HSCT offered a curative option for adult patients with AML harboring +8.

  18. Alexandria's Eastern Harbor, Egypt: Pollen, microscopic charcoal, and the transition from natural to human-modified basin

    Science.gov (United States)

    Stanley, J.-D.; Bernhardt, C.E.

    2010-01-01

    Pollen and microscopic charcoal examined in Holocene sediment core samples record major environmental modifications affecting Alexandria's Eastern Harbor through time. We assess whether such changes on Egypt's coastal margin were influenced primarily by natural, or natural plus human, or primarily human factors. We focus on (1) the times when pollen assemblages and microscopic charcoal content changed in the core, (2) how they changed, and (3) why this occurred. The analysis takes into account the core's stratigraphy, regional climate variability, human history, and local archaeological record. Four pollenmicroscopic charcoal zones are identified. The earliest change occurred at ca. 6000 YBP, during Egypt's earlier Predynastic (Neolithic) period, coinciding with a lithologic break from sand to muddy sand. Pollen during this time indicates a transition to a much drier climate rather than effects of human activity. The second change in pollen occurred 3600-2900 YBP, during a period of continued aridity with no lithologic variation in this core interval. Pollen (cereal taxa, agricultural weeds, grape) and a sharp increase in microscopic charcoal indicate that human activity became prevalent at least 700 y before Alexander the Great's arrival in this region, and these results highlight the transition from a largely natural climatecontrolled environment to one influenced by both climate and anthropogenic activity. The third shift up-core in pollen assemblages is dated at ca. 2300 YBP, at the boundary between a sand and mud unit. It coincides with construction by the Ptolemies of the Heptastadion between Alexandria and Pharos Island. From this time onward, harbor sediment in the nearly enclosed catchment basin indicates a near-continuous record of dominant proximal human activity. ?? 2010 Coastal Education and Research Foundation.

  19. [Pearl Harbor.

    Science.gov (United States)

    Johnson, Jennifer, Ed.

    1992-01-01

    This issue of "Loblolly Magazine" was written in observance of the 50th anniversary of the U.S. entrance into World War II. The publication features interviews conducted by East Texas high school students with Clarence Otterman, one of the few survivors of the crew of the USS Arizona, which was bombed during the attack on Pearl Harbor,…

  20. Human 3β-hydroxysteroid dehydrogenase deficiency seems to affect fertility but may not harbor a tumor risk

    DEFF Research Database (Denmark)

    Burckhardt, Marie-Anne; Udhane, Sameer S; Marti, Nesa

    2015-01-01

    . RESULTS: A 46,XY boy presented at birth with severe undervirilization of the external genitalia. Steroid profiling showed low steroid production for mineralocorticoids, glucocorticoids and sex steroids with typical precursor metabolites for HSD3B2 deficiency. The genetic analysis of the HSD3B2 gene...... enlarged breasts through production of estrogens in the periphery. Testis histology in late puberty revealed primarily a Sertoli-cell-only pattern and only few tubules with arrested spermatogenesis, presence of few Leydig cells in stroma, but no neoplastic changes. CONCLUSIONS: The testis with HSD3B2...... deficiency due to the c.687del27 deletion does not express the defective protein. This patient is unlikely to be fertile and his risk for gonadal malignancy is low. Further studies are needed to obtain firm knowledge on malignancy risk for gonads harboring defects of androgen biosynthesis....

  1. NKT Cell-Deficient Mice Harbor an Altered Microbiota That Fuels Intestinal Inflammation during Chemically Induced Colitis.

    Science.gov (United States)

    Selvanantham, Thirumahal; Lin, Qiaochu; Guo, Cynthia Xinyi; Surendra, Anuradha; Fieve, Stephanie; Escalante, Nichole K; Guttman, David S; Streutker, Catherine J; Robertson, Susan J; Philpott, Dana J; Mallevaey, Thierry

    2016-12-01

    NKT cells are unconventional T cells that respond to self and microbe-derived lipid and glycolipid Ags presented by the CD1d molecule. Invariant NKT (iNKT) cells influence immune responses in numerous diseases. Although only a few studies have examined their role during intestinal inflammation, it appears that iNKT cells protect from Th1-mediated inflammation but exacerbate Th2-mediated inflammation. Studies using iNKT cell-deficient mice and chemically induced dextran sodium sulfate (DSS) colitis have led to inconsistent results. In this study, we show that CD1d-deficient mice, which lack all NKT cells, harbor an altered intestinal microbiota that is associated with exacerbated intestinal inflammation at steady-state and following DSS treatment. This altered microbiota, characterized by increased abundance of the bacterial phyla Proteobacteria, Deferribacteres, and TM7, among which the mucin-eating Mucispirillum, as well as members of the genus Prevotella and segmented filamentous bacteria, was transmissible upon fecal transplant, along with the procolitogenic phenotype. Our results also demonstrate that this proinflammatory microbiota influences iNKT cell function upon activation during DSS colitis. Collectively, alterations of the microbiota have a major influence on colitis outcome and therefore have to be accounted for in such experimental settings and in studies focusing on iNKT cells. Copyright © 2016 by The American Association of Immunologists, Inc.

  2. Transformation of human fetal thymus and spleen lymphocytes by human t-cell leukemia virus type Ι

    Directory of Open Access Journals (Sweden)

    Akagi,Tadaatsu

    1985-04-01

    Full Text Available Co-cultivation of human thymus and spleen lymphocytes, which were obtained from 26-week and 27-week fetuses, with a lethally-irradiated human cord T-cell line harboring human T-cell leukemia virus type Ι(HTLV-Ι resultes in the establishment of T-cell lines positive for adult T-cell leukemia-associated antigens and producing HTLV-Ι. These cell lines had the phenotype of a helper/inducer subset of peripheral T-cells as evidenced by the reactivity with monoclonal antibodies to human T-cells.

  3. Targeting Androgen Receptor/Src Complex Impairs the Aggressive Phenotype of Human Fibrosarcoma Cells

    OpenAIRE

    Gabriella Castoria; Pia Giovannelli; Marzia Di Donato; Ryo Hayashi; Claudio Arra; Ettore Appella; Ferdinando Auricchio; Antimo Migliaccio

    2013-01-01

    BACKGROUND: Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR). EXPERIMENTAL: FINDINGS: We report that the pure anti-androgen Casodex inhibits the...

  4. Human mesenchymal stem cells

    DEFF Research Database (Denmark)

    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....

  5. 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.

  6. Proteomic analysis of HUH-7 cells harboring in vitro-transcribed full-length hepatitis C virus 1b RNA

    Institute of Scientific and Technical Information of China (English)

    Meng XUN; Si-hai ZHAO; Chun-xia CAO; Juan SONG; Ming-ming SHAO; Yong-lie CHU

    2008-01-01

    Aim: The present study examined the differential expression of proteins in HuH-7 cells and HUH-7 cells harboring in vitro-transcribed full-length hepatitis C virus 1b RNA (HuH-7-HCV), and elucidated the cellular responses to HCV replication. Methods: The protein profiles of matched pairs of HuH-7-HCV cells and HUH-7 mock cells were analyzed by 2-D electrophoresis (2DE). Solubilized proteins were separated in the first dimension by isoelectric focusing, and by 12.5% SDS-PAGE in the second dimension. The differential protein expression was analyzed by use of image analysis software to identify candidates for HCV infection-associated proteins. Results: In total, 29 protein spots showed increases and 25 protein spots showed decreases in signal in HuH-7-HCV cell 2DE profiles as compared with HuH-7 mock cells. In the next step, the 10 spots showing the greatest in-crease and the 10 spots showing the greatest decrease were excised from gels and the proteins present were identified by Matrix-Assisted Laser Desorption/Ioniza-tion Time of Flight Mass Spectrometer (MALDI-TOF MS) or MALDI-TOF/TOF MS. In total, 13 proteins were identified successfully. The potential significance of the differential expression due to HCV replication was discussed. Conclusion: Our study identifies changes in the proteome of HuH-7 cells in the presence of HCV replication and yields information of the mechanism of HCV pathogenesis. These results will be useful for the identification of HCV infection-associated proteins that could be molecular targets for treatment.

  7. Mycoplasma contamination revisited: mesenchymal stromal cells harboring Mycoplasma hyorhinis potently inhibit lymphocyte proliferation in vitro.

    Directory of Open Access Journals (Sweden)

    Severin Zinöcker

    Full Text Available BACKGROUND: Mesenchymal stromal cells (MSC have important immunomodulatory effects that can be exploited in the clinical setting, e.g. in patients suffering from graft-versus-host disease after allogeneic stem cell transplantation. In an experimental animal model, cultures of rat T lymphocytes were stimulated in vitro either with the mitogen Concanavalin A or with irradiated allogeneic cells in mixed lymphocyte reactions, the latter to simulate allo-immunogenic activation of transplanted T cells in vivo. This study investigated the inhibitory effects of rat bone marrow-derived MSC subsequently found to be infected with a common mycoplasma species (Mycoplasma hyorhinis on T cell activation in vitro and experimental graft-versus-host disease in vivo. PRINCIPAL FINDINGS: We found that M. hyorhinis infection increased the anti-proliferative effect of MSC dramatically, as measured by both radiometric and fluorimetric methods. Inhibition could not be explained solely by the well-known ability of mycoplasmas to degrade tritiated thymidine, but likely was the result of rapid dissemination of M. hyorhinis in the lymphocyte culture. CONCLUSIONS: This study demonstrates the potent inhibitory effect exerted by M. hyorhinis in standard lymphocyte proliferation assays in vitro. MSC are efficient vectors of mycoplasma infection, emphasizing the importance of monitoring cell cultures for contamination.

  8. Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA.

    Science.gov (United States)

    Lewis, Cody W; Golsteyn, Roy M

    2016-11-16

    We have examined the relationship between checkpoint adaptation (mitosis with damaged DNA) and micronuclei. Micronuclei in cancer cells are linked to genomic change, and may induce chromothripsis (chromosome shattering). We measured the cytotoxicity of the cancer drug cisplatin in M059K (glioma fibroblasts, IC50 15 μM). Nearly 100% of M059K cells were positive for histone γH2AX staining after 48 h treatment with a cytotoxic concentration of cisplatin. The proportion of micronucleated cells, as confirmed by microscopy using DAPI and lamin A/C staining, increased from 24% to 48%, and the total micronuclei in surviving cells accumulated over time. Promoting entry into mitosis with a checkpoint inhibitor increased the number of micronuclei in cells whereas blocking checkpoint adaptation with a Cdk inhibitor reduced the number of micronuclei. Interestingly, some micronuclei underwent asynchronous DNA replication, relative to the main nuclei, as measured by deoxy-bromo-uracil (BrdU) staining. These micronuclei stained positive for histone γH2AX, which was linked to DNA replication, suggesting that micronuclei arise from checkpoint adaptation and that micronuclei may continue to damage DNA. By contrast the normal cell line WI-38 did not undergo checkpoint adaptation when treated with cisplatin and did not show changes in micronuclei number. These data reveal that the production of micronuclei by checkpoint adaptation is part of a process that contributes to genomic change.

  9. Restoration of proper trafficking to the cell surface for membrane proteins harboring cysteine mutations.

    Directory of Open Access Journals (Sweden)

    Angelica Lopez-Rodriguez

    Full Text Available A common phenotype for many genetic diseases is that the cell is unable to deliver full-length membrane proteins to the cell surface. For some forms of autism, hereditary spherocytosis and color blindness, the culprits are single point mutations to cysteine. We have studied two inheritable cysteine mutants of cyclic nucleotide-gated channels that produce achromatopsia, a common form of severe color blindness. By taking advantage of the reactivity of cysteine's sulfhydryl group, we modified these mutants with chemical reagents that attach moieties with similar chemistries to the wild-type amino acids' side chains. We show that these modifications restored proper delivery to the cell membrane. Once there, the channels exhibited normal functional properties. This strategy might provide a unique opportunity to assess the chemical nature of membrane protein traffic problems.

  10. Allogeneic stem cell transplantation for patients harboring T315I BCR-ABL mutated leukemias

    DEFF Research Database (Denmark)

    Nicolini, Franck Emmanuel; Basak, Grzegorz W; Soverini, Simona;

    2011-01-01

    T315I(+) Philadelphia chromosome-positive leukemias are inherently resistant to all licensed tyrosine kinase inhibitors, and therapeutic options remain limited. We report the outcome of allogeneic stem cell transplantation in 64 patients with documented BCR-ABL(T315I) mutations. Median follow...... myeloid leukemia. The occurrence of chronic GVHD had a positive impact on overall survival (P = .047). Transplant-related mortality rates were low. Multivariate analysis identified only blast phase at transplantation (hazard ratio 3.68, P = .0011) and unrelated stem cell donor (hazard ratio 2.98, P = .011......) as unfavorable factors. We conclude that allogeneic stem cell transplantation represents a valuable therapeutic tool for eligible patients with BCR-ABL(T315I) mutation, a tool that may or may not be replaced by third-generation tyrosine kinase inhibitors....

  11. VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt

    Directory of Open Access Journals (Sweden)

    Laura Lafon-Hughes

    2014-10-01

    Full Text Available Poly-ADP-ribose (PAR is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs and degraded by poly-ADP-ribose-glycohydrolase (PARG. Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair. Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt. In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO. PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications.

  12. Dietary restriction-resistant human tumors harboring the PIK3CA-activating mutation H1047R are sensitive to metformin

    Science.gov (United States)

    Cufí, Sílvia; Corominas-Faja, Bruna; Lopez-Bonet, Eugeni; Bonavia, Rosa; Pernas, Sonia; López, Isabel álvarez; Dorca, Joan; Martínez, Susana; López, Norberto Batista; Fernández, Severina Domínguez; Cuyàs, Elisabet; Visa, Joana; Rodríguez-Gallego, Esther; Quirantes-Piné, Rosa; Segura-Carretero, Antonio; Joven, Jorge; Martin-Castillo, Begoña; Menendez, Javier A.

    2013-01-01

    Cancer cells expressing constitutively active phosphatidylinositol-3 kinase (PI3K) are proliferative regardless of the absence of insulin, and they form dietary restriction (DR)-resistant tumors in vivo. Because the binding of insulin to its receptors activates the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling cascade, activating mutations in the PIK3CA oncogene may determine tumor response to DR-like pharmacological strategies targeting the insulin and mTOR pathways. The anti-diabetic drug metformin is a stereotypical DR mimetic that exerts its anti-cancer activity through a dual mechanism involving insulin-related (systemic) and mTOR-related (cell-autonomous) effects. However, it remains unclear whether PIK3CA-activating mutations might preclude the anti-cancer activity of metformin in vivo. To model the oncogenic PIK3CA-driven early stages of cancer, we used the clonal breast cancer cell line MCF10DCIS.com, which harbors the gain-of-function H1047R hot-spot mutation in the catalytic domain of the PI3KCA gene and has been shown to form DR-refractory xenotumors. To model PIK3CA-activating mutations in late stages of cancer, we took advantage of the isogenic conversion of a PIK3CA-wild-type tumor into a PIK3CA H1047R-mutated tumor using the highly metastatic colorectal cancer cell line SW48. MCF10DCIS.com xenotumors, although only modestly affected by treatment with oral metformin (approximately 40% tumor growth inhibition), were highly sensitive to the intraperitoneal (i.p.) administration of metformin, the anti-cancer activity of which increased in a time-dependent manner and reached >80% tumor growth inhibition by the end of the treatment. Metformin treatment via the i.p. route significantly reduced the proliferation factor mitotic activity index (MAI) and decreased tumor cellularity in MCF10DCIS.com cancer tissues. Whereas SW48-wild-type (PIK3CA+/+) cells rapidly formed metformin-refractory xenotumors in mice, ad libitum access to water containing

  13. Docetaxel for non small cell lung cancer harboring the activated EGFR mutation with T790M at initial presentation

    Directory of Open Access Journals (Sweden)

    Yamane H

    2013-03-01

    Full Text Available Hiromichi Yamane,1 Nobuaki Ochi,1 Masayuki Yasugi,2 Takayuki Tabayashi,1 Tomoko Yamagishi,1 Yasumasa Monobe,3 Akiko Hisamoto,4 Katsuyuki Kiura,4 Nagio Takigawa1 1Department of General Internal Medicine 4, Kawasaki Medical School, Okayama, Japan; 2Department of Respiratory Medicine, National Hospital Organization Fukuyama Medical Center, Fukuyama, Japan; 3Department of Pathology, Kawasaki Medical School Kawasaki Hospital, Okayama, Japan; 4Department of Hematology, Oncology, and Respiratory Medicine, Okayama University Graduate School of Medicine, Okayama, Japan Abstract: A 72-year-old woman was referred to our hospital with Stage IV non-small-cell lung cancer (NSCLC. Chest computed tomography revealed a mass in the upper lobe of the right lung, with pleural effusion. Cytologic examination identified adenocarcinoma cells in the right pleural effusion. Furthermore, both a deletion mutation in exon 19 and a threonine–methionine substitution mutation at position 790 in exon 20 (T790M were detected in the epidermal growth factor receptors (EGFR in the malignant cells. As systemic chemotherapy consisting of carboplatin and pemetrexed or erlotinib proved ineffective, docetaxel monotherapy was initiated as a third-line treatment. Following salvage chemotherapy, her Eastern Cooperative Oncology Group performance status improved from 3 to 1, with tumor regression over 5 months. To the best of our knowledge, this is the first report of successful docetaxel treatment for a patient with NSCLC harboring the T790M EGFR-activating mutation identified before treatment with EGFR tyrosine kinase inhibitors. Keywords: non-small-cell lung cancer, EGFR mutation, pretreatment mutation, T790M, docetaxel

  14. Gene expression pattern in transmitochondrial cytoplasmic hybrid cells harboring type 2 diabetes-associated mitochondrial DNA haplogroups.

    Directory of Open Access Journals (Sweden)

    Seungwoo Hwang

    Full Text Available Decreased mitochondrial function plays a pivotal role in the pathogenesis of type 2 diabetes mellitus (T2DM. Recently, it was reported that mitochondrial DNA (mtDNA haplogroups confer genetic susceptibility to T2DM in Koreans and Japanese. Particularly, mtDNA haplogroup N9a is associated with a decreased risk of T2DM, whereas haplogroups D5 and F are associated with an increased risk. To examine functional consequences of these haplogroups without being confounded by the heterogeneous nuclear genomic backgrounds of different subjects, we constructed transmitochondrial cytoplasmic hybrid (cybrid cells harboring each of the three haplogroups (N9a, D5, and F in a background of a shared nuclear genome. We compared the functional consequences of the three haplogroups using cell-based assays and gene expression microarrays. Cell-based assays did not detect differences in mitochondrial functions among the haplogroups in terms of ATP generation, reactive oxygen species production, mitochondrial membrane potential, and cellular dehydrogenase activity. However, differential expression and clustering analyses of microarray data revealed that the three haplogroups exhibit a distinctive nuclear gene expression pattern that correlates with their susceptibility to T2DM. Pathway analysis of microarray data identified several differentially regulated metabolic pathways. Notably, compared to the T2DM-resistant haplogroup N9a, the T2DM-susceptible haplogroup F showed down-regulation of oxidative phosphorylation and up-regulation of glycolysis. These results suggest that variations in mtDNA can affect the expression of nuclear genes regulating mitochondrial functions or cellular energetics. Given that impaired mitochondrial function caused by T2DM-associated mtDNA haplogroups is compensated by the nuclear genome, we speculate that defective nuclear compensation, under certain circumstances, might lead to the development of T2DM.

  15. Impact of physical size on gefitinib efficacy in patients with non-small cell lung cancer harboring EGFR mutations.

    Science.gov (United States)

    Ichihara, Eiki; Hotta, Katsuyuki; Takigawa, Nagio; Kudo, Kenichiro; Kato, Yuka; Honda, Yoshihiro; Hayakawa, Hiromi; Minami, Daisuke; Sato, Akiko; Tabata, Masahiro; Tanimoto, Mitsune; Kiura, Katsuyuki

    2013-09-01

    Gefitinib is an essential drug for the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) gene mutations. The approved dosage is 250 mg/body/day without adjustment for physical size such as body surface area (BSA), and the impact of physical size on the efficacy of gefitinib has not been evaluated. Here, we sought to clarify this issue using a retrospective cohort. We reviewed the medical records of patients with consecutive advanced NSCLC harboring EGFR mutations who underwent gefitinib monotherapy at Okayama University Hospital. In total, 101 patients were included in this study, and the median BSA in this cohort was 1.5 m(2). The median progression-free survival (PFS) of the patients with higher BSA (≥1.5 m(2)) was significantly worse than that of those with lower BSA (BSA on PFS (hazards ratio, 2.34; 95% confidence interval, 1.78-2.89; p = 0.002). By contrast, no significant association between BSA and PFS was observed in those undergoing cytotoxic chemotherapy (4.0 vs. 5.1 months; p = 0.989, log-rank test), suggesting that BSA is a predictive, rather than a prognostic, marker for gefitinib therapy in EGFR-mutated NSCLC. In conclusion, BSA affected PFS in patients with EGFR-mutated NSCLC who underwent gefitinib monotherapy, suggesting the need for appraisal of BSA-based dose adjustment, even for this molecular target-based therapy.

  16. Cecum lymph node dendritic cells harbor slow-growing bacteria phenotypically tolerant to antibiotic treatment.

    Directory of Open Access Journals (Sweden)

    Patrick Kaiser

    2014-02-01

    Full Text Available In vivo, antibiotics are often much less efficient than ex vivo and relapses can occur. The reasons for poor in vivo activity are still not completely understood. We have studied the fluoroquinolone antibiotic ciprofloxacin in an animal model for complicated Salmonellosis. High-dose ciprofloxacin treatment efficiently reduced pathogen loads in feces and most organs. However, the cecum draining lymph node (cLN, the gut tissue, and the spleen retained surviving bacteria. In cLN, approximately 10%-20% of the bacteria remained viable. These phenotypically tolerant bacteria lodged mostly within CD103⁺CX₃CR1⁻CD11c⁺ dendritic cells, remained genetically susceptible to ciprofloxacin, were sufficient to reinitiate infection after the end of the therapy, and displayed an extremely slow growth rate, as shown by mathematical analysis of infections with mixed inocula and segregative plasmid experiments. The slow growth was sufficient to explain recalcitrance to antibiotics treatment. Therefore, slow-growing antibiotic-tolerant bacteria lodged within dendritic cells can explain poor in vivo antibiotic activity and relapse. Administration of LPS or CpG, known elicitors of innate immune defense, reduced the loads of tolerant bacteria. Thus, manipulating innate immunity may augment the in vivo activity of antibiotics.

  17. Different spectra of recurrent gene mutations in subsets of chronic lymphocytic leukemia harboring stereotyped B-cell receptors

    Science.gov (United States)

    Sutton, Lesley-Ann; Young, Emma; Baliakas, Panagiotis; Hadzidimitriou, Anastasia; Moysiadis, Theodoros; Plevova, Karla; Rossi, Davide; Kminkova, Jana; Stalika, Evangelia; Pedersen, Lone Bredo; Malcikova, Jitka; Agathangelidis, Andreas; Davis, Zadie; Mansouri, Larry; Scarfò, Lydia; Boudjoghra, Myriam; Navarro, Alba; Muggen, Alice F.; Yan, Xiao-Jie; Nguyen-Khac, Florence; Larrayoz, Marta; Panagiotidis, Panagiotis; Chiorazzi, Nicholas; Niemann, Carsten Utoft; Belessi, Chrysoula; Campo, Elias; Strefford, Jonathan C.; Langerak, Anton W.; Oscier, David; Gaidano, Gianluca; Pospisilova, Sarka; Davi, Frederic; Ghia, Paolo; Stamatopoulos, Kostas; Rosenquist, Richard

    2016-01-01

    We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobulins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations (BIRC3, MYD88, NOTCH1, SF3B1 and TP53) and cytogenetic aberrations, we reveal a subset-biased acquisition of gene mutations. More specifically, the frequency of NOTCH1 mutations was found to be enriched in subsets expressing unmutated immunoglobulin genes, i.e. #1, #6, #8 and #59 (22–34%), often in association with trisomy 12, and was significantly different (Pimmunoglobulin genes). Interestingly, subsets harboring a high frequency of NOTCH1 mutations were found to carry few (if any) SF3B1 mutations. This starkly contrasts with subsets #2 and #3 where, despite their immunogenetic differences, SF3B1 mutations occurred in 45% and 46% of cases, respectively. In addition, mutations within TP53, whilst enriched in subset #1 (16%), were rare in subsets #2 and #8 (both 2%), despite all being clinically aggressive. All subsets were negative for MYD88 mutations, whereas BIRC3 mutations were infrequent. Collectively, this striking bias and skewed distribution of mutations and cytogenetic aberrations within specific chronic lymphocytic leukemia subsets implies that the mechanisms underlying clinical aggressiveness are not uniform, but rather support the existence of distinct genetic pathways of clonal evolution governed by a particular stereotyped B-cell receptor selecting a certain molecular lesion(s). PMID:27198719

  18. Cell encoding recombinant human erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Coastal Harbors Modeling Facility

    Data.gov (United States)

    Federal Laboratory Consortium — The Coastal Harbors Modeling Facility is used to aid in the planning of harbor development and in the design and layout of breakwaters, absorbers, etc.. The goal is...

  20. Human CD8+ T cells mediate protective immunity induced by a human malaria vaccine in human immune system mice.

    Science.gov (United States)

    Li, Xiangming; Huang, Jing; Zhang, Min; Funakoshi, Ryota; Sheetij, Dutta; Spaccapelo, Roberta; Crisanti, Andrea; Nussenzweig, Victor; Nussenzweig, Ruth S; Tsuji, Moriya

    2016-08-31

    A number of studies have shown that CD8+ T cells mediate protective anti-malaria immunity in a mouse model. However, whether human CD8+ T cells play a role in protection against malaria remains unknown. We recently established human immune system (HIS) mice harboring functional human CD8+ T cells (HIS-CD8 mice) by transduction with HLA-A∗0201 and certain human cytokines using recombinant adeno-associated virus-based gene transfer technologies. These HIS-CD8 mice mount a potent, antigen-specific HLA-A∗0201-restricted human CD8+ T-cell response upon immunization with a recombinant adenovirus expressing a human malaria antigen, the Plasmodium falciparum circumsporozoite protein (PfCSP), termed AdPfCSP. In the present study, we challenged AdPfCSP-immunized HIS-CD8 mice with transgenic Plasmodium berghei sporozoites expressing full-length PfCSP and found that AdPfCSP-immunized (but not naïve) mice were protected against subsequent malaria challenge. The level of the HLA-A∗0201-restricted, PfCSP-specific human CD8+ T-cell response was closely correlated with the level of malaria protection. Furthermore, depletion of human CD8+ T cells from AdPfCSP-immunized HIS-CD8 mice almost completely abolished the anti-malaria immune response. Taken together, our data show that human CD8+ T cells mediate protective anti-malaria immunity in vivo.

  1. Lymph nodes from HIV-infected individuals harbor mature dendritic cells and increased numbers of PD-L1+ conventional dendritic cells.

    Science.gov (United States)

    Carranza, Paloma; Del Río Estrada, Perla M; Díaz Rivera, Dafne; Ablanedo-Terrazas, Yuria; Reyes-Terán, Gustavo

    2016-07-01

    The immune response induced by dendritic cells (DC) during the HIV infection has been of remarkable interest because of the therapeutic potential of DC for vaccine development. However, their beneficial or detrimental contribution in HIV infection remains unclear. The activation state of DC in lymph nodes (LN) is essential to induce T cell responses against HIV. In the present study, we characterized the immunophenotype and function of conventional (cDC) and plasmacytoid (pDC) dendritic cells from peripheral blood (PB) and LN of HIV(+) individuals. We observed that the frequency of PB pDC was decreased and exhibited an immature phenotype; whereas in the LN, activated pDC accumulated (CD40(+) and CD83(+)). In addition, the frequency of PB cDC from HIV(+) individuals was decreased and exhibited an immature phenotype, whereas LN harbored activated and mature cDC (CD40(+), CD83(+), CD80(+) and CD86(+)). However, an increased number of PD-L1(+) cDC was also observed in the LN. Moreover, pDC and cDC were able to produce inflammatory cytokines (IFN-α, TNF-α and IL-12) after TLR stimulation. These findings suggests that LN cDC expressing PD-L1 from HIV(+) individuals may negatively impact the generation of HIV-specific T cells and that DC might be contributing to tissue chronic immune activation.

  2. Hodgkin and Reed-Sternberg cells harbor alterations in the major tumor suppressor pathways and cell-cycle checkpoints: analyses using tissue microarrays.

    Science.gov (United States)

    García, Juan F; Camacho, Francisca I; Morente, Manuel; Fraga, Máximo; Montalbán, Carlos; Alvaro, Tomás; Bellas, Carmen; Castaño, Angel; Díez, Ana; Flores, Teresa; Martin, Carmen; Martinez, Miguel A; Mazorra, Francisco; Menárguez, Javier; Mestre, Maria J; Mollejo, Manuela; Sáez, Ana I; Sánchez, Lydia; Piris, Miguel A

    2003-01-15

    Tumoral cells in Hodgkin lymphoma (HL) display an increased growth fraction and diminished apoptosis, implying a profound disturbance of the cell cycle and apoptosis regulation. However, limitations of molecular techniques have prevented the analysis of the tumor suppressor pathways and cell-cycle checkpoints. Tissue microarray (TMA) is a powerful tool for analyzing a large number of molecular variables in a large series of tumors, although the feasibility of this technique has not yet been demonstrated in heterogeneous tumors. The expression of 29 genes regulating the cell cycle and apoptosis were analyzed by immunohistochemistry and in situ hybridization in 288 HL biopsies using TMA. The sensitivity of the technique was validated by comparing the results with those obtained in standard tissue sections. The results revealed multiple alterations in different pathways and checkpoints, including G1/S and G2/M transition and apoptosis. Striking findings were the overexpression of cyclin E, CDK2, CDK6, STAT3, Hdm2, Bcl2, Bcl-X(L), survivin, and NF-kappaB proteins. A multiparametric analysis identified proteins associated with increased growth fraction (Hdm2, p53, p21, Rb, cyclins A, B1, D3, and E, CDK2, CDK6, SKP2, Bcl-X(L), survivin, STAT1, and STAT3), and proteins associated with apoptosis (NF-kappaB, STAT1, and RB). The analysis also demonstrated that Epstein-Barr virus (EBV)-positive cases displayed a characteristic profile, confirming the pathogenic role of EBV in HL. Survival probability depends on multiple biologic factors, including overexpression of Bcl2, p53, Bax, Bcl-X(L), MIB1, and apoptotic index. In conclusion, Hodgkin and Reed-Sternberg cells harbor concurrent and overlapping alterations in the major tumor suppressor pathways and cell-cycle checkpoints. This appears to determine the viability of the tumoral cells and the clinical outcome.

  3. Disrupted cell cycle control in cultured endometrial cells from patients with endometriosis harboring the progesterone receptor polymorphism PROGINS.

    Science.gov (United States)

    D'Amora, Paulo; Maciel, Thiago Trovati; Tambellini, Rodrigo; Mori, Marcelo A; Pesquero, João Bosco; Sato, Helio; Girão, Manoel João Batista Castello; Guerreiro da Silva, Ismael Dale Cotrim; Schor, Eduardo

    2009-07-01

    Presently, little is understood about how endometriosis is established or maintained, or how genetic factors can predispose women to the disease. Because of the crucial role that the progesterone receptor polymorphism PROGINS plays in predisposing women to the development of endometriosis, we hypothesized that this variant may influence critical steps during endometrial cell metabolism that are involved in the pathogenesis of endometriosis. Eutopic endometria were collected from three sources: women with endometriosis who had a single PROGINS allele (from the progesterone receptor gene); women with endometriosis who had the wild-type progesterone receptor allele; and women without endometriosis who had the wild-type allele. Cells prepared from the eutopic endometria of these women were stimulated with both estradiol and progesterone, and then examined for cell proliferation, viability, and apoptosis. The cells from women with endometriosis that carried the PROGINS allele demonstrated increased proliferation, greater viability, and decreased apoptosis following progesterone treatment. In general, these parameters were very different as compared with those of women with endometriosis but without the PROGINS allele and women in the control group. This result indicates there is a reduced level of progesterone responsiveness in women who carry the PROGINS polymorphism. Because progesterone responsiveness is known to be an important characteristic of women with endometriosis, these data support the contention that the PROGINS polymorphism enhances the endometriosis phenotype.

  4. 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.

  5. Morphodynamic changes as an impact of human intervention at the Ras El-Bar-Damietta Harbor coast, NW Damietta Promontory, Nile Delta, Egypt

    Science.gov (United States)

    El-Asmar, Hesham M.; Taha, Maysa M. N.; El-Sorogy, Abdelbaset S.

    2016-12-01

    Due to the absence of a national strategic plan for coastal management, the Nile Delta coast is no longer described as a fully dissipative, divergent, low-gradient beach face composed of fine to very fine sand. Instead, new patterns have emerged depending on rock type, geomorphology of the coast, direction of the shoreline in relation to waves and current, and the implemented defense measures. This study attempts to record the morphodynamic changes which occurred due to human intervention. Landsat satellite images acquired for the periods of time of 1973, 1984, 1989, 2003, and 2015 are used together with geomorphologic observations in order to monitor the changes along the coastal strip between Ras El-Bar and Damietta Harbor. This study reveals two beach segments; one of which lies to the east, it is protected with detached breakwater system, and shows average shoreline accretions of +4.73 myr-1, +5.0 myr-1, and +0.89myr-1 during the periods of 1984-1998, 1998-2003, and 2003-2015 respectively. This segment still has the geomorphologic imprints of the dissipative beach, wave divergence, low-gradient beach face, fine grained sand and spilling breakers. The second is to the west, between the detached breakwaters and the eastern jetty of the Damietta Harbor. It is an erosional segment with shoreline retreat of -7.43 myr-1, -10.90 myr-1, and -3.11myr-1 for the same periods. This segment shows intermediate "d" beach or intermediate-reflective, wave convergence, rip currents, with the characteristic steep sloped and cuspate beach face, cliffy, reworked sediments of coarse grained sands, mud clasts, discoidal gravels, shelly beach, and plunging breakings. The presence of convergent waves along this segment confirms the concept of an emergence of a new wave pattern of reversed eddy which enhances the steepness of the beach face, accelerates erosion, and increases the possibility of drowning of swimmers at Ras El-Bar resort. Under such circumstances the plunge step

  6. 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...

  7. Efficient Generation of Gene-Modified Pigs Harboring Precise Orthologous Human Mutation via CRISPR/Cas9-Induced Homology-Directed Repair in Zygotes.

    Science.gov (United States)

    Zhou, Xiaoyang; Wang, Lulu; Du, Yinan; Xie, Fei; Li, Liang; Liu, Yu; Liu, Chuanhong; Wang, Shiqiang; Zhang, Shibing; Huang, Xingxu; Wang, Yong; Wei, Hong

    2016-01-01

    Precise genetic mutation of model animals is highly valuable for functional investigation of human mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-induced homology-directed repair (HDR) is usually used for precise genetic mutation, being limited by the relatively low efficiency compared with that of non-homologous end joining (NHEJ). Although inhibition of NHEJ was shown to enhance HDR-derived mutation, in this work, without inhibition of NHEJ, we first generated gene-modified pigs harboring precise orthologous human mutation (Sox10 c.A325>T) via CRISPR/Cas9-induced HDR in zygotes using single-strand oligo DNA (ssODN) as template with an efficiency as high as 80%, indicating that pig zygotes exhibited high activities of HDR relative to NHEJ and were highly amendable to genetic mutation via CIRSPR/Cas9-induced HDR. Besides, we found a higher concentration of ssODN remarkably reduced HDR-derived mutation in pig zygotes, suggesting a possible balance for optimal HDR-derived mutation in zygotes between the excessive accessibility to HDR templates and the activities of HDR relative to NHEJ which appeared to be negatively correlated to ssODN concentration. In addition, the HDR-derived mutation, as well as those from NHEJ, extensively integrated into various tissues including gonad of founder pig without detected off-targeting, suggesting CRISPR/Cas9-induced HDR in zygotes is a reliable approach for precise genetic mutation in pigs.

  8. Acute myeloid leukemia of donor origin after allogeneic stem cell transplantation from a sibling who harbors germline XPD and XRCC3 homozygous polymorphisms

    Directory of Open Access Journals (Sweden)

    da Silva Dayse A

    2011-09-01

    Full Text Available Abstract A 54-year-old woman was diagnosed with infiltrative ductal breast carcinoma. Two years after treatment, the patient developed an acute myeloid leukemia (AML which harbored del(11q23 in 8% of the blast cells. The patient was submitted for allogeneic stem cell transplantation (aSCT from her HLA-compatible sister. Ten months after transplantation, she relapsed with an AML with basophilic maturation characterized by CD45low CD33high, CD117+, CD13-/+, HLA Drhigh, CD123high, and CD203c+ blast cells lacking expression of CD7, CD10, CD34, CD15, CD14, CD56, CD36, CD64, and cytoplasmic tryptase. Karyotype analysis showed the emergence of a new clone with t(2;14 and FISH analysis indicated the presence of MLL gene rearrangement consistent with del(11q23. Interestingly, AML blast cell DNA tested with microsatellite markers showed the same pattern as the donor's, suggesting that this AML emerged from donor cells. Additionally, polymorphisms of the XPA, XPD, XRCC1, XRCC3 and RAD51 DNA repair genes revealed three unfavorable alleles with low DNA repair capacity. In summary, we report the first case of AML involving XPD and XRCC3 polymorphisms from donor origin following allogeneic stem cell transplantation and highlight the potential need for careful analysis of DNA repair gene polymorphisms in selecting candidate donors prior to allogeneic stem cell transplantation.

  9. Pearl Harbor Biological Survey

    Science.gov (United States)

    1974-08-30

    Distribution of Porifera (Wet Weight in Grams) Collected in Piling Samples from Pearl Harbor, Oahu 2.4-26 2.4-7. Syllidae 2.4-28 2.4-8. Cirratulidae...COLLECTED FROM PEARL HARBOR m .-. Sped es/Group Porifera Demospongiae Cnidaria Hydrozoa Hydrdda Anthozoa Actlnarla Stolchactlnldae Radianthus...and abundance data for 113 taxa (species, genera and higher taxa) are provided in Table 2.4-4. Wet weights are listed for all sponges ( porifera ). The

  10. Gefitinib analogue V1801 induces apoptosis of T790M EGFR-harboring lung cancer cells by up-regulation of the BH-3 only protein Noxa.

    Directory of Open Access Journals (Sweden)

    Bo Zhang

    Full Text Available Treatment of non-small cell lung cancer (NSCLC with drugs targeting the epidermal growth factor receptor (EGFR, e.g., gefitinib and erlotinib, will eventually fail because of the development of secondary mutations such as T790M in EGFR. Strategies to overcome this resistance are therefore an urgent need. In this study, we synthesized a dozen of novel gefitinib analogues and evaluated their effects on L858R/T790M-EGFR harboring NSCLC cells, and reported that one of these gefitinib mimetics, N-(2-bromo-5-(trifluoromethyl phenyl-6-methoxy-7-(3-(piperidin-1-ylpropoxyquinazolin-4-amine (hereafter, V1801, triggered apoptosis of the NSCLC cells and overcame gefitinib-resistance in mice inoculated with NCI-H1975 cells. Though V1801 only moderately inhibited EGFR kinase activity, it markedly induced the expression of the BH3-only protein Noxa, and Noxa silencing significantly reduced V1801-induced apoptosis of NCI-H1975 cells. It is showed that V1801 interfered with the expression of the transcription factor c-Myc and the extracellular signal regulated kinase (Erk pathway. V1801 in combination with proteasome inhibitor bortezomib exerted enhanced cytotoxicity in NCI-H1975 cells possibly due to potentiated induction of Noxa expression. These data indicate that gefinitib analogues with weak EGFR inhibitory activity may overcome drug-resistance via activation of BH-3 only pro-apoptotic proteins, and V1801 may have therapeutic potentials for NSCLC.

  11. 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...

  12. Inducers & Organizers in Human Fetal and Adult Lymphoid Tissues : the characterization of RORC+ innate lymphoid cells and RANKL+ marginal reticular cells in human fetal and adult secondary lymphoid organs

    NARCIS (Netherlands)

    K. Hoorweg (Kerim)

    2014-01-01

    markdownabstract__Abstract__ The human immune system harbors the potential to generate novel lymphoid organs within inflamed tissues during disease. These tertiary lymphoid organs (TLOs) resemble lymph nodes and can contain segregated T and B cell areas, germinal centers, high endothelial venules a

  13. Human HDAC7 Harbors a Class IIa Histone Deacetylase-specific Zinc Binding Motif and Cryptic Deacetylase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Schuetz, Anja; Min, Jinrong; Allali-Hassani, Abdellah; Schapira, Matthieu; Shuen, Michael; Loppnau, Peter; Mazitschek, Ralph; Kwiatkowski, Nick P.; Lewis, Timothy A.; Maglathin, Rebecca L.; McLean, Thomas H.; Bochkarev, Alexey; Plotnikov, Alexander N.; Vedadi, Masoud; Arrowsmith, Cheryl H. (MIT); (Toronto)

    2010-10-18

    Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators.

  14. Engineering targeted chromosomal amplifications in human breast epithelial cells.

    Science.gov (United States)

    Springer, Simeon; Yi, Kyung H; Park, Jeenah; Rajpurohit, Anandita; Price, Amanda J; Lauring, Josh

    2015-07-01

    Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including "sawtooth" patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

  15. The preferred substrates for transglutaminase 2 in a complex wheat gluten digest are Peptide fragments harboring celiac disease T-cell epitopes.

    Directory of Open Access Journals (Sweden)

    Siri Dørum

    Full Text Available BACKGROUND: Celiac disease is a T-cell mediated chronic inflammatory disorder of the gut that is induced by dietary exposure to gluten proteins. CD4+ T cells of the intestinal lesion recognize gluten peptides in the context of HLA-DQ2.5 or HLA-DQ8 and the gluten derived peptides become better T-cell antigens after deamidation catalyzed by the enzyme transglutaminase 2 (TG2. In this study we aimed to identify the preferred peptide substrates of TG2 in a heterogeneous proteolytic digest of whole wheat gluten. METHODS: A method was established to enrich for preferred TG2 substrates in a complex gluten peptide mixture by tagging with 5-biotinamido-pentylamine. Tagged peptides were isolated and then identified by nano-liquid chromatography online-coupled to tandem mass spectrometry, database searching and final manual data validation. RESULTS: We identified 31 different peptides as preferred substrates of TG2. Strikingly, the majority of these peptides were harboring known gluten T-cell epitopes. Five TG2 peptide substrates that were predicted to bind to HLA-DQ2.5 did not contain previously characterized sequences of T-cell epitopes. Two of these peptides elicited T-cell responses when tested for recognition by intestinal T-cell lines of celiac disease patients, and thus they contain novel candidate T-cell epitopes. We also found that the intact 9mer core sequences of the respective epitopes were not present in all peptide substrates. Interestingly, those epitopes that were represented by intact forms were frequently recognized by T cells in celiac disease patients, whereas those that were present in truncated versions were infrequently recognized. CONCLUSION: TG2 as well as gastrointestinal proteolysis play important roles in the selection of gluten T-cell epitopes in celiac disease.

  16. The human gut and groundwater harbor non-photosynthetic bacteria belonging to a new candidate phylum sibling to Cyanobacteria.

    Science.gov (United States)

    Di Rienzi, Sara C; Sharon, Itai; Wrighton, Kelly C; Koren, Omry; Hug, Laura A; Thomas, Brian C; Goodrich, Julia K; Bell, Jordana T; Spector, Timothy D; Banfield, Jillian F; Ley, Ruth E

    2013-10-01

    Cyanobacteria were responsible for the oxygenation of the ancient atmosphere; however, the evolution of this phylum is enigmatic, as relatives have not been characterized. Here we use whole genome reconstruction of human fecal and subsurface aquifer metagenomic samples to obtain complete genomes for members of a new candidate phylum sibling to Cyanobacteria, for which we propose the designation 'Melainabacteria'. Metabolic analysis suggests that the ancestors to both lineages were non-photosynthetic, anaerobic, motile, and obligately fermentative. Cyanobacterial light sensing may have been facilitated by regulators present in the ancestor of these lineages. The subsurface organism has the capacity for nitrogen fixation using a nitrogenase distinct from that in Cyanobacteria, suggesting nitrogen fixation evolved separately in the two lineages. We hypothesize that Cyanobacteria split from Melainabacteria prior or due to the acquisition of oxygenic photosynthesis. Melainabacteria remained in anoxic zones and differentiated by niche adaptation, including for symbiosis in the mammalian gut. DOI:http://dx.doi.org/10.7554/eLife.01102.001.

  17. The human gut and groundwater harbor non-photosynthetic bacteria belonging to a new candidate phylum sibling to Cyanobacteria

    Science.gov (United States)

    Di Rienzi, Sara C; Sharon, Itai; Wrighton, Kelly C; Koren, Omry; Hug, Laura A; Thomas, Brian C; Goodrich, Julia K; Bell, Jordana T; Spector, Timothy D; Banfield, Jillian F; Ley, Ruth E

    2013-01-01

    Cyanobacteria were responsible for the oxygenation of the ancient atmosphere; however, the evolution of this phylum is enigmatic, as relatives have not been characterized. Here we use whole genome reconstruction of human fecal and subsurface aquifer metagenomic samples to obtain complete genomes for members of a new candidate phylum sibling to Cyanobacteria, for which we propose the designation ‘Melainabacteria’. Metabolic analysis suggests that the ancestors to both lineages were non-photosynthetic, anaerobic, motile, and obligately fermentative. Cyanobacterial light sensing may have been facilitated by regulators present in the ancestor of these lineages. The subsurface organism has the capacity for nitrogen fixation using a nitrogenase distinct from that in Cyanobacteria, suggesting nitrogen fixation evolved separately in the two lineages. We hypothesize that Cyanobacteria split from Melainabacteria prior or due to the acquisition of oxygenic photosynthesis. Melainabacteria remained in anoxic zones and differentiated by niche adaptation, including for symbiosis in the mammalian gut. DOI: http://dx.doi.org/10.7554/eLife.01102.001 PMID:24137540

  18. Mucosal-associated invariant T cells from induced pluripotent stem cells:A novel approach for modeling human diseases

    Institute of Scientific and Technical Information of China (English)

    Chie; Sugimoto; Hiroyoshi; Fujita; Hiroshi; Wakao

    2016-01-01

    Mice have frequently been used to model human diseases involving immune dysregulation such as autoimmune and inflammatory diseases.These models help elucidatethe mechanisms underlying the disease and in the development of novel therapies.However,if mice are deficient in certain cells and/or effectors associated with human diseases,how can their functions be investigated in this species?Mucosal-associated invariant T(MAIT)cells,a novel innate-like T cell family member,are a good example.MAIT cells are abundant in humans but scarce in laboratory mice.MAIT cells harbor an invariant T cell receptor and recognize nonpeptidic antigens vitamin B2metabolites from bacteria and yeasts.Recent studies have shown that MAIT cells play a pivotal role in human diseases such as bacterial infections and autoimmune and inflammatory diseases.MAIT cells possess granulysin,a human-specific effector molecule,but granulysin and its homologue are absent in mice.Furthermore,MAIT cells show poor proliferation in vitro.To overcome these problems and further our knowledge of MAIT cells,we have established a method to expand MAIT cells via induced pluripotent stem cells(iP SCs).In this review,we describe recent advances in the field of MAIT cell research and our approach for human disease modeling with iP SCderived MAIT cells.

  19. Immunohistochemical characterization of selected cell markers for the detection of hematopoietic cells in formalin-fixed, paraffin wax-embedded lymphoid tissues of harbor seals (Phoca vitulina) and walruses (Odobenus rosmarus rosmarus).

    Science.gov (United States)

    Seibel, H; Stimmer, L; Siebert, U; Beineke, A

    2010-10-15

    To facilitate a detailed investigation of pinniped lymphoid organs, 30 monoclonal antibodies (mAb) as well as eight polyclonal antibodies (pAb) of different species specificities directed against cell antigens of the hematopoietic system were tested for immunohistochemical cross-reactivity on formalin-fixed, paraffin wax-embedded tissues of harbor seals (Phoca vitulina) and a walrus (Odobenus rosmarus rosmarus). Six monoclonal and eight polyclonal antibodies showed specific immunoreactivities. Lymphocytes were immunolabeled by an anti-CD3 pAb, anti-Foxp3 mAb and anti-CD79 alpha mAb, while plasma cell subpopulations were recognized by anti-IgA pAb, anti-IgG pAb and anti-IgM pAb as well as by anti-kappa- and anti-lambda light chain pAb. Cells of the histiocytic lineage were recognized by lysozyme-, myeloid/histiocyte antigen-, and CD68-specific markers. Furthermore, dendritic cell-like cells were detected by an anti-S100 protein pAb. The MHC class II antigen was labeled on the majority of immune cells of the harbor seal and walrus using a bovine mAb. Mast cells were stained by an anti-mast cell tryptase mAb. Thus, using these antibodies from various species, it is now possible to determine phenotypical changes in lymphoid organs and detect different leukocyte subsets involved in inflammatory responses in archived tissue samples of these pinniped species.

  20. Targeting pancreatic progenitor cells in human embryonic stem cell differentiation for the identification of novel cell surface markers.

    Science.gov (United States)

    Fishman, Bettina; Segev, Hanna; Kopper, Oded; Nissenbaum, Jonathan; Schulman, Margarita; Benvenisty, Nissim; Itskovitz-Eldor, Joseph; Kitsberg, Danny

    2012-09-01

    New sources of beta cells are needed in order to develop cell therapies for patients with diabetes. An alternative to forced expansion of post-mitotic beta cells is the induction of differentiation of stem-cell derived progenitor cells that have a natural self-expansion capacity into insulin-producing cells. In order to learn more about these progenitor cells at different stages along the differentiation process in which they become progressively more committed to the final beta cell fate, we took the approach of identifying, isolating and characterizing stage specific progenitor cells. We generated human embryonic stem cell (HESC) clones harboring BAC GFP reporter constructs of SOX17, a definitive endoderm marker, and PDX1, a pancreatic marker, and identified subpopulations of GFP expressing cells. Using this approach, we isolated a highly enriched population of pancreatic progenitor cells from hESCs and examined their gene expression with an emphasis on the expression of stage-specific cell surface markers. We were able to identify novel molecules that are involved in the pancreatic differentiation process, as well as stage-specific cell markers that may serve to define (alone or in combination with other markers) a specific pancreatic progenitor cell. These findings may help in optimizing conditions for ultimately generating and isolating beta cells for transplantation therapy.

  1. 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....

  2. Assessing the Safety of Human Pluripotent Stem Cells and Their Derivatives for Clinical Applications

    Directory of Open Access Journals (Sweden)

    Peter W. Andrews

    2017-07-01

    Full Text Available Pluripotent stem cells may acquire genetic and epigenetic variants during culture following their derivation. At a conference organized by the International Stem Cell Initiative, and held at The Jackson Laboratory, Bar Harbor, Maine, October 2016, participants discussed how the appearance of such variants can be monitored and minimized and, crucially, how their significance for the safety of therapeutic applications of these cells can be assessed. A strong recommendation from the meeting was that an international advisory group should be set up to review the genetic and epigenetic changes observed in human pluripotent stem cell lines and establish a framework for evaluating the risks that they may pose for clinical use.

  3. Genetically engineered T cells bearing chimeric nanoconstructed receptors harboring TAG-72-specific camelid single domain antibodies as targeting agents

    DEFF Research Database (Denmark)

    Sharifzadeh, Zahra; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad A

    2013-01-01

    Despite the preclinical success of adoptive therapy with T cells bearing chimeric nanoconstructed antigen receptors (CARs), certain limitations of this therapeutic approach such as the immunogenicity of the antigen binding domain, the emergence of tumor cell escape variants and the blocking capac...... to reverse multiple tumor immune evasion mechanisms, avoid CAR immunogenicity, and overcome problems in cancer gene therapy with engineered nanoconstructs....

  4. Impaired TGF-beta induced growth inhibition contributes to the increased proliferation rate of neural stem cells harboring mutant p53

    DEFF Research Database (Denmark)

    Kumar, P.; Naumann, U.; Aigner, L.;

    2015-01-01

    Gliomas have been classified according to their histological properties. However, their respective cells of origin are still unknown. Neural progenitor cells (NPC) from the subventricular zone (SVZ) can initiate tumors in murine models of glioma and are likely cells of origin in the human disease...

  5. Narrow harbors. Few joint ventures will find haven in the investment-interest safe harbor.

    Science.gov (United States)

    Lepper, G J; Swoboda, J

    1991-12-01

    Investors and potential investors had hoped for meaningful guidance from the safe harbor regulations on appropriate structures for healthcare joint ventures. Unfortunately, the narrowly drawn final investment-interest safe harbor offers relatively little meaningful guidance or protection for the vast majority of such ventures. The Illegal Remuneration Statute (also known as the fraud and abuse statute) was first enacted in 1972 to prohibit members of the healthcare community from exchanging patient referrals for any kind of remuneration. In 1987 Congress instructed the secretary of Health and Human Services to create "safe harbors" for legitimate payment practices that, although they may violate the statute's strict prohibition, will be protected from prosecution. The investment-interest safe harbor has garnered the most attention. It provides two safe harbors, one for investments in large entities and one for investments in small entities. Both safe harbors contain onerous threshold requirements and other restrictions that diminish the usefulness of the safe harbor for all but a very few ventures. In addition, the Office of the Inspector General has created other obstacles to forming and preserving "safe" healthcare business ventures, including a refusal to "grandfather" or create a "safe harbor restructuring period" for existing business arrangements. Because most existing or planned joint ventures do not qualify for the investment-interest safe harbor, investors are forced to make their business decisions on the basis of the same factors used before publication of the safe harbor regulations. Such analysis will continue to focus on factors that demonstrate organizations' intent in making payments to investors as a return on investments.

  6. Nitrilase-catalyzed conversion of (R,S)-mandelonitrile by immobilized recombinant Escherichia coli cells harboring nitrilase.

    Science.gov (United States)

    Zhang, Xin-Hong; Liu, Zhi-Qiang; Xue, Ya-Ping; Xu, Ming; Zheng, Yu-Guo

    2016-07-01

    (R)-(-)-Mandelic acid (R-MA) is widely used both as a versatile intermediate for pharmaceuticals and a resolving agent in chiral resolution processes. In the current study, to improve the stability of operation, recombinant Escherichia coli cells expressing nitrilase from Alcaligenes faecalis were immobilized for the enantioselective hydrolysis of (R,S)-mandelonitrile to R-MA. Different immobilization methods including entrapment matrices, entrapment matrices cross-linked by cross-linking and polymerization agents, and direct cross-linking cells using glutaraldehyde (GA) or bionic silicon were investigated. To facilitate industrial solid-liquid separation, the direct cross-linking recombinant E. coli cells using diatomite/GA/polyethyleneimine with 135.95% relative activity compared with free cells was chosen using water as the reaction medium. The operational stability of the immobilized cells was obviously superior to that of free cells, without significant activity loss after 28 cycles of batch reaction and the successive production of R-MA could reach 1.88 M. Moreover, the immobilized cells showed good storage stability with about 52% relative activity after storing for 30 days at 4 °C. Therefore, the immobilized biocatalyst is very promising for upscale production of optically pure R-MA with high performance and low cost.

  7. Metabolically active CD4+ T cells expressing Glut1 and OX40 preferentially harbor HIV during in vitro infection.

    Science.gov (United States)

    Palmer, Clovis S; Duette, Gabriel A; Wagner, Marc C E; Henstridge, Darren C; Saleh, Suah; Pereira, Candida; Zhou, Jingling; Simar, David; Lewin, Sharon R; Ostrowski, Matias; McCune, Joseph M; Crowe, Suzanne M

    2017-09-11

    High Glut1 surface expression is associated with increased glycolytic activity in activated CD4+ T cells. PI3K activation measured by p-Akt and OX40 is elevated in CD4+Glut1+ T cells from HIV+ subjects. TCR engagement of CD4+Glut1+ T cells from HIV+ subjects demonstrate hyper-responsive PI3K-mTOR signalling. High basal Glut1 and OX40 on CD4+ T cells from combination antiretroviral therapy (cART)-treated HIV+ patients represent a sufficiently metabolically active state permissive for HIV infection in vitro without external stimuli. The majority of CD4+OX40+ T cells express Glut1, thus OX40 rather than Glut1 itself may facilitate HIV infection. Furthermore, infection of CD4+ T cells is limited by p110γ PI3K inhibition. Modulating glucose metabolism may limit cellular activation and prevent residual HIV replication in 'virologically suppressed' cART-treated HIV+ persons. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  8. Medicago truncatula and Glomus intraradices gene expression in cortical cells harboring arbuscules in the arbuscular mycorrhizal symbiosis

    Directory of Open Access Journals (Sweden)

    Tang Yuhong

    2009-01-01

    Full Text Available Abstract Background Most vascular flowering plants have the capacity to form symbiotic associations with arbuscular mycorrhizal (AM fungi. The symbiosis develops in the roots where AM fungi colonize the root cortex and form arbuscules within the cortical cells. Arbuscules are enveloped in a novel plant membrane and their establishment requires the coordinated cellular activities of both symbiotic partners. The arbuscule-cortical cell interface is the primary functional interface of the symbiosis and is of central importance in nutrient exchange. To determine the molecular events the underlie arbuscule development and function, it is first necessary to identify genes that may play a role in this process. Toward this goal we used the Affymetrix GeneChip® Medicago Genome Array to document the M. truncatula transcript profiles associated with AM symbiosis, and then developed laser microdissection (LM of M. truncatula root cortical cells to enable analyses of gene expression in individual cell types by RT-PCR. Results This approach led to the identification of novel M. truncatula and G. intraradices genes expressed in colonized cortical cells and in arbuscules. Within the arbuscule, expression of genes associated with the urea cycle, amino acid biosynthesis and cellular autophagy was detected. Analysis of gene expression in the colonized cortical cell revealed up-regulation of a lysine motif (LysM-receptor like kinase, members of the GRAS transcription factor family and a symbiosis-specific ammonium transporter that is a likely candidate for mediating ammonium transport in the AM symbiosis. Conclusion Transcript profiling using the Affymetrix GeneChip® Medicago Genome Array provided new insights into gene expression in M. truncatula roots during AM symbiosis and revealed the existence of several G. intraradices genes on the M. truncatula GeneChip®. A laser microdissection protocol that incorporates low-melting temperature Steedman's wax, was

  9. 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

  10. Different spectra of recurrent gene mutations in subsets of chronic lymphocytic leukemia harboring stereotyped B-cell receptors

    DEFF Research Database (Denmark)

    Sutton, Lesley-Ann; Young, Emma; Baliakas, Panagiotis

    2016-01-01

    We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobulins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations...... in subsets #2 and #8 (both 2%), despite all being clinically aggressive. All subsets were negative for MYD88 mutations, whereas BIRC3 mutations were infrequent. Collectively, this striking bias and skewed distribution of mutations and cytogenetic aberrations within specific chronic lymphocytic leukemia...... subsets implies that the mechanisms underlying clinical aggressiveness are not uniform, but rather support the existence of distinct genetic pathways of clonal evolution governed by a particular stereotyped B-cell receptor selecting a certain molecular lesion(s)....

  11. Port and Harbor Security

    Energy Technology Data Exchange (ETDEWEB)

    Saito, T; Guthmuller, H; DeWeert, M

    2004-12-15

    Port and Harbor Security is a daunting task to which optics and photonics offers significant solutions. We are pleased to report that the 2005 Defense and Security Symposium (DSS, Orlando, FL) will include reports on active and passive photonic systems operating from both airborne and subsurface platforms. In addition to imaging techniques, there are various photonic applications, such as total internal reflection fluorescence (TIRF), which can be used to ''sniff'' for traces of explosives or contaminants in marine. These non-imaging technologies are beyond the scope of this article, but will also be represented at DSS 2005. We encourage colleagues to join our technical group to help us to make our ports and harbors safer and more secure.

  12. 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.

  13. Toward an ideal animal model to trace donor cell fates after stem cell therapy: production of stably labeled multipotent mesenchymal stem cells from bone marrow of transgenic pigs harboring enhanced green fluorescence protein gene.

    Science.gov (United States)

    Hsiao, F S H; Lian, W S; Lin, S P; Lin, C J; Lin, Y S; Cheng, E C H; Liu, C W; Cheng, C C; Cheng, P H; Ding, S T; Lee, K H; Kuo, T F; Cheng, C F; Cheng, W T K; Wu, S C

    2011-11-01

    The discovery of postnatal mesenchymal stem cells (MSC) with their general multipotentiality has fueled much interest in the development of cell-based therapies. Proper identification of transplanted MSC is crucial for evaluating donor cell distribution, differentiation, and migration. Lack of an efficient marker of transplanted MSC has precluded our understanding of MSC-related regenerative studies, especially in large animal models such as pigs. In the present study, we produced transgenic pigs harboring an enhanced green fluorescent protein (EGFP) gene. The pigs provide a reliable and reproducible source for obtaining stable EGFP-labeled MSC, which is very useful for donor cell tracking after transplantation. The undifferentiated EGFP-tagged MSC expressed a greater quantity of EGFP while maintaining MSC multipotentiality. These cells exhibited homogeneous surface epitopes and possessed classic trilineage differentiation potential into osteogenic, adipogenic, and chondrogenic lineages, with robust EGFP expression maintained in all differentiated progeny. Injection of donor MSC can dramatically increase the thickness of infarcted myocardium and improve cardiac function in mice. Moreover, the MSC, with their strong EGFP expression, can be easily distinguished from the background autofluorescence in myocardial infarcts. We demonstrated an efficient, effective, and easy way to identify MSC after long-term culture and transplantation. With the transgenic model, we were able to obtain stem or progenitor cells in earlier passages compared with the transfection of traceable markers into established MSC. Because the integration site of the transgene was the same for all cells, we lessened the potential for positional effects and the heterogeneity of the stem cells. The EGFP-transgenic pigs may serve as useful biomedical and agricultural models of somatic stem cell biology.

  14. Pristimerin induces apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation by blocking NF-κB signaling and depleting Bcr-Abl

    Directory of Open Access Journals (Sweden)

    Cao Qi

    2010-05-01

    Full Text Available Abstract Background Chronic myelogenous leukemia (CML is characterized by the chimeric tyrosine kinase Bcr-Abl. Bcr-Abl-T315I is the notorious point mutation that causes resistance to imatinib and the second generation tyrosine kinase inhibitors, leading to poor prognosis. CML blasts have constitutive p65 (RelA NF-κB transcriptional activity, and NF-κB may be a potential target for molecular therapies in CML that may also be effective against CML cells with Bcr-Abl-T315I. Results In this report, we discovered that pristimerin, a quinonemethide triterpenoid isolated from Celastraceae and Hippocrateaceae, inhibited growth and induced apoptosis in CML cells, including the cells harboring Bcr-Abl-T315I mutation. Additionally, pristimerin inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Pristimerin blocked the TNFα-induced IκBα phosphorylation, translocation of p65, and expression of NF-κB-regulated genes. Pristimerin inhibited two steps in NF-κB signaling: TAK1→IKK and IKK→IκBα. Pristimerin potently inhibited two pairs of CML cell lines (KBM5 versus KBM5-T315I, 32D-Bcr-Abl versus 32D-Bcr-Abl-T315I and primary cells from a CML patient with acquired resistance to imatinib. The mRNA and protein levels of Bcr-Abl in imatinib-sensitive (KBM5 or imatinib-resistant (KBM5-T315I CML cells were reduced after pristimerin treatment. Further, inactivation of Bcr-Abl by imatinib pretreatment did not abrogate the TNFα-induced NF-κB activation while silencing p65 by siRNA did not affect the levels of Bcr-Abl, both results together indicating that NF-κB inactivation and Bcr-Abl inhibition may be parallel independent pathways. Conclusion To our knowledge, this is the first report to show that pristimerin is effective in vitro and in vivo against CML cells, including those with the T315I mutation. The mechanisms may involve inhibition of NF-κB and Bcr-Abl. We concluded that pristimerin could be a lead compound for

  15. Elevated PC responsive B cells and anti-PC antibody production in transgenic mice harboring anti-PC immunoglobulin genes.

    Science.gov (United States)

    Pinkert, C A; Manz, J; Linton, P J; Klinman, N R; Storb, U

    1989-12-01

    The rearrangement of heavy and light chain immunoglobulin genes is necessary for the production of functional antibody molecules. The myeloma MOPC 167 produces specific antibodies to the antigen phosphorylcholine (PC), which is present on bacterial surfaces, fungi and other environmental contaminants. Rearranged heavy and light chain immunoglobulin genes cloned from MOPC 167 were microinjected into mouse eggs. Within the resulting transgenic mice, expression of the transgenes were limited to lymphoid tissues. Transgenic mice produced elevated levels of anti-PC antibodies constitutively, at 16 days of age, when normal non-transgenic mice were not fully immunocompetent. A triggering antigenic stimulus was not necessary to evoke anti-PC immunoglobulin production. Additionally, the frequency of PC-responsive B cells in these transgenic mice was further increased upon specific immunization.

  16. Human heredity and politics: A comparative institutional study of the Eugenics Record Office at Cold Spring Harbor (United States), the Kaiser Wilhelm Institute for Anthropology, Human Heredity, and Eugenics (Germany), and the Maxim Gorky Medical Genetics Institute (USSR).

    Science.gov (United States)

    Adams, Mark B; Allen, Garland E; Weiss, Sheila Faith

    2005-01-01

    Despite the fact that much has been written in recent years about the science of heredity under the Third Reich, there is as yet no satisfying analysis of two central questions: What, if anything, was peculiarly "Nazi" about human genetics under National Socialism? How, under whatever set of causes, did at least some of Germany's most well-known and leading biomedical practioners become engaged in entgrenzte Wissenschaft (science without moral boundaries)? This paper attempts to provide some answers to these two questions comparing three institutes that studied eugenics and human heredity in the 1920s and 1930s: the Eugenics Record Office at Cold Spring Harbor, New York, directed by Charles B. Davenport; the Kaiser Wilhelm Institute for Anthropology, Human Heredity and Eugenics, in Berlin, directed by Eugen Fischer; and the Maxim Gorky Medical Genetics Institute in Moscow, directed by Solomon G. Levit. The institutes are compared on the basis of the kind and quality of their research in eugenics and medical genetics, organizational structure, leadership, patronage (private or state), and the economic-social-political context in which they functioned.

  17. Anodic biofilms in microbial fuel cells harbor low numbers of higher-power-producing bacteria than abundant genera

    Energy Technology Data Exchange (ETDEWEB)

    Kiely, Patrick D.; Call, Douglas F.; Yates, Matthew D.; Regan, John M.; Logan, Bruce E. [Pennsylvania State Univ., University Park, PA (United States). Dept. of Civil and Environmental Engineering

    2010-09-15

    Microbial fuel cell (MFC) anode communities often reveal just a few genera, but it is not known to what extent less abundant bacteria could be important for improving performance. We examined the microbial community in an MFC fed with formic acid for more than 1 year and determined using 16S rRNA gene cloning and fluorescent in situ hybridization that members of the Paracoccus genus comprised most ({proportional_to}30%) of the anode community. A Paracoccus isolate obtained from this biofilm (Paracoccus denitrificans strain PS-1) produced only 5.6 mW/m{sup 2}, whereas the original mixed culture produced up to 10 mW/m{sup 2}. Despite the absence of any Shewanella species in the clone library, we isolated a strain of Shewanella putrefaciens (strain PS-2) from the same biofilm capable of producing a higher-power density (17.4 mW/m{sup 2}) than the mixed culture, although voltage generation was variable. Our results suggest that the numerical abundance of microorganisms in biofilms cannot be assumed a priori to correlate to capacities of these predominant species for high-power production. Detailed screening of bacterial biofilms may therefore be needed to identify important strains capable of high-power generation for specific substrates. (orig.)

  18. Anodic biofilms in microbial fuel cells harbor low numbers of higher-power-producing bacteria than abundant genera

    KAUST Repository

    Kiely, Patrick D.

    2010-07-15

    Microbial fuel cell (MFC) anode communities often reveal just a few genera, but it is not known to what extent less abundant bacteria could be important for improving performance. We examined the microbial community in an MFC fed with formic acid for more than 1 year and determined using 16S rRNA gene cloning and fluorescent in situ hybridization that members of the Paracoccus genus comprised most (~30%) of the anode community. A Paracoccus isolate obtained from this biofilm (Paracoccus denitrificans strain PS-1) produced only 5.6 mW/m 2, whereas the original mixed culture produced up to 10 mW/m 2. Despite the absence of any Shewanella species in the clone library, we isolated a strain of Shewanella putrefaciens (strain PS-2) from the same biofilm capable of producing a higher-power density (17.4 mW/m2) than the mixed culture, although voltage generation was variable. Our results suggest that the numerical abundance of microorganisms in biofilms cannot be assumed a priori to correlate to capacities of these predominant species for high-power production. Detailed screening of bacterial biofilms may therefore be needed to identify important strains capable of high-power generation for specific substrates. © 2010 Springer-Verlag.

  19. Cold Spring Harbor symposia on quantitative biology

    Energy Technology Data Exchange (ETDEWEB)

    1989-01-01

    This volume contains the first part of the proceeding of the 53rd Cold Springs Harbor Symposium on Quantitative Biology. This years topic was Immune Recognition. Part 1, this volume, contains papers prepared by presenters of the sessions entitled Introduction, Lymphocyte Development and Receptor Selection, and Recognition by Antibodies, Antigen Recognition by T cells. (DT)

  20. Adipogenic human adenovirus Ad-36 induces commitment, differentiation, and lipid accumulation in human adipose-derived stem cells

    DEFF Research Database (Denmark)

    Pasarica, Magdalena; Mashtalir, Nazar; McAllister, Emily J

    2008-01-01

    , the effect of the virus on commitment, differentiation, and lipid accumulation was investigated in vitro in primary human adipose-derived stem/stromal cells (hASC). Ad-36 infected hASC in a time- and dose-dependent manner. Even in the presence of osteogenic media, Ad-36-infected hASC showed significantly...... and the accumulation of its extracellular fraction. hASC from subjects harboring Ad-36 DNA in their adipose tissue due to natural infection had significantly greater ability to differentiate compared with Ad-36 DNA-negative counterparts, which offers a proof of concept. Thus, Ad-36 has the potential to induce...

  1. 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

  2. Repair at single targeted DNA double-strand breaks in pluripotent and differentiated human cells.

    Directory of Open Access Journals (Sweden)

    Hua Fung

    Full Text Available Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR or nonhomologous end-joining (NHEJ. For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny.

  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. Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells

    Directory of Open Access Journals (Sweden)

    Hiroshi Sato

    2017-01-01

    Full Text Available Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV. Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

  6. Targeting androgen receptor/Src complex impairs the aggressive phenotype of human fibrosarcoma cells.

    Science.gov (United States)

    Castoria, Gabriella; Giovannelli, Pia; Di Donato, Marzia; Hayashi, Ryo; Arra, Claudio; Appella, Ettore; Auricchio, Ferdinando; Migliaccio, Antimo

    2013-01-01

    Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR). We report that the pure anti-androgen Casodex inhibits the growth of HT1080 cell xenografts in immune-depressed mice, revealing a novel role of AR in fibrosarcoma progression. In HT1080 cultured cells EGF, but not androgens, robustly increases DNA synthesis. Casodex abolishes the EGF mitogenic effect, implying a crosstalk between EGFR and AR. The mechanism underlying this crosstalk has been analyzed using an AR-derived small peptide, S1, which prevents AR/Src tyrosine kinase association and androgen-dependent Src activation. Present findings show that in HT1080 cells EGF induces AR/Src Association, and the S1 peptide abolishes both the assembly of this complex and Src activation. The S1 peptide inhibits EGF-stimulated DNA synthesis, cell matrix metalloproteinase-9 (MMP-9) secretion and invasiveness of HT1080 cells. Both Casodex and S1 peptide also prevent DNA synthesis and migration triggered by EGF in various human cancer-derived cells (prostate, breast, colon and pancreas) that express AR. This study shows that targeting the AR domain involved in AR/Src association impairs EGF signaling in human fibrosarcoma HT1080 cells. The EGF-elicited processes inhibited by the peptide (DNA synthesis, MMP-9 secretion and invasiveness) cooperate in increasing the aggressive phenotype of HT1080 cells. Therefore, AR represents a new potential therapeutic target in human fibrosarcoma, as supported by Casodex inhibition of HT1080 cell xenografts. The extension of these findings in various human cancer-derived cell lines highlights the conservation of this process across divergent cancer

  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. New hPSC-based human models to study pediatric Acute Megakaryoblastic Leukemia harboring the fusion oncogene RBM15-MKL1

    Directory of Open Access Journals (Sweden)

    Verónica Ayllón

    2017-03-01

    Full Text Available Pediatric Acute Megakaryoblastic Leukemia not associated to Down Syndrome (non-DS AMKL is a rare disease with a dismal prognosis. Around 15% of patients carry the chromosomal translocation t(1;22 that originates the fusion oncogene RBM15-MKL1, which is linked to an earlier disease onset (median of 6 months of age and arises in utero. Here we report the generation of two hPSC cell lines constitutively expressing the oncogene RBM15-MKL1, resulting in an increased expression of known RBM15-MKL1 gene targets. These cell lines represent new disease models of pediatric AMKL to study the impact of the RBM15-MKL1 oncogene on human embryonic hematopoietic development.

  9. 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.

  10. Satellite cells in human skeletal muscle plasticity.

    Science.gov (United States)

    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.

  11. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.

    Science.gov (United States)

    Natsume, Toyoaki; Kiyomitsu, Tomomi; Saga, Yumiko; Kanemaki, Masato T

    2016-04-01

    Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function.

  12. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors

    Directory of Open Access Journals (Sweden)

    Toyoaki Natsume

    2016-04-01

    Full Text Available Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function.

  13. Interferon-γ-induced upregulation of immunoproteasome subunit assembly overcomes bortezomib resistance in human hematological cell lines

    OpenAIRE

    2014-01-01

    Background Despite encouraging results with the proteasome inhibitor bortezomib in the treatment of hematologic malignancies, emergence of resistance can limit its efficacy, hence calling for novel strategies to overcome bortezomib-resistance. We previously showed that bortezomib-resistant human leukemia cell lines expressed significantly lower levels of immunoproteasome at the expense of constitutive proteasomes, which harbored point mutations in exon 2 of the PSMB5 gene encoding the β5 subu...

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Prey capture by harbor porpoises

    DEFF Research Database (Denmark)

    Miller, Lee; Verfuss, Ursula

    2009-01-01

      The harbor porpoise (Phocoena phocoena) is a small toothed whale living mostly in coastal waters.  There are large, but unknown, numbers in the inner Danish waters. Four are in captivity at Fjord & Bælt, Kerteminde, Denmark, one of which was born here in 2006. Harbor porpoises use their ultraso...

  19. Network meta-analysis of erlotinib, gefitinib, afatinib and icotinib in patients with advanced non-small-cell lung cancer harboring EGFR mutations.

    Directory of Open Access Journals (Sweden)

    Wenhua Liang

    Full Text Available BACKGROUND: Several EGFR-tyrosine kinase inhibitors (EGFR-TKIs including erlotinib, gefitinib, afatinib and icotinib are currently available as treatment for patients with advanced non-small-cell lung cancer (NSCLC who harbor EGFR mutations. However, no head to head trials between these TKIs in mutated populations have been reported, which provides room for indirect and integrated comparisons. METHODS: We searched electronic databases for eligible literatures. Pooled data on objective response rate (ORR, progression free survival (PFS, overall survival (OS were calculated. Appropriate networks for different outcomes were established to incorporate all evidences. Multiple-treatments comparisons (MTCs based on Bayesian network integrated the efficacy and specific toxicities of all included treatments. RESULTS: Twelve phase III RCTs that investigated EGFR-TKIs involving 1821 participants with EGFR mutation were included. For mutant patients, the weighted pooled ORR and 1-year PFS of EGFR-TKIs were significant superior to that of standard chemotherapy (ORR: 66.6% vs. 30.9%, OR 5.46, 95%CI 3.59 to 8.30, P<0.00001; 1-year PFS: 42.9% vs. 9.7%, OR 7.83, 95%CI 4.50 to 13.61; P<0.00001 through direct meta-analysis. In the network meta-analyses, no statistically significant differences in efficacy were found between these four TKIs with respect to all outcome measures. Trend analyses of rank probabilities revealed that the cumulative probabilities of being the most efficacious treatments were (ORR, 1-year PFS, 1-year OS, 2-year OS: erlotinib (51%, 38%, 14%, 19%, gefitinib (1%, 6%, 5%, 16%, afatinib (29%, 27%, 30%, 27% and icotinib (19%, 29%, NA, NA, respectively. However, afatinib and erlotinib showed significant severer rash and diarrhea compared with gefitinib and icotinib. CONCLUSIONS: The current study indicated that erlotinib, gefitinib, afatinib and icotinib shared equivalent efficacy but presented different efficacy-toxicity pattern for EGFR

  20. Synergistic growth inhibition of cancer cells harboring the RET/PTC1 oncogene by staurosporine and rotenone involves enhanced cell death

    Indian Academy of Sciences (India)

    António Pedro Gonçalves; Arnaldo Videira; Valdemar Máximo; Paula Soares

    2011-09-01

    TPC-1 is a highly proliferative thyroid papillary carcinoma-derived cell line. These cells express the RET/PTC1 fusion protein, whose isoforms are characterized in this work. The bacterial alkaloid staurosporine and the plant extract rotenone are death-inducing drugs that have an inhibitory synergistic effect on the growth of TPC-1 cells. We show that this synergism is accompanied by an enhancement of the induction of cell death. Staurosporine alone induces cell cycle arrest in G1, whereas rotenone induces arrest in G2/M. We suggest that this additive pressure may drive cells to die, resulting in the synergistic interaction of the drug combination. These data emphasize the potential use of the staurosporine plus rotenone combination as an anticancer tool.

  1. 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.

  2. Nucleic acid-based polymers effective against hepatitis B Virus infection in patients don’t harbor immunostimulatory properties in primary isolated liver cells

    Science.gov (United States)

    Real, Catherine Isabell; Werner, Melanie; Paul, Andreas; Gerken, Guido; Schlaak, Joerg Friedrich; Vaillant, Andrew; Broering, Ruth

    2017-01-01

    Nucleic acid polymers (NAPs) block the release of subviral particles from hepatocytes, a mechanism consistent with their antiviral activity against hepatitis B virus (HBV) in patients. Analysis of immunostimulatory properties of NAPs were conducted with several NAP species: REP 2006, the prototypic degenerate NAP [dN]40, containing TLR9-stimulatory CpG; REP 2055 a clinically active NAP with a sequence [dAdC]20 devoid of CpG content; REP 2139 (also clinically active) and REP 2165 (REP 2055 analogues further rendered immunologically inactive by replacing cytidine with 5-methylcytidine and incorporating 2′-O methylation of riboses). These analyses revealed pro-inflammatory responses in human peripheral blood mononuclear cells with REP 2006 and with REP 2139 and REP 2165 only at high dose but displayed no significant antiviral activity. In primary isolated human hepatocytes and liver sinusoidal endothelial cells no significant inflammatory or antiviral responses were detected for any NAPs. In human Kupffer cells pro-inflammatory activity was observed with REP 2006 and REP 2055, whereas a weak but significant induction of interferon genes was only observed with REP 2006 at the highest concentration. We therefore hypothesize that the antiviral activity of NAPs optimized to treat HBV infection in patients cannot be explained by direct induction of innate antiviral responses. PMID:28272460

  3. Obtaining freshly isolated and cultured mesenchymal stem cells from human adipose tissue.

    Science.gov (United States)

    Boquest, Andrew C; Collas, Philippe

    2012-01-01

    The stromal compartment of adipose tissue harbors mesenchymal stem cells (MSCs) (also called stromal stem cells) that display extensive proliferative capacity and multilineage differentiation potential. Such cells offer a practical avenue of generating patient-matched tissue for use in regenerative medicine. It is relatively easy to isolate these cells from adipose tissue in large enough quantities (tens of millions) to allow for their clinical use in a native, uncultured form. Alternatively, MSCs from adipose tissue can be expanded and differentiated into the desired tissue type in vitro using straightforward cell culture techniques. In this chapter, we outline procedures for isolating large numbers of highly purified MSCs from human adipose tissue in their native, uncultured form and methods for their subsequent expansion and differentiation in vitro.

  4. 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

  5. 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...

  6. Targeting of the human coagulation factor IX gene at rDNA locus of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Xionghao Liu

    Full Text Available BACKGROUND: Genetic modification is a prerequisite to realizing the full potential of human embryonic stem cells (hESCs in human genetic research and regenerative medicine. Unfortunately, the random integration methods that have been the primary techniques used keep creating problems, and the primary alternative method, gene targeting, has been effective in manipulating mouse embryonic stem cells (mESCs but poorly in hESCs. METHODOLOGY/PRINCIPAL FINDINGS: Human ribosomal DNA (rDNA repeats are clustered on the short arm of acrocentric chromosomes. They consist of approximately 400 copies of the 45S pre-RNA (rRNA gene per haploid. In the present study, we targeted a physiological gene, human coagulation factor IX, into the rDNA locus of hESCs via homologous recombination. The relative gene targeting efficiency (>50% and homologous recombination frequency (>10(-5 were more than 10-fold higher than those of loci targeted in previous reports. Meanwhile, the targeted clones retained both a normal karyotype and the main characteristics of ES cells. The transgene was found to be stably and ectopically expressed in targeted hESCs. CONCLUSION/SIGNIFICANCE: This is the first targeting of a human physiological gene at a defined locus on the hESC genome. Our findings indicate that the rDNA locus may serve as an ideal harbor for transgenes in hESCs.

  7. 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...

  8. 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.

  9. Energy metabolism in human pluripotent stem cells and their differentiated counterparts.

    Directory of Open Access Journals (Sweden)

    Sandra Varum

    Full Text Available Human pluripotent stem cells have the ability to generate all cell types present in the adult organism, therefore harboring great potential for the in vitro study of differentiation and for the development of cell-based therapies. Nonetheless their use may prove challenging as incomplete differentiation of these cells might lead to tumoregenicity. Interestingly, many cancer types have been reported to display metabolic modifications with features that might be similar to stem cells. Understanding the metabolic properties of human pluripotent stem cells when compared to their differentiated counterparts can thus be of crucial importance. Furthermore recent data has stressed distinct features of different human pluripotent cells lines, namely when comparing embryo-derived human embryonic stem cells (hESCs and induced pluripotent stem cells (IPSCs reprogrammed from somatic cells.We compared the energy metabolism of hESCs, IPSCs, and their somatic counterparts. Focusing on mitochondria, we tracked organelle localization and morphology. Furthermore we performed gene expression analysis of several pathways related to the glucose metabolism, including glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA cycle. In addition we determined oxygen consumption rates (OCR using a metabolic extracellular flux analyzer, as well as total intracellular ATP levels by high performance liquid chromatography (HPLC. Finally we explored the expression of key proteins involved in the regulation of glucose metabolism.Our results demonstrate that, although the metabolic signature of IPSCs is not identical to that of hESCs, nonetheless they cluster with hESCs rather than with their somatic counterparts. ATP levels, lactate production and OCR revealed that human pluripotent cells rely mostly on glycolysis to meet their energy demands. Furthermore, our work points to some of the strategies which human pluripotent stem cells may use to maintain high

  10. Sediment toxicity in Savannah Harbor

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — Savannah Harbor, located near the mouth of the Savannah River, Georgia and South Carolina, is impacted by industrial and municipal effluents. Contaminants released...

  11. Alaska Harbor Seal Glacial Surveys

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Floating glacial ice serves as a haul-out substrate for a significant number (10-15%) of Alaskan harbor seals, and thus surveying tidewater glacial fjords is an...

  12. 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.

  13. 2007 China Harbor Ten People

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    @@ 2007 China Harbor Ten People elected the entrepreneurs who contributed a lot to port economy and enterprises this year trough their talent management.These ten people embody their social responsibility,professional skills,creative ability,and charming personality.Bearing full confidence in China's port economy,the port entrepreneurs are brave enough to explore a brand new area,so as to promote harbor economic development.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Analysis of the role of homology arms in gene-targeting vectors in human cells.

    Directory of Open Access Journals (Sweden)

    Ayako Ishii

    Full Text Available Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ, a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4, the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.

  1. 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

  2. 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.

  3. 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.

  4. Genomic instability and tumorigenic induction in immortalized human bronchial epithelial cells by heavy ions

    Science.gov (United States)

    Hei, T. K.; Piao, C. Q.; Wu, L. J.; Willey, J. C.; Hall, E. J.

    1998-11-01

    Carcinogenesis is postulated to be a progressive multistage process characterized by an increase in genomic instability and clonal selection with each mutational event endowing a selective growth advantage. Genomic instability as manifested by the amplification of specific gene fragments is common among tumor and transformed cells. In the present study, immortalized human bronchial (BEP2D) cells were irradiated with graded doses of either 1GeV/nucleon 56Fe ions or 150 keV/μm alpha particles. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Tumorigenic cells showed neither ras mutations nor deletion in the p16 tumor suppressor gene. In contrast, they harbored mutations in the p53 gene and over-expressed cyclin D1. Genomic instability among transformed cells at various stage of the carcinogenic process was examined based on frequencies of PALA resistance. Incidence of genomic instability was highest among established tumor cell lines relative to transformed, non-tumorigenic and control cell lines. Treatment of BEP2D cells with a 4 mM dose of the aminothiol WR-1065 significantly reduced their neoplastic transforming response to 56Fe particles. This model provides an opportunity to study the cellular and molecular mechanisms involved in malignant transformation of human epithelial cells by heavy ions.

  5. Human cancer cells with stem cell-like phenotype exhibit enhanced sensitivity to the cytotoxicity of IL-2 and IL-15 activated natural killer cells.

    Science.gov (United States)

    Yin, Tao; Wang, Guoping; He, Sisi; Liu, Qin; Sun, Jianhong; Wang, Yongsheng

    2016-02-01

    Tumors harbor a population of cancer stem cells (CSCs) which can drive tumor progression and therapeutical resistance. Nature killer (NK) cells are best known for their ability to directly recognize and kill malignant cells. However, the susceptibility of cancer stem cells to NK cells is not fully understood. Here we demonstrated that human CD44+CD24- breast CSCs were shown enhanced sensitivity to IL-2 and IL-15 activated NK cells. CD44+CD24- CSCs expressed higher levels of NKG2D ligands ULBP1, ULBP2 and MICA. Blockade assay showed that the sensitivity of CSCs to NK cells-mediated lysis was mainly dependent on NKG2D. Furthermore, redox oxygen species (ROS)-low tumor cells were more sensitive to NK cells. The presence of antioxidant enzymes inhibitor L-S,R-buthionine sulfoximine or H2O2 retarded the cytotoxicity of NK cells to CD44+CD24- CSCs. In addition, NK cells could readily target CD133+ colonal CSCs. Our findings provide novel targets for NK cells-based immunotherapy and are of great importance for translational medicine.

  6. 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...

  7. 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.

  8. 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.

  9. MIO-M1 cells and similar muller glial cell lines derived from adult human retina exhibit neural stem cell characteristics.

    Science.gov (United States)

    Lawrence, Jean M; Singhal, Shweta; Bhatia, Bhairavi; Keegan, David J; Reh, Thomas A; Luthert, Philip J; Khaw, Peng T; Limb, Gloria Astrid

    2007-08-01

    Growing evidence suggests that glial cells may have a role as neural precursors in the adult central nervous system. Although it has been shown that Müller cells exhibit progenitor characteristics in the postnatal chick and rat retinae, their progenitor-like role in developed human retina is unknown. We first reported the Müller glial characteristics of the spontaneously immortalized human cell line MIO-M1, but recently we have derived similar cell lines from the neural retina of several adult eye donors. Since immortalization is one of the main properties of stem cells, we investigated whether these cells expressed stem cell markers. Cells were grown as adherent monolayers, responded to epidermal growth factor, and could be expanded indefinitely without growth factors under normal culture conditions. They could be frozen and thawed without losing their characteristics. In the presence of extracellular matrix and fibroblast growth factor-2 or retinoic acid, they acquired neural morphology, formed neurospheres, and expressed neural stem cell markers including betaIII tubulin, Sox2, Pax6, Chx10, and Notch 1. They also expressed markers of postmitotic retinal neurons, including peripherin, recoverin, calretinin, S-opsin, and Brn3. When grafted into the subretinal space of dystrophic Royal College of Surgeons rats or neonatal Lister hooded rats, immortalized cells migrated into the retina, where they expressed various markers of retinal neurons. These observations indicate that adult human neural retina harbors a population of cells that express both Müller glial and stem cell markers and suggest that these cells may have potential use for cell-based therapies to restore retinal function. Disclosure of potential conflicts of interest is found at the end of this article.

  10. 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.

  11. Myeloid Dysregulation in a Human Induced Pluripotent Stem Cell Model of PTPN11-Associated Juvenile Myelomonocytic Leukemia

    Directory of Open Access Journals (Sweden)

    Sonia Mulero-Navarro

    2015-10-01

    Full Text Available Somatic PTPN11 mutations cause juvenile myelomonocytic leukemia (JMML. Germline PTPN11 defects cause Noonan syndrome (NS, and specific inherited mutations cause NS/JMML. Here, we report that hematopoietic cells differentiated from human induced pluripotent stem cells (hiPSCs harboring NS/JMML-causing PTPN11 mutations recapitulated JMML features. hiPSC-derived NS/JMML myeloid cells exhibited increased signaling through STAT5 and upregulation of miR-223 and miR-15a. Similarly, miR-223 and miR-15a were upregulated in 11/19 JMML bone marrow mononuclear cells harboring PTPN11 mutations, but not those without PTPN11 defects. Reducing miR-223’s function in NS/JMML hiPSCs normalized myelogenesis. MicroRNA target gene expression levels were reduced in hiPSC-derived myeloid cells as well as in JMML cells with PTPN11 mutations. Thus, studying an inherited human cancer syndrome with hiPSCs illuminated early oncogenesis prior to the accumulation of secondary genomic alterations, enabling us to discover microRNA dysregulation, establishing a genotype-phenotype association for JMML and providing therapeutic targets.

  12. Identification of Multipotent Stem Cells in Human Brain Tissue Following Stroke.

    Science.gov (United States)

    Tatebayashi, Kotaro; Tanaka, Yasue; Nakano-Doi, Akiko; Sakuma, Rika; Kamachi, Saeko; Shirakawa, Manabu; Uchida, Kazutaka; Kageyama, Hiroto; Takagi, Toshinori; Yoshimura, Shinichi; Matsuyama, Tomohiro; Nakagomi, Takayuki

    2017-06-01

    Perivascular regions of the brain harbor multipotent stem cells. We previously demonstrated that brain pericytes near blood vessels also develop multipotency following experimental ischemia in mice and these ischemia-induced multipotent stem cells (iSCs) can contribute to neurogenesis. However, it is essential to understand the traits of iSCs in the poststroke human brain for possible applications in stem cell-based therapies for stroke patients. In this study, we report for the first time that iSCs can be isolated from the poststroke human brain. Putative iSCs were derived from poststroke brain tissue obtained from elderly stroke patients requiring decompressive craniectomy and partial lobectomy for diffuse cerebral infarction. Immunohistochemistry showed that these iSCs were localized near blood vessels within poststroke areas containing apoptotic/necrotic neurons and expressed both the stem cell marker nestin and several pericytic markers. Isolated iSCs expressed these same markers and demonstrated high proliferative potential without loss of stemness. Furthermore, isolated iSCs expressed other stem cell markers, such as Sox2, c-myc, and Klf4, and differentiated into multiple cells in vitro, including neurons. These results show that iSCs, which are likely brain pericyte derivatives, are present within the poststroke human brain. This study suggests that iSCs can contribute to neural repair in patients with stroke.

  13. Fibronectin production by human mammary cells

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. 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.

  15. 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.

  16. The microcell mediated transfer of human chromosome 8 into highly metastatic rat liver cancer cell line C5F

    Institute of Scientific and Technical Information of China (English)

    Hu Liu; Sheng-Long Ye; Jiong Yang; Zhao-You Tang; Yin-Kun Liu; Lun-Xiu Qin; Shuang-Jian Qiu; Rui-Xia Sun

    2003-01-01

    AIM: Our previous research on the surgical samples of primary liver cancer with CGH showed that the loss of human chromosome 8p had correlation with the metastatic phenotype of liver cancer. In order to seek the functional evidence that there could be a metastatsis suppressor gene (s) for liver cancer on human chromosome 8, we tried to transfer normal human chromosome 8 into rat liver cancer cell line C5F, which had high metastatic potential to lung.METHODS: Human chromosome 8 randomly marked with neo gene was introduced into C5F cell line by MMCT and positive microcell hybrids were screened by double selections of G418 and HAT. Single cell isolation cloning was applied to clone microcell hybrids. Finally, STS-PCR and WCP-FISH were used to confirm the introduction.RESULTS: Microcell hybrids resistant to HAT and G418 were obtained and 15 clones were obtained by single-cell isolation cloning. STS-PCR and WCP-FISH proved that human chromosome 8 had been successfully introduced into rat liver cancer cell line C5F. STS-PCR detected a random loss in the chromosome introduced and WCP-FISH found a consistent recombination of the introduced human chromosome with the rat chromosome.CONCLUSION: The successful introduction of human chromosome 8 into highly metastatic rat liver cancer cell line builds the basis for seeking functional evidence of a metastasis suppressor gene for liver cancer harboring on human chromosome 8 and its subsequent cloning.

  17. The CK2 alpha/CK2 beta interface of human protein kinase CK2 harbors a binding pocket for small molecules

    DEFF Research Database (Denmark)

    Raaf, Jennifer; Brunstein, Elena; Issinger, Olaf-Georg;

    2008-01-01

    , selective CK2 inhibitors are required. An often-used CK2 inhibitor is 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). In a complex structure with human CK2 alpha, DRB binds to the canonical ATP cleft, but additionally it occupies an allosteric site that can be alternatively filled by glycerol....... Inhibition kinetic studies corroborate the dual binding mode of the inhibitor. Structural comparisons reveal a surprising conformational plasticity of human CK2 alpha around both DRB binding sites. After local rearrangement, the allosteric site serves as a CK2 beta interface. This opens the potential...

  18. 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

  19. 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.

  20. WNT/β-Catenin signaling pathway regulates non-tumorigenesis of human embryonic stem cells co-cultured with human umbilical cord mesenchymal stem cells

    Science.gov (United States)

    Chang, Yu-Hsun; Chu, Tang-Yuan; Ding, Dah-Ching

    2017-01-01

    Human pluripotent stem cells harbor hope in regenerative medicine, but have limited application in treating clinical diseases due to teratoma formation. Our previous study has indicated that human umbilical cord mesenchymal stem cells (HUCMSC) can be adopted as non-teratogenenic feeders for human embryonic stem cells (hESC). This work describes the mechanism of non-tumorigenesis of that feeder system. In contrast with the mouse embryonic fibroblast (MEF) feeder, HUCMSC down-regulates the WNT/β-catenin/c-myc signaling in hESC. Thus, adding β-catenin antagonist (FH535 or DKK1) down-regulates β-catenin and c-myc expressions, and suppresses tumorigenesis (3/14 vs. 4/4, p = 0.01) in hESC fed with MEF, while adding the β-catenin enhancer (LiCl or 6-bromoindirubin-3′-oxime) up-regulates the expressions, and has a trend (p = 0.056) to promote tumorigenesis (2/7 vs. 0/21) in hESC fed with HUCMSC. Furthermore, FH535 supplement does not alter the pluripotency of hESC when fed with MEF, as indicated by the differentiation capabilities of the three germ layers. Taken together, this investigation concludes that WNT/β-catenin/c-myc pathway causes the tumorigenesis of hESC on MEF feeder, and β-catenin antagonist may be adopted as a tumor suppressor. PMID:28157212

  1. Cloning and Sequencing of Protein Kinase cDNA from Harbor Seal (Phoca vitulina Lymphocytes

    Directory of Open Access Journals (Sweden)

    Jennifer C. C. Neale

    2004-01-01

    Full Text Available Protein kinases (PKs play critical roles in signal transduction and activation of lymphocytes. The identification of PK genes provides a tool for understanding mechanisms of immunotoxic xenobiotics. As part of a larger study investigating persistent organic pollutants in the harbor seal and their possible immunomodulatory actions, we sequenced harbor seal cDNA fragments encoding PKs. The procedure, using degenerate primers based on conserved motifs of human protein tyrosine kinases (PTKs, successfully amplified nine phocid PK gene fragments with high homology to human and rodent orthologs. We identified eight PTKs and one dual (serine/threonine and tyrosine kinase. Among these were several PKs important in early signaling events through the B- and T-cell receptors (FYN, LYN, ITK and SYK and a MAP kinase involved in downstream signal transduction. V-FGR, RET and DDR2 were also expressed. Sequential activation of protein kinases ultimately induces gene transcription leading to the proliferation and differentiation of lymphocytes critical to adaptive immunity. PKs are potential targets of bioactive xenobiotics, including persistent organic pollutants of the marine environment; characterization of these molecules in the harbor seal provides a foundation for further research illuminating mechanisms of action of contaminants speculated to contribute to large-scale die-offs of marine mammals via immunosuppression.

  2. 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.

  3. Characterization of epidemic IncI1-Iγ plasmids harboring ambler class A and C genes in Escherichia coli and Salmonella enterica from animals and humans

    NARCIS (Netherlands)

    Smith, Hilde; Bossers, Alex; Harders, Frank; Wu, Guanghui; Woodford, Neil; Schwarz, Stefan; Guerra, Beatriz; Rodríguez, Irene; Essen-Zandbergen, Van Alieda; Brouwer, Michael; Mevius, Dik

    2015-01-01

    The aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained from Escherichia coli and Salmonella enterica isolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spect

  4. Characterization of epidemic IncI1-Iγ plasmids harboring ambler class A and C genes in Escherichia coli and Salmonella enterica from animals and humans

    NARCIS (Netherlands)

    Smith, Hilde; Bossers, Alex; Harders, Frank; Wu, Guanghui; Woodford, Neil; Schwarz, Stefan; Guerra, Beatriz; Rodríguez, Irene; van Essen-Zandbergen, Alieda; Brouwer, Michael; Mevius, Dik

    2015-01-01

    The aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained from Escherichia coli and Salmonella enterica isolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum

  5. Targeting androgen receptor/Src complex impairs the aggressive phenotype of human fibrosarcoma cells.

    Directory of Open Access Journals (Sweden)

    Gabriella Castoria

    Full Text Available BACKGROUND: Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR. EXPERIMENTAL: FINDINGS: We report that the pure anti-androgen Casodex inhibits the growth of HT1080 cell xenografts in immune-depressed mice, revealing a novel role of AR in fibrosarcoma progression. In HT1080 cultured cells EGF, but not androgens, robustly increases DNA synthesis. Casodex abolishes the EGF mitogenic effect, implying a crosstalk between EGFR and AR. The mechanism underlying this crosstalk has been analyzed using an AR-derived small peptide, S1, which prevents AR/Src tyrosine kinase association and androgen-dependent Src activation. Present findings show that in HT1080 cells EGF induces AR/Src Association, and the S1 peptide abolishes both the assembly of this complex and Src activation. The S1 peptide inhibits EGF-stimulated DNA synthesis, cell matrix metalloproteinase-9 (MMP-9 secretion and invasiveness of HT1080 cells. Both Casodex and S1 peptide also prevent DNA synthesis and migration triggered by EGF in various human cancer-derived cells (prostate, breast, colon and pancreas that express AR. CONCLUSION: This study shows that targeting the AR domain involved in AR/Src association impairs EGF signaling in human fibrosarcoma HT1080 cells. The EGF-elicited processes inhibited by the peptide (DNA synthesis, MMP-9 secretion and invasiveness cooperate in increasing the aggressive phenotype of HT1080 cells. Therefore, AR represents a new potential therapeutic target in human fibrosarcoma, as supported by Casodex inhibition of HT1080 cell xenografts. The extension of these findings in various human cancer-derived cell lines

  6. 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].

  7. Perspectives for induced pluripotent stem cell technology: new insights into human physiology involved in somatic mosaicism.

    Science.gov (United States)

    Nagata, Naoki; Yamanaka, Shinya

    2014-01-31

    Induced pluripotent stem cell technology makes in vitro reprogramming of somatic cells from individuals with various genetic backgrounds possible. By applying this technology, it is possible to produce pluripotent stem cells from biopsy samples of arbitrarily selected individuals with various genetic backgrounds and to subsequently maintain, expand, and stock these cells. From these induced pluripotent stem cells, target cells and tissues can be generated after certain differentiation processes. These target cells/tissues are expected to be useful in regenerative medicine, disease modeling, drug screening, toxicology testing, and proof-of-concept studies in drug development. Therefore, the number of publications concerning induced pluripotent stem cells has recently been increasing rapidly, demonstrating that this technology has begun to infiltrate many aspects of stem cell biology and medical applications. In this review, we discuss the perspectives of induced pluripotent stem cell technology for modeling human diseases. In particular, we focus on the cloning event occurring through the reprogramming process and its ability to let us analyze the development of complex disease-harboring somatic mosaicism.

  8. The role of mitochondria in the radiation-induced bystander effect in human lymphoblastoid cells.

    Science.gov (United States)

    Rajendran, Sountharia; Harrison, Scott H; Thomas, Robert A; Tucker, James D

    2011-02-01

    Cells without intact mitochondrial DNA have been shown to lack the bystander effect, which is an energy-dependent process. We hypothesized that cells harboring mutations in mitochondrial genes responsible for ATP synthesis would show a decreased bystander effect compared to normal cells. Radiation-induced bystander effects were analyzed in two normal and four mitochondrial mutant human lymphoblastoid cells. Medium from previously irradiated cells (conditioned medium) was transferred to unirradiated cells from the respective cell lines and evaluated for the bystander effect using the cytokinesis-block micronucleus assay. Unlike normal cells that were used as a control, mitochondrial mutant cells neither generated nor responded to the bystander signals. The bystander effect was inhibited in normal cells by adding the mitochondrial inhibitors rotenone and oligomycin to the culture medium. Time-controlled blocking of the bystander effect by inhibitors was found to occur either for prolonged exposure to the inhibitor prior to irradiation with an immediate and subsequent removal of the inhibitors or immediate post-application of the inhibitor. Adding the inhibitors just prior to irradiation and removing them immediately after irradiation was uneventful. Fully functional mitochondrial metabolic capability may therefore be essential for the bystander effect.

  9. 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...

  10. Human umbilical cord-derived mesenchymal stem cells can secrete insulin in vitro and in vivo.

    Science.gov (United States)

    Boroujeni, Zahra Niki; Aleyasin, Ahmad

    2014-01-01

    Diabetes mellitus is characterized by autoimmune destruction of pancreatic beta cells, leading to decreased insulin production. Differentiation of mesenchymal stem cells (MSCs) into insulin-producing cells offers novel ways of diabetes treatment. MSCs can be isolated from the human umbilical cord tissue and differentiate into insulin-secreting cells. Human umbilical cord-derived stem cells (hUDSCs) were obtained after birth, selected by plastic adhesion, and characterized by flow cytometric analysis. hUDSCs were transduced with nonintegrated lentivirus harboring PDX1 (nonintegrated LV-PDX1) and was cultured in differentiation medium in 21 days. Pancreatic duodenum homeobox protein-1 (PDX1) is a transcription factor in pancreatic development. Significant expressions of PDX1, neurogenin3 (Ngn3), glucagon, glucose transporter2 (Glut2), and somatostatin were detected by quantitative RT-PCR (P insulin proteins were shown by immunocytochemistry analysis. Insulin secretion of hUDSCs(PDX1+) in the high-glucose medium was 1.8 μU/mL. They were used for treatment of diabetic rats and could decrease the blood glucose level from 400 mg/dL to a normal level in 4 days. In conclusion, our results demonstrated that hUDSCs are able to differentiate into insulin-producing cells by transduction with nonintegrated LV-PDX1. These hUDSCs(PDX1+) have the potential to be used as a viable resource in cell-based gene therapy of type 1 diabetes.

  11. 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.

  12. Ca2+ signaling in HEK-293 and skeletal muscle cells expressing recombinant ryanodine receptors harboring malignant hyperthermia and central core disease mutations.

    Science.gov (United States)

    Brini, Marisa; Manni, Sabrina; Pierobon, Nicola; Du, Guo Guang; Sharma, Parveen; MacLennan, David H; Carafoli, Ernesto

    2005-04-15

    Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca(2+) release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca(2+) pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca(2+) homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca(2+) revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca(2+) transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca(2+) derangements of different degrees account for the specific cellular phenotypes of the two disorders.

  13. 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

  14. 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.

  15. [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.

  16. 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....

  17. 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.

  18. CLOSTRIDIUM SPORE ATTACHMENT TO HUMAN CELLS

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. 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. 

  20. Cell pattern in adult human corneal endothelium.

    Directory of Open Access Journals (Sweden)

    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.

  1. Merkel cell distribution in the human eyelid

    Directory of Open Access Journals (Sweden)

    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.

  2. 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

  3. 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.

  4. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. [Establishment of stably expressed human RANTES gene in prunella vulgaris cell clone].

    Science.gov (United States)

    Zeng, Qing-Ping; Feng, Li-Ling; Yang, Rui-Yi; Chen, Zhu-Hua

    2003-03-01

    To express interesting human genes in herbal cells for boosting their specific pharmacological activities, RANTES gene cloned from human peripheral blood lymphocyte (PBL) mRNA was introduced into A. tumefaciens strain LBA4404 harboring pAL4404 plasmid via tumor-inducing (Ti) plasmid-derived intermediate expression vector pROKII. In vitro cultured P. vulgaris cells were transformed by leaf-disk cocultivation procedure. Integration of RANTES gene in the genome of transformed cells was confirmed by Southern blotting, and expression of RANTES gene in transformed cells was analyzed by RT-PCR amplification, Western blotting and enzyme-linked immunosorbent assay (ELISA). The peroxidase activity of PBL was utilized as a detection index of cellular chemotropism induction by recombinant RANTES. The results have shown the RANTES gene was integrated in transgenic P. vulgaris cells, and RANTES gene-stably expressed cell clones were available, which could pave the way to obtain transgenic P. vulgaris plants demonstrating specific pharmacological activities.

  12. [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.

  13. 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.

  14. 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...

  15. The human epidermal growth factor receptor (EGFR gene in European patients with advanced colorectal cancer harbors infrequent mutations in its tyrosine kinase domain

    Directory of Open Access Journals (Sweden)

    Delvenne Philippe

    2011-10-01

    Full Text Available Abstract Background The epidermal growth factor receptor (EGFR, a member of the ErbB family of receptors, is a transmembrane tyrosine kinase (TK activated by the binding of extracellular ligands of the EGF-family and involved in triggering the MAPK signaling pathway, which leads to cell proliferation. Mutations in the EGFR tyrosine kinase domain are frequent in non-small-cell lung cancer (NSCLC. However, to date, only very few, mainly non-European, studies have reported rare EGFR mutations in colorectal cancer (CRC. Methods We screened 236 clinical tumor samples from European patients with advanced CRC by direct DNA sequencing to detect potential, as yet unknown mutations, in the EGFR gene exons 18 to 21, mainly covering the EGFR TK catalytic domain. Results EGFR sequences showed somatic missense mutations in exons 18 and 20 at a frequency of 2.1% and 0.4% respectively. Somatic SNPs were also found in exons 20 and 21 at a frequency of about 3.1% and 0.4% respectively. Of these mutations, four have not yet been described elsewhere. Conclusions These mutation frequencies are higher than in a similarly sized population characterized by Barber and colleagues, but still too low to account for a major role played by the EGFR gene in CRC.

  16. Human blood BDCA-1 dendritic cells differentiate into Langerhans-like cells with thymic stromal lymphopoietin and TGF-β.

    Science.gov (United States)

    Martínez-Cingolani, Carolina; Grandclaudon, Maximilien; Jeanmougin, Marine; Jouve, Mabel; Zollinger, Raphaël; Soumelis, Vassili

    2014-10-09

    The ontogeny of human Langerhans cells (LCs) remains poorly characterized, in particular the nature of LC precursors and the factors that may drive LC differentiation. Here we report that thymic stromal lymphopoietin (TSLP), a keratinocyte-derived cytokine involved in epithelial inflammation, cooperates with transforming growth factor (TGF)-β for the generation of LCs. We show that primary human blood BDCA-1(+), but not BDCA-3(+), dendritic cells (DCs) stimulated with TSLP and TGF-β harbor a typical CD1a(+)Langerin(+) LC phenotype. Electron microscopy established the presence of Birbeck granules, an intracellular organelle specific to LCs. LC differentiation was not observed from tonsil BDCA-1(+) and BDCA-3(+) subsets. TSLP + TGF-β LCs had a mature phenotype with high surface levels of CD80, CD86, and CD40. They induced a potent CD4(+) T-helper (Th) cell expansion and differentiation into Th2 cells with increased production of tumor necrosis factor-α and interleukin-6 compared with CD34-derived LCs. Our findings establish a novel LC differentiation pathway from BDCA-1(+) blood DCs with potential implications in epithelial inflammation. Therapeutic targeting of TSLP may interfere with tissue LC repopulation from circulating precursors.

  17. Construction and characterization of adenoviral vectors for the delivery of TALENs into human cells.

    Science.gov (United States)

    Holkers, Maarten; Cathomen, Toni; Gonçalves, Manuel A F V

    2014-09-01

    Transcription activator-like effector nucleases (TALENs) are designed to cut the genomic DNA at specific chromosomal positions. The resulting DNA double strand break activates cellular repair pathways that can be harnessed for targeted genome modifications. TALENs thus constitute a powerful tool to interrogate the function of DNA sequences within complex genomes. Moreover, their high DNA cleavage activity combined with a low cytotoxicity make them excellent candidates for applications in human gene therapy. Full exploitation of these large and repeat-bearing nucleases in human cell types will benefit largely from using the adenoviral vector (AdV) technology. The genetic stability and the episomal nature of AdV genomes in conjunction with the availability of a large number of AdV serotypes able to transduce various human cell types make it possible to achieve high-level and transient expression of TALENs in numerous target cells, regardless of their mitotic state. Here, we describe a set of protocols detailing the rescue, propagation and purification of TALEN-encoding AdVs. Moreover, we describe procedures for the characterization and quantification of recombinant viral DNA present in the resulting AdV preparations. The protocols are preceded by information about their underlying principles and applied in the context of second-generation capsid-modified AdVs expressing TALENs targeted to the AAVS1 "safe harbor" locus on human chromosome 19.

  18. 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.

  19. PARP Inhibitors in Clinical Use Induce Genomic Instability in Normal Human Cells.

    Directory of Open Access Journals (Sweden)

    Shuhei Ito

    Full Text Available Poly(ADP-ribose polymerases (PARPs are the first proteins involved in cellular DNA repair pathways to be targeted by specific inhibitors for clinical benefit. Tumors harboring genetic defects in homologous recombination (HR, a DNA double-strand break (DSB repair pathway, are hypersensitive to PARP inhibitors (PARPi. Early phase clinical trials with PARPi have been promising in patients with advanced BRCA1 or BRCA2-associated breast, ovary and prostate cancer and have led to limited approval for treatment of BRCA-deficient ovary cancer. Unlike HR-defective cells, HR-proficient cells manifest very low cytotoxicity when exposed to PARPi, although they mount a DNA damage response. However, the genotoxic effects on normal human cells when agents including PARPi disturb proficient cellular repair processes have not been substantially investigated. We quantified cytogenetic alterations of human cells, including primary lymphoid cells and non-tumorigenic and tumorigenic epithelial cell lines, exposed to PARPi at clinically relevant doses by both sister chromatid exchange (SCE assays and chromosome spreading. As expected, both olaparib and veliparib effectively inhibited poly-ADP-ribosylation (PAR, and caused marked hypersensitivity in HR-deficient cells. Significant dose-dependent increases in SCEs were observed in normal and non-tumorigenic cells with minimal residual PAR activity. Clinically relevant doses of the FDA-approved olaparib led to a marked increase of SCEs (5-10-fold and chromatid aberrations (2-6-fold. Furthermore, olaparib potentiated SCE induction by cisplatin in normal human cells. Our data have important implications for therapies with regard to sustained genotoxicity to normal cells. Genomic instability arising from PARPi warrants consideration, especially if these agents will be used in people with early stage cancers, in prevention strategies or for non-oncologic indications.

  20. 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.

  1. 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....

  2. 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.

  3. Surgidero de Batabanó Harbor, Cuba

    NARCIS (Netherlands)

    Hopmans, R.; Van Kessel, L.; Lendering, K.; Oud, M.; Tromp, R.

    2011-01-01

    The harbor of Surgidero de Batabano is a harbor that lies in the Gulf of Batabano in the South-Western part of Cuba. It serves as a connection between the main land of Cuba and the islands 'Isla de la Juventud' and Cayo Largo. The Batabano harbor suffers from sediment accretion. The accretion of sed

  4. Surgidero de Batabanó Harbor, Cuba

    NARCIS (Netherlands)

    Hopmans, R.; Van Kessel, L.; Lendering, K.; Oud, M.; Tromp, R.

    2011-01-01

    The harbor of Surgidero de Batabano is a harbor that lies in the Gulf of Batabano in the South-Western part of Cuba. It serves as a connection between the main land of Cuba and the islands 'Isla de la Juventud' and Cayo Largo. The Batabano harbor suffers from sediment accretion. The accretion of

  5. Harbor Expansion Facilitates Crude and Petrochemicals Transportation

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    @@ Douwei Harbor attracting petrochem investment Substantial progress has been made in the preliminary preparation of Douwei Harbor project in Hui'an, Fujian Province. It is one of the major four transfer ports in China planned by the Ministry of Transportation. A number of projects, with a total investment approaching 10 billion yuan,will come under construction in the harbor zone.

  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. Army Engineer Divers: First In Port-Au-Prince Harbor

    Science.gov (United States)

    2010-12-01

    America . Shortly after the quake hit, United States Southern Command (SOUTHCOM) diverted the USNS Grasp and the 544th to Port-au-Prince. Once there...Prince harbor. Pe- troleum floated on top of the water, and numerous marine hazards like jellyfish , human waste, and debris floated be- neath the

  8. 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.

  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. 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

  11. 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...

  12. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells.

    Science.gov (United States)

    Holkers, Maarten; Maggio, Ignazio; Liu, Jin; Janssen, Josephine M; Miselli, Francesca; Mussolino, Claudio; Recchia, Alessandra; Cathomen, Toni; Gonçalves, Manuel A F V

    2013-03-01

    The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform.

  13. 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.

  14. Tuberin and PRAS40 are anti-apoptotic gatekeepers during early human amniotic fluid stem-cell differentiation.

    Science.gov (United States)

    Fuchs, Christiane; Rosner, Margit; Dolznig, Helmut; Mikula, Mario; Kramer, Nina; Hengstschläger, Markus

    2012-03-01

    Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products.

  15. Increased expression of the PRL-3 gene in human oral squamous cell carcinoma and dysplasia tissues.

    Science.gov (United States)

    Hassan, Nur Mohammad Monsur; Hamada, Jun-ichi; Kameyama, Takeshi; Tada, Mitsuhiro; Nakagawa, Koji; Yoshida, Shoko; Kashiwazaki, Haruhiko; Yamazaki, Yutaka; Suzuki, Yukiko; Sasaki, Akira; Nagatsuka, Hitoshi; Inoue, Nobuo; Moriuchi, Tetsuya

    2011-01-01

    Phosphatase of regenerating liver (PRL) belongs to a class of the protein tyrosine phosphatase family, which is known so far to consist of 3 members, PRL-1, PRL-2, and PRL-3. The aim of this study was to uncover the role of PRL genes in development of oral malignancy. We analyzed expression levels of the 3 PRL genes in 50 human oral squamous cell carcinomas (OSCCs), 11 dysplasia and 12 normal mucosa tissues by a real-time RT-PCR method. PRL-3 but not PRL-1 or PRL-2 expressions were significantly higher in OSCC and dysplasia than in normal mucosa tissues. Additionally, PRL-3 expressions were significantly higher in OSCC tissues harboring dominant-negative p53 or recessive p53 mutation than in those harboring wild-type p53. These results suggest that PRL-3 plays a role in oral cancer development and can be useful as a marker of pre-malignant and malignant lesion of oral mucosa.

  16. Knowledge based recognition of harbor target

    Institute of Scientific and Technical Information of China (English)

    Zhu Bing; Li Jinzong; Cheng Aijun

    2006-01-01

    A fast knowledge based recognition method of the harbor target in large gray remote-sensing image is presented. First, the distributed features and the inherent feature are analyzed according to the knowledge of harbor targets; then, two methods for extracting the candidate region of harbor are devised in accordance with different sizes of the harbors; after that, thresholds are used to segment the land and the sea with strategies of the segmentation error control; finally, harbor recognition is implemented according to its inherent character (semi-closed region of seawater).

  17. Next-generation EGFR/HER tyrosine kinase inhibitors for the treatment of patients with non-small-cell lung cancer harboring EGFR mutations: a review of the evidence

    Directory of Open Access Journals (Sweden)

    Wang X

    2016-09-01

    Full Text Available Xiaochun Wang,1,2 David Goldstein,3 Philip J Crowe,1,2 Jia-Lin Yang1,2 1Department of Surgery, 2Sarcoma and Nanooncology Group, Adult Cancer Program, Lowy Cancer Research Centre, 3Department of Medical Oncology, Prince of Wales Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia Abstract: Tyrosine kinase inhibitors (TKIs against human epidermal growth factor receptor (EGFR/HER family have been introduced into the clinic to treat cancers, particularly non-small-cell lung cancer (NSCLC. There have been three generations of the EGFR/HER-TKIs. First-generation EGFR/HER-TKIs, binding competitively and reversibly to the ATP-binding site of the EGFR TK domain, show a significant breakthrough treatment in selected NSCLC patients with activating EGFR mutations (actEGFRm EGFRL858R and EGFRDel19, in terms of safety, efficacy, and quality of life. However, all those responders inevitably develop acquired resistance within 12 months, because of the EGFRT790M mutation, which prevents TKI binding to ATP-pocket of EGFR by steric hindrance. The second-generation EGFR/HER-TKIs were developed to prolong and maintain more potent response as well as overcome the resistance to the first-generation EGFR/HER-TKIs. They are different from the first-generation EGFR/HER-TKIs by covalently binding to the ATP-binding site, irreversibly blocking enzymatic activation, and targeting EGFR/HER family members, including EGFR, HER2, and HER4. Preclinically, these compounds inhibit the enzymatic activation for actEGFRm, EGFRT790M, and wtEGFR. The second-generation EGFR/HER-TKIs improve overall survival in cancer patients with actEGFRm in a modest way. However, they are not clinically active in overcoming EGFRT790M resistance, mainly because of dose-limiting toxicity due to simultaneous inhibition against wtEGFR. The third-generation EGFR/HER-TKIs selectively and irreversibly target EGFRT790M and actEGFRm while sparing wtEGFR. They yield

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Cold spring harbor symposia on quantitative biology

    Energy Technology Data Exchange (ETDEWEB)

    1988-01-01

    For many decades, it has been clear that cells have a multitude of ways of sensing their environment and converting a plethora of external signals into measured intracellular responses. Now we realize that many first messengers do not act directly through second messengers, but instead work at the genetic level by binding to cytoplasmically located receptors, which can then bind to DNA and turn on or off the functioning of specific genes. Today, we refer to the way that external signals are passed through various cellular components as signal transduction processes, with receptors and their associated molecules known as biological transducers. Because most transducer molecules are present in very limited amounts, their study at the biochemical level until recently was at best difficult, and hypothesis as to how they functioned were almost impossible to test rigorously. Today, recombinant DNA techniques have dramatically changed the picture. Even very rare receptors are now open to analyses if their respective genes can be cloned, and virtually every month, the amino acid sequence of a new key biological transducer is established. The time was thus appropriate last June to hold a Cold Spring Harbor Symposium on the Molecular Biology of Signal Transduction. The final program consisted of 119 speakers, who spoke before an audience of 439, the largest ever yet to attend a Cold spring Harbor Symposium. This volume contains 61 papers. Individual papers are indexed separately on the energy data base.

  3. Cold spring harbor symposia on quantitative biology

    Energy Technology Data Exchange (ETDEWEB)

    1988-01-01

    For many decades, it has been clear that cells have a multitude of ways of sensing their environment and converting a plethora of external signals into measured intracellular responses. Now we realize that many first messengers do not act directly through second messengers, but instead work at the genetic level by binding to cytoplasmically located receptors, which can then bind to DNA and turn on or off the functioning of specific genes. Today, we refer to the way that external signals are passed through various cellular components as signal transduction processes, with receptors and their associated molecules known as biological transducers. Because most transducer molecules are present in very limited amounts, their study at the biochemical level until recently was at best difficult, and hypotheses as to how they functioned were almost impossible to test rigorously. Today, recombinant DNA techniques have dramatically changed the picture. Even very rare receptors are now open to analysis if their respective genes can be cloned, and virtually every month the amino acid sequence of a new key biological transducer is established. The time was thus appropriate last June to hold a Cold Spring Harbor Symposium on the Molecular Biology of Signal Transduction. The final program consisted of 119 speakers, who spoke before an audience of 439, the largest ever yet to attend a Cold Spring Harbor Symposium. This volume contains 54 papers. Individual papers are indexed separately on the energy data base.

  4. 33 CFR 80.1122 - Channel Islands Harbor, CA.

    Science.gov (United States)

    2010-07-01

    ...) A line drawn from Channel Islands Harbor South Jetty Light 2 to Channel Islands Harbor Breakwater South Light 1. (b) A line drawn from Channel Islands Harbor Breakwater North Light to Channel Islands Harbor North Jetty Light 5....

  5. 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.

  6. 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.

  7. 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.

  8. Dopamine receptor repertoire of human granulosa cells

    Directory of Open Access Journals (Sweden)

    Kunz Lars

    2007-10-01

    Full Text Available Abstract Background High levels of dopamine (DA were described in human ovary and recently evidence for DA receptors in granulosa and luteal cells has been provided, as well. However, neither the full repertoire of ovarian receptors for DA, nor their specific role, is established. Human granulosa cells (GCs derived from women undergoing in vitro fertilization (IVF are an adequate model for endocrine cells of the follicle and the corpus luteum and were therefore employed in an attempt to decipher their DA receptor repertoire and functionality. Methods Cells were obtained from patients undergoing IVF and examined using cDNA-array, RT-PCR, Western blotting and immunocytochemistry. In addition, calcium measurements (with FLUO-4 were employed. Expression of two DA receptors was also examined by in-situ hybridization in rat ovary. Effects of DA on cell viability and cell volume were studied by using an ATP assay and an electronic cell counter system. Results We found members of the two DA receptor families (D1- and D2 -like associated with different signaling pathways in human GCs, namely D1 (as expected and D5 (both are Gs coupled and linked to cAMP increase and D2, D4 (Gi/Gq coupled and linked to IP3/DAG. D3 was not found. The presence of the trophic hormone hCG (10 IU/ml in the culture medium for several days did not alter mRNA (semiquantitative RT-PCR or protein levels (immunocytochemistry/Western blotting of D1,2,4,5 DA receptors. Expression of prototype receptors for the two families, D1 and D2, was furthermore shown in rat granulosa and luteal cells by in situ hybridization. Among the DA receptors found in human GCs, D2 expression was marked both at mRNA and protein levels and it was therefore further studied. Results of additional RT-PCR and Western blots showed two splice variants (D2L, D2S. Irrespective of these variants, D2 proved to be functional, as DA raised intracellular calcium levels. This calcium mobilizing effect of DA was observed

  9. Genomic and expression array profiling of chromosome 20q amplicon in human colon cancer cells

    Directory of Open Access Journals (Sweden)

    Carter Jennifer

    2005-01-01

    Full Text Available Background: Gain of the q arm of chromosome 20 in human colorectal cancer has been associated with poorer survival time and has been reported to increase in frequency from adenomas to metastasis. The increasing frequency of chromosome 20q amplification during colorectal cancer progression and the presence of this amplification in carcinomas of other tissue origin has lead us to hypothesize that 20q11-13 harbors one or more genes which, when over expressed promote tumor invasion and metastasis. Aims: Generate genomic and expression profiles of the 20q amplicon in human cancer cell lines in order to identify genes with increased copy number and expression. Materials and Methods: Utilizing genomic sequencing clones and amplification mapping data from our lab and other previous studies, BAC/ PAC tiling paths spanning the 20q amplicon and genomic microarrays were generated. Array-CGH on the custom array with human cancer cell line DNAs was performed to generate genomic profiles of the amplicon. Expression array analysis with RNA from these cell lines using commercial oligo microarrays generated expression profiles of the amplicon. The data were then combined in order to identify genes with increased copy number and expression. Results: Over expressed genes in regions of increased copy number were identified and a list of potential novel genetic tumor markers was assembled based on biological functions of these genes Conclusions: Performing high-resolution genomic microarray profiling in conjunction with expression analysis is an effective approach to identify potential tumor markers.

  10. Nuclear reprogramming of luminal-like breast cancer cells generates Sox2-overexpressing cancer stem-like cellular states harboring transcriptional activation of the mTOR pathway

    Science.gov (United States)

    Corominas-Faja, Bruna; Cufí, Sílvia; Oliveras-Ferraros, Cristina; Cuyàs, Elisabet; López-Bonet, Eugeni; Lupu, Ruth; Alarcón, Tomás; Vellon, Luciano; Iglesias, Juan Manuel; Leis, Olatz; Martín, Ángel G; Vazquez-Martin, Alejandro; Menendez, Javier A

    2013-01-01

    Energy metabolism plasticity enables stemness programs during the reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state. This relationship may introduce a new era in the understanding of Warburg’s theory on the metabolic origin of cancer at the level of cancer stem cells (CSCs). Here, we used Yamanaka’s stem cell technology in an attempt to create stable CSC research lines in which to dissect the transcriptional control of mTOR—the master switch of cellular catabolism and anabolism—in CSC-like states. The rare colonies with iPSC-like morphology, obtained following the viral transduction of the Oct4, Sox2, Klf4, and c-Myc (OSKM) stemness factors into MCF-7 luminal-like breast cancer cells (MCF-7/Rep), demonstrated an intermediate state between cancer cells and bona fide iPSCs. MCF-7/Rep cells notably overexpressed SOX2 and stage-specific embryonic antigen (SSEA)-4 proteins; however, other stemness-related markers (OCT4, NANOG, SSEA-1, TRA-1–60, and TRA-1–81) were found at low to moderate levels. The transcriptional analyses of OSKM factors confirmed the strong but unique reactivation of the endogenous Sox2 stemness gene accompanied by the silencing of the exogenous Sox2 transgene in MCF-7/Rep cells. Some but not all MCF-7/Rep cells acquired strong alkaline phosphatase (AP) activity compared with MCF-7 parental cells. SOX2-overexpressing MCF-7/Rep cells contained drastically higher percentages of CD44+ and ALDEFLUOR-stained ALDHbright cells than MCF-7 parental cells. The overlap between differentially expressed mTOR signaling-related genes in 3 different SOX2-overexpressing CSC-like cell lines revealed a notable downregulation of 3 genes, PRKAA1 (which codes for the catalytic α 1 subunit of AMPK), DDIT4/REDD1 (a stress response gene that operates as a negative regulator of mTOR), and DEPTOR (a naturally occurring endogenous inhibitor of mTOR activity). The insulin-receptor gene (INSR) was differentially upregulated in MCF-7/Rep

  11. Mouse T-cells restrict replication of human immunodeficiency virus at the level of integration

    Directory of Open Access Journals (Sweden)

    Goffinet Christine

    2008-07-01

    Full Text Available Abstract Background The development of an immunocompetent, genetically modified mouse model to study HIV-1 pathogenesis and to test antiviral strategies has been hampered by the fact that cells from native mice do not or only inefficiently support several steps of the HIV-1 replication cycle. Upon HIV-1 infection, mouse T-cell lines fail to express viral proteins, but the underlying replication barrier has thus far not been unambiguously identified. Here, we performed a kinetic and quantitative assessment of consecutive steps in the early phase of the HIV-1 replication cycle in T-cells from mice and humans. Results Both T-cell lines and primary T-cells from mice harbor a severe post-entry defect that is independent of potential species-specTR transactivation. Reverse transcription occurred efficiently following VSV-G-mediated entry of virions into mouse T-cells, and abundant levels of 2-LTR circles indicated successful nuclear import of the pre-integration complex. To probe the next step in the retroviral replication cycle, i.e. the integration of HIV-1 into the host cell genome, we established and validated a nested real-time PCR to specifically quantify HIV-1 integrants exploiting highly repetitive mouse B1 elements. Importantly, we demonstrate that the frequency of integrant formation is diminished 18- to > 305-fold in mouse T-cell lines compared to a human counterpart, resulting in a largely abortive infection. Moreover, differences in transgene expression from residual vector integrants, the transcription off which is cyclin T1-independent, provided evidence for an additional, peri-integrational deficit in certain mouse T-cell lines. Conclusion In contrast to earlier reports, we find that mouse T-cells efficiently support early replication steps up to and including nuclear import, but restrict HIV-1 at the level of chromosomal integration.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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...

  18. 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.

  19. 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.

  20. "Curing" of plasmid DNA in acetogen using microwave or applying an electric pulse improves cell growth and metabolite production as compared to the plasmid-harboring strain.

    Science.gov (United States)

    Berzin, Vel; Kiriukhin, Michael; Tyurin, Michael

    2013-03-01

    Plasmid-free acetogen Clostridium sp. MT962 electrotransformed with a small cryptic plasmid pMT351 was used to develop time- and cost-effective methods for plasmid elimination. Elimination of pMT351 restored production of acetate and ethanol to the levels of the plasmid-free strain with no dry cell weight changes. Destabilizing cell membrane via microwave at 2.45 GHz, or exposure to a single 12 ms square electric pulse at 35 kV cm⁻¹, eliminated pMT351 in 42-47 % of cells. Plasmid elimination with a single square electric pulse required 10 versus 0.1 J needed to introduce the same 3,202-bp plasmid into the cells as calculated per cell sample of Clostridium sp. MT962. Microwave caused visible changes in repPCR pattern and increased ethanol production at the expense of acetate. This is the first report on microwave of microwave ovens, wireless routers, and mobile devices causing chromosomal DNA aberrations in microbes along with carbon flux change.

  1. Activation of neural progenitor cells in human eyes with proliferative vitreoretinopathy.

    Science.gov (United States)

    Johnsen, Erik O; Frøen, Rebecca C; Albert, Réka; Omdal, Bente K; Sarang, Zsolt; Berta, András; Nicolaissen, Bjørn; Petrovski, Goran; Moe, Morten C

    2012-05-01

    passages. In conclusion, the adult human eye may harbor two different populations of neuroepithelial stem/progenitor cells; a non-glial population located in the proximal pars plana around peripheral cysts in addition to a population with Müller glia characteristics. Yet, we only found that the glial population was able to respond to retinal injury by targeted migration into the vitreous.

  2. 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...

  3. 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.

  4. 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.

  5. 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.

  6. Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer.

    Science.gov (United States)

    Feng, Xiujing; Cao, Shaoxian; Wang, Huili; Meng, Chunhua; Li, Jingxin; Jiang, Jin; Qian, Yong; Su, Lei; He, Qiang; Zhang, Qingxiao

    2015-02-01

    Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat β-lactoglobulin (βLG) promoter, the hα-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60-70%) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0%, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.

  7. Infection of xenotransplanted human cell lines by murine retroviruses: A lesson brought back to light by XMRV

    Directory of Open Access Journals (Sweden)

    Heidi Anne Hempel

    2013-06-01

    Full Text Available Infection of xenotransplanted human cells by xenotropic retroviruses is a known phenomenon in the scientific literature, with examples cited since the early 1970’s. However, arguably, until recently, the importance of this phenomenon had not been largely recognized. The emergence and subsequent debunking of Xenotropic Murine leukemia virus-Related Virus (XMRV as a cell culture contaminant as opposed to a potential pathogen in several human diseases, notably prostate cancer and Chronic Fatigue Syndrome, highlighted a potential problem of murine endogenous gammaretroviruses infecting commonly used human cell lines. Subsequent to the discovery of XMRV, many additional cell lines that underwent xenotransplantation in mice have been shown to harbor murine gammaretroviruses. Such retroviral infection poses the threat of not only confounding experiments performed in these cell lines via virus-induced changes in cellular behavior but also the potential infection of other cell lines cultured in the same laboratory. Thus, the possibility of xenotropic retroviral infection of cell lines may warrant additional precautions, such as periodic testing for retroviral sequences in cell lines cultured in the laboratory.

  8. 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.

  9. MicroRNA-143 replenishment re-sensitizes colorectal cancer cells harboring mutant, but not wild-type, KRAS to paclitaxel treatment.

    Science.gov (United States)

    Fei, Bing-Yuan; Wang, Xiu-Ying; Fang, Xue-Dong

    2016-05-01

    Colorectal cancer (CRC) global incidence is one of the highest among cancers. The KRAS gene has been shown as a robust biomarker for poor prognosis and drug resistance. MicroRNA-143 (miR-143) and let-7 are families of tumor suppressor microRNAs that are often downregulated in CRC, especially with coexistent KRAS mutations. In order to evaluate if miR-143 and/or let-7b replenishment would re-sensitize CRC cells to paclitaxel treatment, we investigated in effect of miR-143 and let-7b replenishments on sensitivity to paclitaxel treatment in KRAS mutant LoVo and wild-type SW48 CRC cell lines. Our results showed that miR-143, but not let-7b, increased sensitization of KRAS mutant tumor cells to paclitaxel. Furthermore, transfection of miR-143, but not let-7b, mimic negatively regulated the expression of mutant but not wild-type KRAS. Combination of miR-143 mimic and paclitaxel induced the onset of apoptosis, and reverted in vitro metastatic properties (migration and invasion) in KRAS mutant tumor cells. MiR-143 thus can be used as a chemosensitizer for the treatment of KRAS mutant tumors and warrants further investigations in in vitro and pre-clinical in vivo models.

  10. 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....

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Transcription activator-like effector nuclease (TALEN-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC and neural stem cell (NSC lines.

    Directory of Open Access Journals (Sweden)

    Trevor Cerbini

    Full Text Available Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%-58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs and neural stem cells (NSCs. The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells.

  16. Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines.

    Science.gov (United States)

    Cerbini, Trevor; Funahashi, Ray; Luo, Yongquan; Liu, Chengyu; Park, Kyeyoon; Rao, Mahendra; Malik, Nasir; Zou, Jizhong

    2015-01-01

    Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs) targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%-58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells.

  17. Markers of T Cell Senescence in Humans

    Directory of Open Access Journals (Sweden)

    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.

  18. Prognosis of acute myeloid leukemia harboring monosomal karyotype in patients treated with or without allogeneic hematopoietic cell transplantation after achieving complete remission

    Science.gov (United States)

    Yanada, Masamitsu; Kurosawa, Saiko; Yamaguchi, Takuhiro; Yamashita, Takuya; Moriuchi, Yukiyoshi; Ago, Hiroatsu; Takeuchi, Jin; Nakamae, Hirohisa; Taguchi, Jun; Sakura, Toru; Takamatsu, Yasushi; Waki, Fusako; Yokoyama, Hiroki; Watanabe, Masato; Emi, Nobuhiko; Fukuda, Takahiro

    2012-01-01

    To evaluate the prognostic impact of monosomal karyotype on post-remission outcome in acute myeloid leukemia, we retrospectively analyzed 2,099 patients who had achieved complete remission. Monosomal karyotype was noted in 73 patients (4%). Of these, the probability of overall survival from first complete remission was 14% at four years, which was significantly lower than that reported in patients without monosomal karyotype, primarily due to a high relapse rate (86%). Monosomal karyotype remained significantly associated with worse overall survival among patients with unfavorable cytogenetics or complex karyotype, and even in patients who underwent allogeneic hematopoietic cell transplantation during first complete remission. These findings confirm that monosomal karyotype has a significantly adverse effect on post-remission outcome in patients with acute myeloid leukemia treated with and without allogeneic hematopoietic cell transplantation in first complete remission, emphasizing the need for the development of alternative therapies for this patient population. PMID:22180431

  19. 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

  20. 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.

  1. Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells.

    Science.gov (United States)

    Liu, Tuoen; Krysiak, Kilannin; Shirai, Cara Lunn; Kim, Sanghyun; Shao, Jin; Ndonwi, Matthew; Walter, Matthew J

    2017-01-01

    Myelodysplastic syndromes (MDS) are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q) cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q) cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q) compared to non-del(5q) MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS.

  2. Chromosome loss caused by DNA fragmentation induced in main nuclei and micronuclei of human lymphoblastoid cells treated with colcemid.

    Science.gov (United States)

    Yamamoto, Mika; Wakata, Akihiro; Aoki, Yoshinobu; Miyamae, Yoichi; Kodama, Seiji

    2014-04-01

    Aneuploidy, a change in the number of chromosomes, plays an essential role in tumorigenesis. Our previous study demonstrated that a loss of a whole chromosome is induced in human lymphocytes by colcemid, a well-known aneugen. Here, to clarify the mechanism for colcemid-induced chromosome loss, we investigated the relationship between chromosome loss and DNA fragmentation in human lymphoblastoid cells treated with colcemid (an aneugen) compared with methyl methanesulfonate (MMS; a clastogen). We analyzed the number of fluorescence in situ hybridization (FISH) signals targeted for a whole chromosome 2 in cytokinesis-blocked binucleated TK6 cells and WTK-1 cells treated with colcemid and MMS, and concurrently detected DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results revealed that DNA fragmentation occurred in 60% of all binucleated TK6 cells harboring colcemid-induced chromosome loss (30% of micronuclei and 30% of main nuclei). DNA fragmentation was observed in colcemid-induced micronuclei containing a whole chromosome but not in MMS-induced micronuclei containing chromosome fragments. In contrast, colcemid-induced nondisjunction had no effect on induction of DNA fragmentation, suggesting that DNA fragmentation was triggered by micronuclei containing a whole chromosome but not by micronuclei containing chromosome fragments or nondisjunction. In addition, the frequency of binucleated cells harboring chromosome loss with DNA fragmentation in micronuclei or main nuclei was higher in wild-type p53 TK6 cells than in mutated-p53 WTK-1 cells treated with colcemid. Taken together, these present and previous results suggest that colcemid-induced chromosome loss is caused by DNA fragmentation, which is triggered by a micronucleus with a whole chromosome and controlled by the p53-dependent pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Submarine harbor navigation using image data

    Science.gov (United States)

    Stubberud, Stephen C.; Kramer, Kathleen A.

    2017-01-01

    The process of ingress and egress of a United States Navy submarine is a human-intensive process that takes numerous individuals to monitor locations and for hazards. Sailors pass vocal information to bridge where it is processed manually. There is interest in using video imaging of the periscope view to more automatically provide navigation within harbors and other points of ingress and egress. In this paper, video-based navigation is examined as a target-tracking problem. While some image-processing methods claim to provide range information, the moving platform problem and weather concerns, such as fog, reduce the effectiveness of these range estimates. The video-navigation problem then becomes an angle-only tracking problem. Angle-only tracking is known to be fraught with difficulties, due to the fact that the unobservable space is not the null space. When using a Kalman filter estimator to perform the tracking, significant errors arise which could endanger the submarine. This work analyzes the performance of the Kalman filter when angle-only measurements are used to provide the target tracks. This paper addresses estimation unobservability and the minimal set of requirements that are needed to address it in this complex but real-world problem. Three major issues are addressed: the knowledge of navigation beacons/landmarks' locations, the minimal number of these beacons needed to maintain the course, and update rates of the angles of the landmarks as the periscope rotates and landmarks become obscured due to blockage and weather. The goal is to address the problem of navigation to and from the docks, while maintaining the traversing of the harbor channel based on maritime rules relying solely on the image-based data. The minimal number of beacons will be considered. For this effort, the image correlation from frame to frame is assumed to be achieved perfectly. Variation in the update rates and the dropping of data due to rotation and obscuration is considered

  4. 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.

  5. CRSMP Potential Harbor Borrow Sites 2012

    Data.gov (United States)

    California Department of Resources — Harbor locations as identified originally in the California Shoreline Database compiled by Noble Consultants (Jon Moore) for California Department of Boating and...

  6. 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.

  7. Putative alternative polyadenylation (APA) events in the early interaction of Salmonella enterica Typhimurium and human host cells.

    Science.gov (United States)

    Afonso-Grunz, Fabian

    2015-12-01

    The immune response of epithelial cells upon infection is mediated by changing activity levels of a variety of proteins along with changes in mRNA, and also ncRNA abundance. Alternative polyadenylation (APA) represents a mechanism that diversifies gene expression similar to alternative splicing. T-cell activation, neuronal activity, development and several human diseases including viral infections involve APA, but at present it remains unclear if this mechanism is also implicated in the response to bacterial infections. Our recently published study of interacting Salmonella enterica Typhimurium and human host cells includes genome-wide expression profiles of human epithelial cells prior and subsequent to infection with the invasive pathogen. The generated dataset (GEO accession number: GSE61730) covers several points of time post infection, and one of these interaction stages was additionally profiled with MACE-based dual 3'Seq, which allows for identification of polyadenylation (PA) sites. The present study features the polyadenylation landscape in early interacting cells based on this data, and provides a comparison of the identified PA sites with those of a corresponding 3P-Seq dataset of non-interacting cells. Differential PA site usage of FTL, PRDX1 and VAPA results in transcription of mRNA isoforms with distinct sets of miRNA and protein binding sites that influence processing, localization, stability, and translation of the respective mRNA. APA of these candidate genes consequently harbors the potential to modulate the host cell response to bacterial infection.

  8. Putative alternative polyadenylation (APA events in the early interaction of Salmonella enterica Typhimurium and human host cells

    Directory of Open Access Journals (Sweden)

    Fabian Afonso-Grunz

    2015-12-01

    Full Text Available The immune response of epithelial cells upon infection is mediated by changing activity levels of a variety of proteins along with changes in mRNA, and also ncRNA abundance. Alternative polyadenylation (APA represents a mechanism that diversifies gene expression similar to alternative splicing. T-cell activation, neuronal activity, development and several human diseases including viral infections involve APA, but at present it remains unclear if this mechanism is also implicated in the response to bacterial infections. Our recently published study of interacting Salmonella enterica Typhimurium and human host cells includes genome-wide expression profiles of human epithelial cells prior and subsequent to infection with the invasive pathogen. The generated dataset (GEO accession number: GSE61730 covers several points of time post infection, and one of these interaction stages was additionally profiled with MACE-based dual 3'Seq, which allows for identification of polyadenylation (PA sites. The present study features the polyadenylation landscape in early interacting cells based on this data, and provides a comparison of the identified PA sites with those of a corresponding 3P-Seq dataset of non-interacting cells. Differential PA site usage of FTL, PRDX1 and VAPA results in transcription of mRNA isoforms with distinct sets of miRNA and protein binding sites that influence processing, localization, stability, and translation of the respective mRNA. APA of these candidate genes consequently harbors the potential to modulate the host cell response to bacterial infection.

  9. Patient affected by neurofibromatosis type 1 and thyroid C-cell hyperplasia harboring pathogenic germ-line mutations in both NF1 and RET genes.

    Science.gov (United States)

    Ercolino, Tonino; Lai, Roberta; Giachè, Valentino; Melchionda, Salvatore; Carella, Massimo; Delitala, Alessandro; Mannelli, Massimo; Fanciulli, Giuseppe

    2014-02-25

    Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease with an estimated incidence of 1 in 3000/3500 live births. NF1 is caused by a mutation in a gene which encodes a protein known as neurofibromin. In up to 5% of cases, NF1 is associated with pheochromocytomas. RET proto-oncogene encodes a member of the receptor tyrosine kinase family involved in the normal development or the neoplastic growth of neural crest cell lineages. Germ-line RET mutations account for cases of Multiple Endocrine Neoplasia type 2 (MEN2), an autosomal dominant genetic syndrome where medullary thyroid carcinoma (MTC) is the major and more clinically severe feature, with nearly complete penetrance. C-cell hyperplasia (CCH) is described in MEN2 patients, and it has been implicated as the precursor of in situ MTC. Patients with RET mutations develop pheochromocytomas in 50% of cases. Rarely, patients with NF1 have been found to present, in addition to the NF1 clinical picture, other lesions, such as parathyroid hyperplasia/adenoma and/or medullary thyroid carcinoma. In spite of the presence of these MEN2 lesions, in none of these patients mutations of gene RET have been found so far. In this report, we describe the first case of a patient affected by a germ-line mutation in both NF1 and RET genes.

  10. 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.

  11. 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 ...

  12. 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.

  13. Prey capture by harbor porpoises

    DEFF Research Database (Denmark)

    Miller, Lee

    2008-01-01

    their ultrasonic clicks as biosonar for orientation and detection of prey (mostly smaller pelagic and bottom dwelling fish), and for communication.  For studying wild animals, hydrophone arrays [Villadsgaard et al. J.Exp.Biol. 210 (2007)] and acoustic (time/depth) tags [Akamatsu et al. Deep Sea Research II 54...... (2007)] have been used.  For studying captive animals, arrays and video techniques [Verfuss et al. J.Exp.Biol. 208 (2005)] as well as miniature acoustic-behavioral tags [Deruiter et al. JASA 123 (2008)] have been used.  While searching for prey, harbor porpoises use clicks at long intervals (~50 ms......) that progressively decrease when closing on an object.  After detecting the prey, the click interval stabilizes and then becomes progressively shorter while approaching the prey.  The sequence ends in a terminal, high repetition rate buzz (~500 clicks/s) just before capturing the prey (a video will be shown...

  14. 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.

  15. Evidence of distinct tumour-propagating cell populations with different properties in primary human hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Federico Colombo

    Full Text Available BACKGROUND AND AIMS: Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC compartments within human hepatocellular carcinoma (HCC. METHODS: After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ/⁻ mice. RESULTS: The primary cell populations (hcc-1, -2 and -3 and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8 differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features. CONCLUSIONS: Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution.

  16. 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.

  17. 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.

  18. Periodic Inspections of Cleveland Harbor East Breakwater, Ohio, and Burns Harbor North Breakwater, Indiana

    Science.gov (United States)

    2015-05-01

    lwd. The authorized channel depth is -30 ft lwd at the entrance and -28 ft lwd in the Harbor. Original construction of Burns Harbor was completed in...and Burns Harbor North Breakwater, Indiana C oa st al a n d H yd ra u lic s La b or at or y Glenn B. Myrick, Jeffrey A. Melby, and...Breakwater, Ohio, and Burns Harbor North Breakwater, Indiana Glenn B. Myrick, Jeffrey A. Melby, and Elizabeth C. Burg Coastal and Hydraulics

  19. 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.

  20. 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

  1. 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...

  2. 33 CFR 100.109 - Winter Harbor Lobster Boat Race, Winter Harbor, ME.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Winter Harbor Lobster Boat Race, Winter Harbor, ME. 100.109 Section 100.109 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF... Lobster Boat Race, Winter Harbor, ME. (a) Regulated area. The regulated area includes all waters of...

  3. 75 FR 78601 - Drawbridge Operation Regulation; Gulf Intracoastal Waterway, New Orleans Harbor, Inner Harbor...

    Science.gov (United States)

    2010-12-16

    ... Orleans Harbor, Inner Harbor Navigation Canal, New Orleans, Orleans Parish, LA AGENCY: Coast Guard, DHS... Seeber/Claiborne Avenue) ] vertical lift bridge across the Inner Harbor Navigational Canal, mile 0.9, (Gulf Intracoastal Waterway mile 6.7 East of Harvey Lock), at New Orleans, Orleans Parish,...

  4. 76 FR 8653 - Drawbridge Operation Regulation; Gulf Intracoastal Waterway, New Orleans Harbor, Inner Harbor...

    Science.gov (United States)

    2011-02-15

    ... Orleans Harbor, Inner Harbor Navigation Canal, New Orleans, Orleans Parish, LA AGENCY: Coast Guard, DHS... Seeber/Claiborne Avenue) vertical lift bridge across the Inner Harbor Navigational Canal, mile 0.9, (Gulf Intracoastal Waterway mile 6.7 East of Harvey Lock), at New Orleans, Orleans Parish, Louisiana. This...

  5. 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.

  6. 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.

  7. 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

  8. 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 from the donor material and are not a result of contamination. Karyotypic analyses demonstrate that reprogramming of human cells by defined factors does not induce, or require, chromosomal abnormalities. Finally, we provide evidence that human iPS cells can be induced to differentiate along lineages representative of the three embryonic germ layers indicating the pluripotency of these cells. Our findings are an important step toward manipulating somatic human cells to generate an unlimited supply of patient-specific pluripotent stem cells. In the future, the use of defined factors to change cell fate may be the key to routine nuclear reprogramming of human somatic cells.

  9. Embryo genome profiling by single-cell sequencing for successful preimplantation genetic diagnosis in a family harboring COL4A1 c.1537G>A; p.G513S mutation

    Science.gov (United States)

    Patel, Nayana H.; Bhadarka, Harsha K.; Patel, Kruti B.; Vaniawala, Salil N.; Acharya, Arpan; Mukhopadhyaya, Pratap N.; Sodagar, Nilofar R.

    2016-01-01

    CONTEXT: Genetic profiling of embryos (also known as preimplantation genetic diagnosis) before implantation has dramatically enhanced the success quotient of in vitro fertilization (IVF) in recent times. The technology helps in avoiding selective pregnancy termination since the baby is likely to be free of the disease under consideration. AIM: Screening of embryos free from c.1537G>A; p.G513S mutation within the COL4A1 gene for which the father was known in before be in heterozygous condition. SUBJECTS AND METHODS: Processing of trophectoderm biopsies was done from twelve embryos for c.1537G>A; p.G513S mutation within the COL4A1 gene. DNA extracted from isolated cells were subjected to whole genome amplification using an isothermal amplification and strand displacement technology. Oligonucleotide primers bracketing the mutation were synthesized and used to amplify 162 base pairs (bp) polymerase chain reaction amplicons originating from each embryo which were subsequently sequenced to detect the presence or absence of the single base polymorphism. RESULTS: Three out of 12 embryos interrogated in this study were found to be normal while 9 were found to harbor the mutation in heterozygous condition. Implantation of one of the normal embryos following by chorionic villus sampling at 11th week of pregnancy indicated that the baby was free from c.1537G>A; p.G513S mutation within the COL4A1 gene. CONCLUSIONS: Single-cell sequencing is a helpful tool for preimplantation embryo profiling. This is the first report from India describing the birth of a normal child through IVF procedure where a potential pathogenic COL4A1 allele was avoided using this technology. PMID:27803589

  10. Combination of the somatic cell nuclear transfer method and RNAi technology for the production of a prion gene-knockdown calf using plasmid vectors harboring the U6 or tRNA promoter.

    Science.gov (United States)

    Wongsrikeao, Pimprapar; Sutou, Shizuyo; Kunishi, Miho; Dong, Ya Juan; Bai, Xuejin; Otoi, Takeshige

    2011-01-01

    By combining RNAi technology with SCNT method, we attempted to produce transgenic calves with knocked down bPRNP for technological assessments. The respective utilities of type II (tRNA) and type III (hU6) Pol III promoters in mediating plasmid vector-based RNAi for the production of a bPRNP-knockdown calf were compared. Plasmid harboring DNA for siRNA expression was introduced stably into the genome of primary cultured bovine cells. By inserting the transgenic cell into an enucleated bovine egg, SCNT embryos were produced. The ability for SCNT embryos to develop to blastocysts was higher in hU6 based vector groups (44-53%) than in a tRNA group (32%). In all, 30 hU6-embryos and 12 tRNA-embryos were transferred to 11 recipients. Only tRNA-embryos were able to impregnate recipients (6 out of 11 transfers), resulting in four aborted fetuses, one stillbirth, and one live-born calf. The expression of EGFP, a marker, was detected in all six. The bPRNP transcript levels in the nervous tissues (brain, cerebellum, spinal bulb, and spinal cord) from the calf, which was killed 20 days after birth, were reduced to 35% of those of the control calf on average, as determined by qRT-PCR. The PrPC levels, as estimated by western blot were reduced to 86% on average in the nervous tissues. These findings suggest that SCNT technology remains immature, that the tRNA promoter is useful, and that RNAi can significantly reduce PRNP mRNA levels, but insufficient reduction of PrPC levels exists in cattle under these conditions.

  11. PAH- and PCB-induced Alterations of Protein Tyrosine Kinase and Cytokine Gene Transcription in Harbor Seal (Phoca Vitulina PBMC

    Directory of Open Access Journals (Sweden)

    Jennifer C. C. Neale

    2005-01-01

    Full Text Available Mechanisms underlying in vitro immunomodulatory effects of polycyclic aromatic hydrocarbons (PAHs and polychlorinated biphenyls (PCBs were investigated in harbor seal peripheral leukocytes, via real-time PCR. We examined the relative genetic expression of the protein tyrosine kinases (PTKs Fyn and Itk, which play a critical role in T cell activation, and IL-2, a cytokine of central importance in initiating adaptive immune responses. IL-1, the macrophage-derived pro-inflammatory cytokine of innate immunity, was also included as a measure of macrophage function. Harbor seal PBMC were exposed to the prototypic immunotoxic PAH benzo[a]pyrene (BaP, 3,3',4,4',5,5'-hexachlorobiphenyl (CB-169, a model immunotoxic PCB, or DMSO (vehicle control. Exposure of Con A-stimulated harbor seal PBMC to both BaP and CB-169 produced significantly altered expression in all four targets relative to vehicle controls. The PTKs Fyn and Itk were both up-regulated following exposure to BaP and CB-169. In contrast, transcripts for IL-2 and IL-1 were decreased relative to controls by both treatments. Our findings are consistent with those of previous researchers working with human and rodent systems and support a hypothesis of contaminant-altered lymphocyte function mediated (at least in part by disruption of T cell receptor (TCR signaling and cytokine production.

  12. Clinicopathologic characteristics andtherapeutic responses ofChinese patients withnon-small cell lung cancer who harbor an anaplastic lymphoma kinase rearrangement

    Institute of Scientific and Technical Information of China (English)

    ShaFu; HaiYunWang; FangWang; MaYanHuang; LingDeng; XiaoZhang; ZuLuYe; JianYong Shao

    2015-01-01

    Introduction:The rearrangement of the anaplastic lymphoma kinase (ALK) gene accounts for approximately 1%–6%of lung adenocarcinoma cases and deifnes a molecular subgroup of tumors characterized by clinical sensitivity toALK inhibitors such as crizotinib. This study aimed to identify the relationship betweenALK rearrangement and the clinico‑pathologic characteristics of non‑small cell lung cancer (NSCLC) and to analyze the therapeutic responses of crizotinib and conventional chemotherapy toALK rearrangement in NSCLC patients. Methods:A total of 487 lung cancer patients who underwent testing forALK rearrangement in our department were included in this study.ALK rearrangement was examined by using lfuorescence insitu hybridization (FISH) assay. Results:Among the 487 patients, 44 (9.0%) were diagnosed withALK rearrangement by using FISH assay. In 123 patients with adenocarcinoma who were non‑smokers and of a young age (≤58years old), the frequency ofALK rearrangement was 20.3% (25/123). Short overall survival (OS) was associated with non‑adenocarcinoma tumor type (P=0.006), poorly differentiated tumors (P=0.001), advanced‑stage tumors (P<0.001), smoking history (P=0.008), and wild‑type epidermal growth factor receptor (EGFR) (P=0.008). Moreover, patients with poorly differentiated and advanced‑stage tumors had a shorter time to cancer progression compared with those with well differentiated (P=0.023) and early‑stage tumors (P=0.001), respectively. Conclusions:ALK‑rearranged NSCLC tends to occur in younger individuals who are either non‑smokers or light smokers with adenocarcinoma. Patients withALK rearrangement might beneift fromALK inhibitor therapy.

  13. Patients with Merkel cell carcinoma tumors < or = 1.0 cm in diameter are unlikely to harbor regional lymph node metastasis.

    Science.gov (United States)

    Stokes, Jayme B; Graw, Katherine S; Dengel, Lynn T; Swenson, Brian R; Bauer, Todd W; Slingluff, Craig L; Ledesma, Elihu J

    2009-08-10

    Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine cutaneous malignancy. Current recommendations include offering regional lymph node evaluation by either sentinel lymph node biopsy (SLNB) or complete lymph node dissection (CLND) to all patients with MCC; however, we hypothesized a cohort of low-risk patients may exist for whom regional nodal metastasis would be unlikely. A retrospective review of the Department of Veterans Affairs national health care database was performed. Patients undergoing resection of primary MCC were identified; and demographic, medical, and social history; tumor characteristics; nodal status; and recurrence events were recorded. Between 1995 and 2006, 346 patients were diagnosed with MCC. Of these, 213 underwent resection of the primary lesion and evaluation of the draining lymph node basin. Fifty-four patients (25%) had tumors < or = 1.0 cm in diameter. Average tumor diameter was 0.7 cm, and 63% were located on the head or neck. Only two patients (4%) with tumors < or = 1.0 cm had regional lymph node metastasis, compared with 51 (24%) of 213 patients with tumors more than 1.0 cm (P < .0001). Both patients had clinically evident nodal disease at presentation and underwent CLND. Both have remained recurrence-free for 40 months. Thirteen (25%) of 51 patients with nodal metastasis and tumors more than 1 cm had occult nodal metastasis. In this series, patients with MCC < or = 1.0 cm were unlikely to have regional lymph node metastasis, suggesting that regional nodal evaluation may reasonably be avoided in these patients. However, these data support SLNB for MCC more than 1 cm in diameter.

  14. Madaket Harbor, Nantucket, Massachusetts. Water Resources Improvement.

    Science.gov (United States)

    1977-07-01

    beamc. Tnis material will be re-deposited,, viaj troio it 1-apfro1inr ox prior location. j, MADAKET HARBOR NANTUCKET, MASSACHUSETTS FEASIBILITY...re- colonization of approximately 395 acres by scallops and quahogs. Also, barring any future disruption of the harbor area, the continued use of

  15. 33 CFR 117.549 - Cambridge Harbor.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Cambridge Harbor. 117.549 Section... DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Maryland § 117.549 Cambridge Harbor. The draw of the S342 bridge, mile 0.1 at Cambridge, shall open on signal from 6 a.m. to 8 p.m.; except that, from...

  16. Stereological quantification of mast cells in human synovium

    DEFF Research Database (Denmark)

    Damsgaard, T E; Sørensen, Flemming Brandt; Herlin, T;

    1999-01-01

    Mast cells participate in both the acute allergic reaction as well as in chronic inflammatory diseases. Earlier studies have revealed divergent results regarding the quantification of mast cells in the human synovium. The aim of the present study was therefore to quantify these cells in the human...... synovium, using stereological techniques. Different methods of staining and quantification have previously been used for mast cell quantification in human synovium. Stereological techniques provide precise and unbiased information on the number of cell profiles in two-dimensional tissue sections of......, in this case, human synovium. In 10 patients suffering from osteoarthritis a median of 3.6 mast cells/mm2 synovial membrane was found. The total number of cells (synoviocytes, fibroblasts, lymphocytes, leukocytes) present was 395.9 cells/mm2 (median). The mast cells constituted 0.8% of all the cell profiles...

  17. Technical Challenges in the Derivation of Human Pluripotent Cells

    Directory of Open Access Journals (Sweden)

    Parinya Noisa

    2011-01-01

    Full Text Available It has long been discovered that human pluripotent cells could be isolated from the blastocyst state of embryos and called human embryonic stem cells (ESCs. These cells can be adapted and propagated indefinitely in culture in an undifferentiated manner as well as differentiated into cell representing the three major germ layers: endoderm, mesoderm, and ectoderm. However, the derivation of human pluripotent cells from donated embryos is limited and restricted by ethical concerns. Therefore, various approaches have been explored and proved their success. Human pluripotent cells can also be derived experimentally by the nuclear reprogramming of somatic cells. These techniques include somatic cell nuclear transfer (SCNT, cell fusion and overexpression of pluripotent genes. In this paper, we discuss the technical challenges of these approaches for nuclear reprogramming, involving their advantages and limitations. We will also highlight the possible applications of these techniques in the study of stem cell biology.

  18. Human B cell activating factor (BCAF): production by a human T cell tumor line.

    Science.gov (United States)

    Fevrier, M; Diu, A; Mollier, P; Abadie, A; Olive, D; Mawas, C; Theze, J

    1989-01-01

    In a previous study, we demonstrated that supernatants from human T cell clones stimulated by a pair of anti-CD2 monoclonal antibodies cause resting human B cells to become activated and to proliferate in the absence of any other signals. The activity responsible for these effects was shown to be different from already characterized lymphokines and in particular from IL-2 and IL-4, and was named B Cell Activating Factor or BCAF. In this paper, we describe the production of BCAF by a human T cell tumor line T687 after phorbol myristate acetate (PMA) stimulation; this production can be potentiated by phytohemagglutinin (PHA). We further show that the stimulatory phase can be separated from the secretory phase thereby avoiding contamination of BCAF-containing supernatant by PMA and PHA. Supernatants produced under these conditions do not contain either IL-4 or IFN but contain traces of lymphotoxin and 2 to 10 ng/ml of IL-2. The T687 cell line will allow us to obtain a large volume of supernatant for biochemical study and purification of the molecule(s) responsible for BCAF activity.

  19. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  20. Human Immunodeficiency Syndromes Affecting Human Natural Killer Cell Cytolytic Activity

    OpenAIRE

    Ham, Hyoungjun; Billadeau, Daniel D.

    2014-01-01

    Natural killer (NK) cells are lymphocytes of the innate immune system that secrete cytokines upon activation and mediate the killing of tumor cells and virus-infected cells, especially those that escape the adaptive T cell response caused by the down regulation of MHC-I. The induction of cytotoxicity requires that NK cells contact target cells through adhesion receptors, and initiate activation signaling leading to increased adhesion and accumulation of F-actin at the NK cell cytotoxic synaps...

  1. Human immunodeficiency virus type 1 infection of human uterine epithelial cells: viral shedding and cell contact-mediated infectivity.

    Science.gov (United States)

    Asin, Susana N; Wildt-Perinic, Dunja; Mason, Sarah I; Howell, Alexandra L; Wira, Charles R; Fanger, Michael W

    2003-05-15

    We examined the mechanism of human immunodeficiency virus (HIV) type 1 infection of human uterine epithelial cells to gain a clearer understanding of the events by which HIV-1 infects cells within the female reproductive tract. We demonstrated that these cells can be productively infected by HIV-1 and that infection is associated with viral RNA reverse transcription, DNA transcription, and secretion of infectious virus. Levels of viral DNA and secreted virus decreased gradually after infection. Moreover, virus released by the uterine epithelial cells shortly after infection was able to infect human T cell lines, but virus released later did not. In contrast, human CD4(+) T cell lines were infected after cocultivation with epithelial cells at both early and late stages of infection. These data demonstrated that HIV-1 infects human epithelial cells of upper reproductive tract origin and that productive viral infection of epithelial cells may be an important mechanism of transmission of HIV-1 infection in women.

  2. Effect of human neural progenitor cells on injured spinal cord

    Institute of Scientific and Technical Information of China (English)

    XU Guang-hui; BAI Jin-zhu; CAI Qin-lin; LI Xiao-xia; LI Ling-song; SHEN Li

    2005-01-01

    Objective: To study whether human neural progenitor cells can differentiate into neural cells in vivo and improve the recovery of injured spinal cord in rats.Methods: Human neural progenitor cells were transplanted into the injured spinal cord and the functional recovery of the rats with spinal cord contusion injury was evaluated with Basso-Beattie-Bresnahan (BBB) locomotor scale and motor evoked potentials. Additionally, the differentiation of human neural progenitor cells was shown by immunocytochemistry.Results: Human neural progenitor cells developed into functional cells in the injured spinal cord and improved the recovery of injured spinal cord in both locomotor scores and electrophysiological parameters in rats.Conclusions: Human neural progenitor cells can treat injured spinal cord, which may provide a new cell source for research of clinical application.

  3. Generation of induced pluripotent stem cells from human blood.

    Science.gov (United States)

    Loh, Yuin-Han; Agarwal, Suneet; Park, In-Hyun; Urbach, Achia; Huo, Hongguang; Heffner, Garrett C; Kim, Kitai; Miller, Justine D; Ng, Kitwa; Daley, George Q

    2009-05-28

    Human dermal fibroblasts obtained by skin biopsy can be reprogrammed directly to pluripotency by the ectopic expression of defined transcription factors. Here, we describe the derivation of induced pluripotent stem cells from CD34+ mobilized human peripheral blood cells using retroviral transduction of OCT4/SOX2/KLF4/MYC. Blood-derived human induced pluripotent stem cells are indistinguishable from human embryonic stem cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes, and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human blood will allow the generation of patient-specific stem cells for diseases in which the disease-causing somatic mutations are restricted to cells of the hematopoietic lineage.

  4. A Chemical Probe that Labels Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Nao Hirata

    2014-03-01

    Full Text Available A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1] that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1 and ABCG2 (BCRP, both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

  5. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.

    Directory of Open Access Journals (Sweden)

    Jin Hee Kim

    Full Text Available When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i there are no publicly available cloning vectors harboring a 2A peptide gene and (ii comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.

  6. Enhanced casein kinase II activity in human tumour cell cultures

    DEFF Research Database (Denmark)

    Prowald, K; Fischer, H; Issinger, O G

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...

  7. MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

    NARCIS (Netherlands)

    KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

    1995-01-01

    In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

  8. Derivation and differentiation of haploid human embryonic stem cells.

    Science.gov (United States)

    Sagi, Ido; Chia, Gloryn; Golan-Lev, Tamar; Peretz, Mordecai; Weissbein, Uri; Sui, Lina; Sauer, Mark V; Yanuka, Ofra; Egli, Dieter; Benvenisty, Nissim

    2016-04-07

    Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development.

  9. Human embryonic stem cells and respect for life

    OpenAIRE

    Meyer, J.(CERN, Geneva, Switzerland)

    2000-01-01

    The purpose of this essay is to stimulate academic discussion about the ethical justification of using human primordial stem cells for tissue transplantation, cell replacement, and gene therapy. There are intriguing alternatives to using embryos obtained from elective abortions and in vitro fertilisation to reconstitute damaged or dysfunctional human organs. These include the expansion and transplantation of latent adult progenitor cells.

  10. Abnormalities in human pluripotent cells due to reprogramming mechanisms.

    Science.gov (United States)

    Ma, Hong; Morey, Robert; O'Neil, Ryan C; He, Yupeng; Daughtry, Brittany; Schultz, Matthew D; Hariharan, Manoj; Nery, Joseph R; Castanon, Rosa; Sabatini, Karen; Thiagarajan, Rathi D; Tachibana, Masahito; Kang, Eunju; Tippner-Hedges, Rebecca; Ahmed, Riffat; Gutierrez, Nuria Marti; Van Dyken, Crystal; Polat, Alim; Sugawara, Atsushi; Sparman, Michelle; Gokhale, Sumita; Amato, Paula; Wolf, Don P; Ecker, Joseph R; Laurent, Louise C; Mitalipov, Shoukhrat

    2014-07-10

    Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the 'gold standard', they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies.

  11. Monocytes harboring cytomegalovirus: interactions with endothelial cells, smooth muscle cells, and oxidized low-density lipoprotein. Possible mechanisms for activating virus delivered by monocytes to sites of vascular injury.

    Science.gov (United States)

    Guetta, E; Guetta, V; Shibutani, T; Epstein, S E

    1997-07-01

    Cytomegalovirus (CMV) infection and its periodic reactivation from latency may contribute to atherogenesis and restenosis. It is unknown how CMV is delivered to the vessel wall and is reactivated. We examined the following hypothesis: CMV, present in monocytes recruited to sites of vascular injury, is activated by endothelial cell (EC) or smooth muscle cell (SMC) contact and by oxidized low-density lipoproteins (oxLDLs). The CMV major immediate-early promoter (MIEP) controls immediate-early (IE) gene expression, and thereby viral replication. To determine whether elements of the vessel wall can activate CMV present in monocytes, we transiently transfected the promonocytic cell line HL-60 with a chloramphenicol acetyltransferase reporter gene construct driven by MIEP. MIEP activity increased 1.7 +/- 0.5-fold (P = .02) when the transfected HL-60 cells were cocultured with ECs, 4.5 +/- 1.5-fold when cocultured with SMCs (P = .03), and 2.0 +/- 0.5-fold (P = .01) when exposed to oxLDL. The combination of oxLDL and EC coculture increased MIEP activity over 7-fold. We also found that freshly isolated human monocytes, infected with endothelium-passaged CMV, were capable of transmitting infectious virus to cocultured ECs or SMCs. CMV-related progression of atherosclerosis or restenosis may, at least in part, involve monocyte delivery of the virus to the site of vascular injury, where the vascular milieu, ie, contact with ECs, SMCs, and oxLDL, can contribute to viral reactivation and/or replication by enhancing CMV IE gene expression. The virus may then infect neighboring ECs or SMCs, initiating a cascade of events predisposing to the development of atherogenesis-related processes.

  12. Proteomic analysis of human blastocoel fluid and blastocyst cells

    DEFF Research Database (Denmark)

    Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen

    2013-01-01

    Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the blastocyst and can differentiate into any cell type in the human body. These cells hold a great potential for regenerative medicine, but to obtain enough cells needed for medical treatment, culture is required......, the blastocoel fluid, which is in contact with all the cells in the blastocyst, including hESCs. Fifty-three surplus human blastocysts were donated after informed consent, and blastocoel fluid was isolated by micromanipulation. Using highly sensitive nano-high-pressure liquid chromatography-tandem mass...

  13. 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.

  14. Derivation and characterization of human embryonic stem cells on human amnion epithelial cells.

    Science.gov (United States)

    Lai, Dongmei; Wang, Yongwei; Sun, Jian; Chen, Yifei; Li, Ting; Wu, Yi; Guo, Lihe; Wei, Chunsheng

    2015-05-07

    Culture conditions that support the growth of undifferentiated human embryonic stem cells (hESCs) have already been established using primary human amnion epithelial cells (hAECs) as an alternative to traditional mitotically inactivated mouse embryonic fibroblasts (MEFs). In the present work, inner cell masses (ICM) were isolated from frozen embryos obtained as donations from couples undergoing in vitro fertilization (IVF) treatment and four new hESC lines were derived using hAECs as feeder cells. This feeder system was able to support continuous growth of what were, according to their domed shape and markers, undifferentiated naïve-like hESCs. Their pluripotent potential were also demonstrated by embryoid bodies developing to the expected three germ layers in vitro and the productions of teratoma in vivo. The cell lines retained their karyotypic integrity for over 35 passages. Transmission electron microscopy (TEM) indicated that these newly derived hESCs consisted mostly of undifferentiated cells with large nuclei and scanty cytoplasm. The new hESCs cultured on hAECs showed distinct undifferentiated characteristics in comparison to hESCs of the same passage maintained on MEFs. This type of optimized culture system may provide a useful platform for establishing clinical-grade hESCs and assessing the undifferentiated potential of hESCs.

  15. Characterization of gene rearrangements resulted from genomic structural aberrations in human esophageal squamous cell carcinoma KYSE150 cells.

    Science.gov (United States)

    Hao, Jia-Jie; Gong, Ting; Zhang, Yu; Shi, Zhi-Zhou; Xu, Xin; Dong, Jin-Tang; Zhan, Qi-Min; Fu, Song-Bin; Wang, Ming-Rong

    2013-01-15

    Chromosomal rearrangements and involved genes have been reported to play important roles in the development and progression of human malignancies. But the gene rearrangements in esophageal squamous cell carcinoma (ESCC) remain to be identified. In the present study, array-based comparative genomic hybridization (array-CGH) was performed on the ESCC cell line KYSE150. Eight disrupted genes were detected according to the obviously distinct unbalanced breakpoints. The splitting of these genes was validated by dual-color fluorescence in-situ hybridization (FISH). By using rapid amplification of cDNA ends (RACE), genome walking and sequencing analysis, we further identified gene disruptions and rearrangements. A fusion transcript DTL-1q42.2 was derived from an intrachromosomal rearrangement of chromosome 1. Highly amplified segments of DTL and PTPRD were self-rearranged. The sequences on either side of the junctions possess micro-homology with each other. FISH results indicated that the split DTL and PTPRD were also involved in comprising parts of the derivative chromosomes resulted from t(1q;9p;12p) and t(9;1;9). Further, we found that regions harboring DTL (1q32.3) and PTPRD (9p23) were also splitting in ESCC tumors. The data supplement significant information on the existing genetic background of KYSE150, which may be used as a model for studying these gene rearrangements.

  16. 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

  17. Cell Culture Systems To Study Human Herpesvirus 6A/B Chromosomal Integration.

    Science.gov (United States)

    Gravel, Annie; Dubuc, Isabelle; Wallaschek, Nina; Gilbert-Girard, Shella; Collin, Vanessa; Hall-Sedlak, Ruth; Jerome, Keith R; Mori, Yasuko; Carbonneau, Julie; Boivin, Guy; Kaufer, Benedikt B; Flamand, Louis

    2017-07-15

    Human herpesviruses 6A/B (HHV-6A/B) can integrate their viral genomes in the telomeres of human chromosomes. The viral and cellular factors contributing to HHV-6A/B integration remain largely unknown, mostly due to the lack of efficient and reproducible cell culture models to study HHV-6A/B integration. In this study, we characterized the HHV-6A/B integration efficiencies in several human cell lines using two different approaches. First, after a short-term infection (5 h), cells were processed for single-cell cloning and analyzed for chromosomally integrated HHV-6A/B (ciHHV-6A/B). Second, cells were infected with HHV-6A/B and allowed to grow in bulk for 4 weeks or longer and then analyzed for the presence of ciHHV-6. Using quantitative PCR (qPCR), droplet digital PCR, and fluorescent in situ hybridization, we could demonstrate that HHV-6A/B integrated in most human cell lines tested, including telomerase-positive (HeLa, MCF-7, HCT-116, and HEK293T) and telomerase-negative cell lines (U2OS and GM847). Our results also indicate that inhibition of DNA replication, using phosphonoacetic acid, did not affect HHV-6A/B integration. Certain clones harboring ciHHV-6A/B spontaneously express viral genes and proteins. Treatment of cells with phorbol ester or histone deacetylase inhibitors triggered the expression of many viral genes, including U39, U90, and U100, without the production of infectious virus, suggesting that the tested stimuli were not sufficient to trigger full reactivation. In summary, both integration models yielded comparable results and should enable the identification of viral and cellular factors contributing to HHV-6A/B integration and the screening of drugs influencing viral gene expression, as well as the release of infectious HHV-6A/B from the integrated state.IMPORTANCE The analysis and understanding of HHV-6A/B genome integration into host DNA is currently limited due to the lack of reproducible and efficient viral integration systems. In the

  18. Complete Genome Sequence of a Human-Invasive Salmonella enterica Serovar Typhimurium Strain of the Emerging Sequence Type 213 Harboring a Multidrug Resistance IncA/C Plasmid and a blaCMY-2-Carrying IncF Plasmid.

    Science.gov (United States)

    Silva, Claudia; Calva, Edmundo; Calva, Juan J; Wiesner, Magdalena; Fernández-Mora, Marcos; Puente, José L; Vinuesa, Pablo

    2015-11-12

    Salmonella enterica subsp. enterica serovar Typhimurium strain 33676 was isolated in Mexico City, Mexico, from a patient with a systemic infection, and its complete genome sequence was determined using PacBio single-molecule real-time technology. Strain 33676 harbors an IncF plasmid carrying the extended-spectrum cephalosporin gene blaCMY-2 and a multidrug resistance IncA/C plasmid. Copyright © 2015 Silva et al.

  19. 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.

  20. Human Cell and Tissue Establishment Registration Public Query

    Data.gov (United States)

    U.S. Department of Health & Human Services — This application provides Human Cell and Tissue registration information for registered, inactive, and pre-registered firms. Query options are by Establishment Name,...

  1. Human Cell and Tissue Establishment Registration Public Query

    Data.gov (United States)

    U.S. Department of Health & Human Services — This application provides Human Cell and Tissue registration information for registered, inactive, and pre-registered firms. Query options are by Establishment Name,...

  2. Human tissue legislation in South Africa: Focus on stem cell ...

    African Journals Online (AJOL)

    Human tissue legislation in South Africa: Focus on stem cell research and therapy. ... Related Substances Act, the Consumer Protection Act, the Children's Act and ... human tissue legislation in SA, the legislator has an opportunity to mirror the ...

  3. Human Stem Cell Derived Cardiomyocytes: An Alternative ...

    Science.gov (United States)

    Chemical spills and associated deaths in the US has increased 2.6-fold and 16-fold from 1983 to 2012, respectfully. In addition, the number of chemicals to which humans are exposed to in the environment has increased almost 10-fold from 2001 to 2013 within the US. Internationally, a WHO report on the global composite impact of chemicals on health reported that 16% of the total burden of cardiovascular disease was attributed to environmental chemical exposure with 2.5 million deaths per year. Clearly, the cardiovascular system, at all its various developmental and life stages, represents a critical target organ system that can be adversely affected by existing and emerging chemicals (e.g., engineered nanomaterials) in a variety of environmental media. The ability to assess chemical cardiac risk and safety is critically needed but extremely challenging due to the number and categories of chemicals in commerce, as indicated. This presentation\\session will evaluate the use of adult human stem cell derived cardiomyocytes, and existing platforms, as an alternative model to evaluate environmental chemical cardiac toxicity as well as provide key information for the development of predictive adverse outcomes pathways associated with environmental chemical exposures. (This abstract does not represent EPA policy) Rapid and translatable chemical safety screening models for cardiotoxicity current status for informing regulatory decisions, a workshop sponsored by the Society

  4. Human pancreatic β-cell G1/S molecule cell cycle atlas.

    Science.gov (United States)

    Fiaschi-Taesch, Nathalie M; Kleinberger, Jeffrey W; Salim, Fatimah G; Troxell, Ronnie; Wills, Rachel; Tanwir, Mansoor; Casinelli, Gabriella; Cox, Amy E; Takane, Karen K; Scott, Donald K; Stewart, Andrew F

    2013-07-01

    Expansion of pancreatic β-cells is a key goal of diabetes research, yet induction of adult human β-cell replication has proven frustratingly difficult. In part, this reflects a lack of understanding of cell cycle control in the human β-cell. Here, we provide a comprehensive immunocytochemical "atlas" of G1/S control molecules in the human β-cell. This atlas reveals that the majority of these molecules, previously known to be present in islets, are actually present in the β-cell. More importantly, and in contrast to anticipated results, the human β-cell G1/S atlas reveals that almost all of the critical G1/S cell cycle control molecules are located in the cytoplasm of the quiescent human β-cell. Indeed, the only nuclear G1/S molecules are the cell cycle inhibitors, pRb, p57, and variably, p21: none of the cyclins or cdks necessary to drive human β-cell proliferation are present in the nuclear compartment. This observation may provide an explanation for the refractoriness of human β-cells to proliferation. Thus, in addition to known obstacles to human β-cell proliferation, restriction of G1/S molecules to the cytoplasm of the human β-cell represents an unanticipated obstacle to therapeutic human β-cell expansion.

  5. Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells

    DEFF Research Database (Denmark)

    Anisimov, Sergey V.; Christophersen, Nicolaj S.; Correia, Ana S.

    2011-01-01

    the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early...

  6. Human embryonic stem cell research: ethical and legal issues.

    Science.gov (United States)

    Robertson, J A

    2001-01-01

    The use of human embryonic stem cells to replace damaged cells and tissues promises future hope for the treatment of many diseases. However, many countries now face complex ethical and legal questions as a result of the research needed to develop these cell-replacement therapies. The challenge that must be met is how to permit research on human embryonic tissue to occur while maintaining respect for human life generally.

  7. Induction of genomic instability and activation of autophagy in artificial human aneuploid cells

    Energy Technology Data Exchange (ETDEWEB)

    Ariyoshi, Kentaro [Hirosaki University, Institute of Radiation Emergency Medicine, 66-1 Hon-cho, Hirosaki 036-8564 (Japan); Miura, Tomisato; Kasai, Kosuke; Fujishima, Yohei [Department of Biomedical Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki 036-8564 (Japan); Oshimura, Mitsuo [Chromosome Engineering Research Center (CERC), Tottori University, Nishicho 86, Yonago, Tottori 683-8503 (Japan); Yoshida, Mitsuaki A., E-mail: ariyoshi@hirosaki-u.ac.jp [Hirosaki University, Institute of Radiation Emergency Medicine, 66-1 Hon-cho, Hirosaki 036-8564 (Japan)

    2016-08-15

    Highlights: • Clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. • Increased autophagy was observed in the artificially aneuploid clones. • Inhibition of autophagy resulted in increased genomic instability and DNA damage. • Intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones. - Abstract: Chromosome missegregation can lead to a change in chromosome number known as aneuploidy. Although aneuploidy is a known hallmark of cancer cells, the various mechanisms by which altered gene and/or DNA copy number facilitate tumorigenesis remain unclear. To understand the effect of aneuploidy occurring in non-tumorigenic human breast epithelial cells, we generated clones harboring artificial aneuploidy using microcell-mediated chromosome transfer. Our results demonstrate that clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. Also, the increased autophagy was observed in the artificially aneuploidy clones, and inhibition of autophagy resulted in increased genomic instability and DNA damage. In addition, the intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones, and inhibition of autophagy further increased the production of reactive oxygen species. Together, these results suggest that even a single extraneous chromosome can induce genomic instability, and that autophagy triggered by aneuploidy-induced stress is a mechanism to protect cells bearing abnormal chromosome number.

  8. Epigenetic Regulation of Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Qidong eHu

    2012-11-01

    Full Text Available Recently, there has been tremendous progress in characterizing the transcriptional network regulating hESCs (MacArthur et al., 2009; Loh et al., 2011, including those signaling events mediated by Oct4, Nanog and Sox2. There is growing interest in the epigenetic machinery involved in hESC self-renewal and differentiation. In general, epigenetic regulation includeschromatin reorganization, DNA modification and histone modification, which are not directly related to alterations in DNA sequences. Various protein complexes, includingPolycomb, trithorax, NuRD, SWI/SNF andOct4, have been shown to play critical roles in epigenetic control of hESC maintenance and differentiation. Hence, we will formally review recent advances in unraveling the multifaceted role of epigenetic regulation in hESC self-renewal and induced differentiation, particularly with respect to chromatin remodeling and DNA methylation events. Unraveling the molecular mechanisms underlying the maintenance/differentiation of hESCs and reprogramming of somatic cells will greatly strengthen our capacity to generate various types of cells to treat human diseases.

  9. 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.

  10. Development and characterization of a novel human Waldenström macroglobulinemia cell line: RPCI-WM1, Roswell Park Cancer Institute - Waldenström Macroglobulinemia 1.

    Science.gov (United States)

    Chitta, Kasyapa S; Paulus, Aneel; Ailawadhi, Sikander; Foster, Barbara A; Moser, Michael T; Starostik, Petr; Masood, Aisha; Sher, Taimur; Miller, Kena C; Iancu, Dan M; Conroy, Jeffrey; Nowak, Norma J; Sait, Sheila N; Personett, David A; Coleman, Morton; Furman, Richard R; Martin, Peter; Ansell, Stephen M; Lee, Kelvin; Chanan-Khan, Asher A

    2013-02-01

    Understanding the biology of Waldenström macroglobulinemia is hindered by a lack of preclinical models. We report a novel cell line, RPCI-WM1, from a patient treated for WM. The cell line secretes human immunoglobulin M (h-IgM) with κ-light chain restriction identical to the primary tumor. The cell line has a modal chromosomal number of 46 and harbors chromosomal changes such as deletion of 6q21, monoallelic deletion of 9p21 (CDKN2A), 13q14 (RB1) and 18q21 (BCL-2), with a consistent amplification of 14q32 (immunoglobulin heavy chain; IgH) identical to its founding tumor sample. The clonal relationship is confirmed by identical CDR3 length and single nucleotide polymorphisms as well as a matching IgH sequence of the cell line and founding tumor. Both also harbor a heterozygous, non-synonymous mutation at amino acid 265 in the MYD88 gene (L265P). The cell line expresses most of the cell surface markers present on the parent cells. Overall, RPCI-WM1 represents a valuable model to study Waldenström macroglobulinemia.

  11. Derivation, propagation and differentiation of human embryonic stem cells.

    Science.gov (United States)

    Conley, Brock J; Young, Julia C; Trounson, Alan O; Mollard, Richard

    2004-04-01

    Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug

  12. Alloimmune Responses of Humanized Mice to Human Pluripotent Stem Cell Therapeutics

    Directory of Open Access Journals (Sweden)

    Nigel G. Kooreman

    2017-08-01

    Full Text Available There is growing interest in using embryonic stem cell (ESC and induced pluripotent stem cell (iPSC derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an “allogenized” mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts.

  13. New frontiers in human cell biology and medicine: can pluripotent stem cells deliver?

    Science.gov (United States)

    Goldstein, Lawrence S B

    2012-11-12

    Human pluripotent stem cells provide enormous opportunities to treat disease using cell therapy. But human stem cells can also drive biomedical and cell biological discoveries in a human model system, which can be directly linked to understanding disease or developing new therapies. Finally, rigorous scientific studies of these cells can and should inform the many science and medical policy issues that confront the translation of these technologies to medicine. In this paper, I discuss these issues using amyotrophic lateral sclerosis as an example.

  14. Human Satellite Cell Transplantation and Regeneration from Diverse Skeletal Muscles

    Directory of Open Access Journals (Sweden)

    Xiaoti Xu

    2015-09-01

    Full Text Available Identification of human satellite cells that fulfill muscle stem cell criteria is an unmet need in regenerative medicine. This hurdle limits understanding how closely muscle stem cell properties are conserved among mice and humans and hampers translational efforts in muscle regeneration. Here, we report that PAX7 satellite cells exist at a consistent frequency of 2–4 cells/mm of fiber in muscles of the human trunk, limbs, and head. Xenotransplantation into mice of 50–70 fiber-associated, or 1,000–5,000 FACS-enriched CD56+/CD29+ human satellite cells led to stable engraftment and formation of human-derived myofibers. Human cells with characteristic PAX7, CD56, and CD29 expression patterns populated the satellite cell niche beneath the basal lamina on the periphery of regenerated fibers. After additional injury, transplanted satellite cells robustly regenerated to form hundreds of human-derived fibers. Together, these findings conclusively delineate a source of bona-fide endogenous human muscle stem cells that will aid development of clinical applications.

  15. CD56 marks human dendritic cell subsets with cytotoxic potential

    NARCIS (Netherlands)

    Roothans, D.; Smits, E.; Lion, E.; Tel, J.; Anguille, S.

    2013-01-01

    Human plasmacytoid and myeloid dendritic cells (DCs), when appropriately stimulated, can express the archetypal natural killer (NK)-cell surface marker CD56. In addition to classical DC functions, CD56+ DCs are endowed with an unconventional cytotoxic capacity.

  16. Effects of Human Umbilical Cord Mesenchymal Stem Cells on Human Trophoblast Cell Functions In Vitro

    Directory of Open Access Journals (Sweden)

    Yajing Huang

    2016-01-01

    Full Text Available Trophoblast cell dysfunction is involved in many disorders during pregnancy such as preeclampsia and intrauterine growth restriction. Few treatments exist, however, that target improving trophoblast cell function. Human umbilical cord mesenchymal stem cells (hUCMSCs are capable of self-renewing, can undergo multilineage differentiation, and have homing abilities; in addition, they have immunomodulatory effects and paracrine properties and thus are a prospective source for cell therapy. To identify whether hUCMSCs can regulate trophoblast cell functions, we treated trophoblast cells with hUCMSC supernatant or cocultured them with hUCMSCs. Both treatments remarkably enhanced the migration and invasion abilities of trophoblast cells and upregulated their proliferation ability. At a certain concentration, hUCMSCs also modulated hCG, PIGF, and sEndoglin levels in the trophoblast culture medium. Thus, hUCMSCs have a positive effect on trophoblast cellular functions, which may provide a new avenue for treatment of placenta-related diseases during pregnancy.

  17. Inner Harbor Navigation Canal Basin Velocity Analysis

    Science.gov (United States)

    2014-10-01

    ER D C/ CH L TR -1 4- 12 Inner Harbor Navigation Canal Basin Velocity Analysis Co as ta l a nd H yd ra ul ic s La bo ra to ry...library at http://acwc.sdp.sirsi.net/client/default. ERDC/CHL TR-14-12 October 2014 Inner Harbor Navigation Canal Basin Velocity Analysis...system of levees, gates, and drainage structures in the Inner Harbor Navigation Canal (IHNC) basin and the greater New Orleans, Louisiana, area. Two

  18. ETV6 mutations in early immature human T cell leukemias

    Science.gov (United States)

    Van Vlierberghe, Pieter; Ambesi-Impiombato, Alberto; Perez-Garcia, Arianne; Haydu, J. Erika; Rigo, Isaura; Hadler, Michael; Tosello, Valeria; Della Gatta, Giusy; Paietta, Elisabeth; Racevskis, Janis; Wiernik, Peter H.; Luger, Selina M.; Rowe, Jacob M.; Rue, Montserrat

    2011-01-01

    Early immature T cell acute lymphoblastic leukemias (T-ALLs) account for ∼5–10% of pediatric T-ALLs and are associated with poor prognosis. However, the genetic defects that drive the biology of these tumors remain largely unknown. In this study, analysis of microarray gene expression signatures in adult T-ALL demonstrated a high prevalence of early immature leukemias and revealed a close relationship between these tumors and myeloid leukemias. Many adult immature T-ALLs harbored mutations in myeloid-specific oncogenes and tumor suppressors including IDH1, IDH2, DNMT3A, FLT3, and NRAS. Moreover, we identified ETV6 mutations as a novel genetic lesion uniquely present in immature adult T-ALL. Our results demonstrate that early immature adult T-ALL represents a heterogeneous category of leukemias characterized by the presence of overlapping myeloid and T-ALL characteristics, and highlight the potential role of ETV6 mutations in these tumors. PMID:22162831

  19. Stem Cells: A Renaissance in Human Biology Research.

    Science.gov (United States)

    Wu, Jun; Izpisua Belmonte, Juan Carlos

    2016-06-16

    The understanding of human biology and how it relates to that of other species represents an ancient quest. Limited access to human material, particularly during early development, has restricted researchers to only scratching the surface of this inherently challenging subject. Recent technological innovations, such as single cell "omics" and human stem cell derivation, have now greatly accelerated our ability to gain insights into uniquely human biology. The opportunities afforded to delve molecularly into scarce material and to model human embryogenesis and pathophysiological processes are leading to new insights of human development and are changing our understanding of disease and choice of therapy options.

  20. 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.

  1. Molecular aging and rejuvenation of human muscle stem cells

    DEFF Research Database (Denmark)

    Carlson, Morgan E; Suetta, Charlotte; Conboy, Michael J

    2009-01-01

    Very little remains known about the regulation of human organ stem cells (in general, and during the aging process), and most previous data were collected in short-lived rodents. We examined whether stem cell aging in rodents could be extrapolated to genetically and environmentally variable humans....... Our findings establish key evolutionarily conserved mechanisms of human stem cell aging. We find that satellite cells are maintained in aged human skeletal muscle, but fail to activate in response to muscle attrition, due to diminished activation of Notch compounded by elevated transforming growth...... factor beta (TGF-beta)/phospho Smad3 (pSmad3). Furthermore, this work reveals that mitogen-activated protein kinase (MAPK)/phosphate extracellular signal-regulated kinase (pERK) signalling declines in human muscle with age, and is important for activating Notch in human muscle stem cells. This molecular...

  2. Ultrastructure of interstitial cells in subserosa of human colon

    DEFF Research Database (Denmark)

    Rumessen, Jüri Johannes; Vanderwinden, Jean-Marie; Hansen, Alastair;

    2013-01-01

    We studied the ultrastructure of interstitial cells in the subserosal/adventitial layer in human colon. An interstitial cell type with an ultrastructure intermediate between fibroblast-like cells (FLC) and interstitial cells of Cajal was identified (IC-SS). IC-SS had thin and flattened branching...

  3. Differentiation of neuroepithelia from human embryonic stem cells

    OpenAIRE

    2009-01-01

    We describe the method for in vitro differentiation of neuroepithelial cells from human embryonic stem cells under a chemically defined condition. The protocol is established following the fundamental principle of in vivo neuroectodermal specification. The primitive neuroepithelial cells generated by this protocol can be further induced into neuronal and glia cells with forebrain, mid/hind brain, and spinal cord identities.

  4. Identification of a candidate stem cell in human gallbladder.

    Science.gov (United States)

    Manohar, Rohan; Li, Yaming; Fohrer, Helene; Guzik, Lynda; Stolz, Donna Beer; Chandran, Uma R; LaFramboise, William A; Lagasse, Eric

    2015-05-01

    There are currently no reports of identification of stem cells in human gallbladder. The differences between human gallbladder and intrahepatic bile duct (IHBD) cells have also not been explored. The goals of this study were to evaluate if human fetal gallbladder contains a candidate stem cell population and if fetal gallbladder cells are distinct from fetal IHBD cells. We found that EpCAM+CD44+CD13+ cells represent the cell population most enriched for clonal self-renewal from primary gallbladder. Primary EpCAM+CD44+CD13+ cells gave rise to EpCAM+CD44+CD13+ and EpCAM+CD44+CD13- cells in vitro, and gallbladder cells expanded in vitro exhibited short-term engraftment in vivo. Last, we found that CD13, CD227, CD66, CD26 and CD49b were differentially expressed between gallbladder and IHBD cells cultured in vitro indicating clear phenotypic differences between the two cell populations. Microarray analyses of expanded cultures confirmed that both cell types have unique transcriptional profiles with predicted functional differences in lipid, carbohydrate, nucleic acid and drug metabolism. 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.

  5. Comparative mutagenesis of human cells in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Thilly, W.G.

    1992-05-01

    This report discusses measuring methods of point mutations; high density cell cultures for low dose studies; measurement and sequence determination of mutations in DNA; the mutational spectra of styrene oxide and ethlyene oxide in TK-6 cells; mutational spectrum of Cr in human lymphoblast cells; mutational spectra of radon in TK-6 cells; and the mutational spectra of smokeless tobacco. (CBS)

  6. Comparative mutagenesis of human cells in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Thilly, W.G.

    1992-05-01

    This report discusses measuring methods of point mutations; high density cell cultures for low dose studies; measurement and sequence determination of mutations in DNA; the mutational spectra of styrene oxide and ethlyene oxide in TK-6 cells; mutational spectrum of Cr in human lymphoblast cells; mutational spectra of radon in TK-6 cells; and the mutational spectra of smokeless tobacco. (CBS)

  7. Training human mesenchymal stromal cells for bone tissue engineering applications

    NARCIS (Netherlands)

    Doorn, J.

    2012-01-01

    Human mesenchymal stromal cells (hMSCs) are an interesting source for cell therapies and tissue engineering applications, because these cells are able to differentiate into various target tissues, such as bone, cartilage, fat and endothelial cells. In addition, they secrete a wide array of growth fa

  8. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... working with cells derived from one individual or animal species, only to eventually learn that the cells..., morphology, pathologic or disease-state, hybrid or mixed culture, feeder cells, date of origin, etc), the STR... National Institute of Standards and Technology Identification of Human Cell Lines Project AGENCY: National...

  9. The evolution of human cells in terms of protein innovation.

    Science.gov (United States)

    Sardar, Adam J; Oates, Matt E; Fang, Hai; Forrest, Alistair R R; Kawaji, Hideya; Gough, Julian; Rackham, Owen J L

    2014-06-01

    Humans are composed of hundreds of cell types. As the genomic DNA of each somatic cell is identical, cell type is determined by what is expressed and when. Until recently, little has been reported about the determinants of human cell identity, particularly from the joint perspective of gene evolution and expression. Here, we chart the evolutionary past of all documented human cell types via the collective histories of proteins, the principal product of gene expression. FANTOM5 data provide cell-type-specific digital expression of human protein-coding genes and the SUPERFAMILY resource is used to provide protein domain annotation. The evolutionary epoch in which each protein was created is inferred by comparison with domain annotation of all other completely sequenced genomes. Studying the distribution across epochs of genes expressed in each cell type reveals insights into human cellular evolution in terms of protein innovation. For each cell type, its history of protein innovation is charted based on the genes it expresses. Combining the histories of all cell types enables us to create a timeline of cell evolution. This timeline identifies the possibility that our common ancestor Coelomata (cavity-forming animals) provided the innovation required for the innate immune system, whereas cells which now form the brain of human have followed a trajectory of continually accumulating novel proteins since Opisthokonta (boundary of animals and fungi). We conclude that exaptation of existing domain architectures into new contexts is the dominant source of cell-type-specific domain architectures.

  10. Emergence of a colistin-resistant Escherichia coli clinical isolate harboring mcr-1 in Japan.

    Science.gov (United States)

    Tada, Tatsuya; Uechi, Kohei; Nakasone, Isamu; Shimada, Kayo; Nakamatsu, Masashi; Kirikae, Teruo; Fujita, Jiro

    2017-08-02

    The mcr-1 is a gene encoding a phosphoethanolamine transferase, which confers resistance to colistin by transferring phosphoethanolamine to lipid A. We describe here the emergence of a colistin-resistant Escherichia coli clinical isolate harboring plasmid-mediated mcr-1 in Japan. The isolate belonged to ST5702 and is suspected to come from livestock and transmitted to human. This is the first report of a clinical isolate harboring mcr-1 in Japan. Copyright © 2017. Published by Elsevier Ltd.

  11. Adult human brain cell culture for neuroscience research.

    Science.gov (United States)

    Gibbons, Hannah M; Dragunow, Mike

    2010-06-01

    Studies of the brain have progressed enormously through the use of in vivo and in vitro non-human models. However, it is unlikely such studies alone will unravel the complexities of the human brain and so far no neuroprotective treatment developed in animals has worked in humans. In this review we discuss the use of adult human brain cell culture methods in brain research to unravel the biology of the normal and diseased human brain. The advantages of using adult human brain cells as tools to study human brain function from both historical and future perspectives are discussed. In particular, studies using dissociated cultures of adult human microglia, astrocytes, oligodendrocytes and neurons are described and the applications of these types of study are evaluated. Alternative sources of human brain cells such as adult neural stem cells, induced pluripotent stem cells and slice cultures of adult human brain tissue are also reviewed. These adult human brain cell culture methods could benefit basic research and more importantly, facilitate the translation of basic neuroscience research to the clinic for the treatment of brain disorders. Copyright 2009 Elsevier Ltd. All rights reserved.

  12. Apoptosis of human pancreatic cancer cells induced by Triptolide

    Institute of Scientific and Technical Information of China (English)

    Guo-Xiong Zhou; Xiao-Ling Ding; Jie-Fei Huang; Hong Zhang; Sheng-Bao Wu; Jian-Ping Cheng; Qun Wei

    2008-01-01

    AIM:To investigate apoptosis in human pancreatic cancer ceils induced by Triptolide (TL),and the relationship between this apoptosis and expression of caspase-3' bcl-2 and bax.METHODS:Human pancreatic cancer cell line SW1990 was cultured in DIEM media for this study.MTT assay was used to determine the cell growth inhibitory rate in vitro.Flow cytometry and TUNEL assay were used to detect the apoptosis of human pancreatic cancer cells before and after TL treatment.RT-PCR was used to detect the expression of apoptosis-associated gene caspase-3' bcl-2 and bax.RESULTS:TL inhibited the growth of human pancreatic cancer cells in a dose-and time-dependent manner.TL induced human pancreatic cancer cells to undergo apoptosis with typically apoptotic characteristics.TUNEL assay showed that after the treatment of human pancreatic cancer cells with 40 ng/mL TL for 12 h and 24 h,the apoptotic rates of human pancreatic cancer cells increased significantly.RT-PCR demonstrated that caspase-3 and bax were significantly up-regulated in SW1990 cells treated with TL while bcl-2 mRNA was not.CONCLUSION:TL is able to induce the apoptosis in human pancreatic cancer cells.This apoptosis may be mediated by up-regulating the expression of apoptosisassociated caspase-3 and bax gene.

  13. 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.

  14. Activation of GPR119 Stimulates Human β-Cell Replication and Neogenesis in Humanized Mice with Functional Human Islets

    Science.gov (United States)

    Ansarullah; Free, Colette; Christopherson, Jenica; Chen, Quanhai; Gao, Jie; Liu, Chengyang; Naji, Ali; Rabinovitch, Alex; Guo, Zhiguang

    2016-01-01

    Using humanized mice with functional human islets, we investigated whether activating GPR119 by PSN632408, a small molecular agonist, can stimulate human β-cell regeneration in vivo. Human islets were transplanted under the left kidney capsule of immunodeficient mice with streptozotocin- (STZ-) induced diabetes. The recipient mice were treated with PSN632408 or vehicle and BrdU daily. Human islet graft function in the mice was evaluated by nonfasting glucose levels, oral glucose tolerance, and removal of the grafts. Immunostaining for insulin, glucagon, and BrdU or Ki67 was performed in islet grafts to evaluate α- and β-cell replication. Insulin and CK19 immunostaining was performed to evaluate β-cell neogenesis. Four weeks after human islet transplantation, 71% of PSN632408-treated mice achieved normoglycaemia compared with 24% of vehicle-treated mice. Also, oral glucose tolerance was significantly improved in the PSN632408-treated mice. PSN632408 treatment significantly increased both human α- and β-cell areas in islet grafts and stimulated α- and β-cell replication. In addition, β-cell neogenesis was induced from pancreatic duct cells in the islet grafts. Our results demonstrated that activation of GPR119 increases β-cell mass by stimulating human β-cell replication and neogenesis. Therefore, GPR119 activators may qualify as therapeutic agents to increase human β-cell mass in patients with diabetes. PMID:27413754

  15. Analysis of lead toxicity in human cells

    Directory of Open Access Journals (Sweden)

    Gillis Bruce S

    2012-07-01

    Full Text Available Abstract Background Lead is a metal with many recognized adverse health side effects, and yet the molecular processes underlying lead toxicity are still poorly understood. Quantifying the injurious effects of lead is also difficult because of the diagnostic limitations that exist when analyzing human blood and urine specimens for lead toxicity. Results We analyzed the deleterious impact of lead on human cells by measuring its effects on cytokine production and gene expression in peripheral blood mononuclear cells. Lead activates the secretion of the chemokine IL-8 and impacts mitogen-dependent activation by increasing the secretion of the proinflammatory cytokines IL-6 and TNF-α and of the chemokines IL-8 and MIP1-α in the presence of phytohemagglutinin. The recorded changes in gene expression affected major cellular functions, including metallothionein expression, and the expression of cellular metabolic enzymes and protein kinase activity. The expression of 31 genes remained elevated after the removal of lead from the testing medium thereby allowing for the measurement of adverse health effects of lead poisoning. These included thirteen metallothionein transcripts, three endothelial receptor B transcripts and a number of transcripts which encode cellular metabolic enzymes. Cellular responses to lead correlated with blood lead levels and were significantly altered in individuals with higher lead content resultantly affecting the nervous system, the negative regulation of transcription and the induction of apoptosis. In addition, we identified changes in gene expression in individuals with elevated zinc protoporphyrin blood levels and found that genes regulating the transmission of nerve impulses were affected in these individuals. The affected pathways were G-protein mediated signaling, gap junction signaling, synaptic long-term potentiation, neuropathic pain signaling as well as CREB signaling in neurons. Cellular responses to lead were

  16. Entamoeba lysyl-tRNA synthetase contains a cytokine-like domain with chemokine activity towards human endothelial cells.

    Directory of Open Access Journals (Sweden)

    Manuel Castro de Moura

    2011-11-01

    Full Text Available Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II. This Entamoeba EMAPII-like polypeptide (EELP is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity.

  17. Entamoeba lysyl-tRNA synthetase contains a cytokine-like domain with chemokine activity towards human endothelial cells.

    Directory of Open Access Journals (Sweden)

    Manuel Castro de Moura

    2011-11-01

    Full Text Available Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II. This Entamoeba EMAPII-like polypeptide (EELP is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity.

  18. In vitro proliferation of adult human beta-cells.

    Directory of Open Access Journals (Sweden)

    Sabine Rutti

    Full Text Available A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1 analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide.Human islets from 7 adult cadaveric organ donors were dispersed into single cells. Beta-cells were purified by FACS. Non-sorted cells and the beta-cell enriched ("beta-cells" population were plated on extracellular matrix from rat (804G and human bladder carcinoma cells (HTB9 or bovine corneal endothelial ECM (BCEC. Cells were maintained in culture+/-liraglutide for 4 days in the presence of BrdU.Rare human beta-cell proliferation could be observed either in the purified beta-cell population (0.051±0.020%; 22 beta-cells proliferating out of 84'283 beta-cells counted or in the non-sorted cell population (0.055±0.011%; 104 proliferating beta-cells out of 232'826 beta-cells counted, independently of the matrix or the culture conditions. Liraglutide increased human beta-cell proliferation on BCEC in the non-sorted cell population (0.082±0.034% proliferating beta-cells vs. 0.017±0.008% in control, p<0.05.These results indicate that adult human beta-cell proliferation can occur in vitro but remains an extremely rare event with these donors and particular culture conditions. Liraglutide increases beta-cell proliferation only in the non-sorted cell population and only on BCEC. However, it cannot be excluded that human beta-cells may proliferate to a greater extent in situ in response to natural stimuli.

  19. Extracellular protonation modulates cell-cell interaction mechanics and tissue invasion in human melanoma cells

    Science.gov (United States)

    Hofschröer, Verena; Koch, Kevin Alexander; Ludwig, Florian Timo; Friedl, Peter; Oberleithner, Hans; Stock, Christian; Schwab, Albrecht

    2017-01-01

    Detachment of cells from the primary tumour precedes metastatic progression by facilitating cell release into the tissue. Solid tumours exhibit altered pH homeostasis with extracellular acidification. In human melanoma, the Na+/H+ exchanger NHE1 is an important modifier of the tumour nanoenvironment. Here we tested the modulation of cell-cell-adhesion by extracellular pH and NHE1. MV3 tumour spheroids embedded in a collagen matrix unravelled the efficacy of cell-cell contact loosening and 3D emigration into an environment mimicking physiological confinement. Adhesive interaction strength between individual MV3 cells was quantified using atomic force microscopy and validated by multicellular aggregation assays. Extracellular acidification from pHe7.4 to 6.4 decreases cell migration and invasion but increases single cell detachment from the spheroids. Acidification and NHE1 overexpression both reduce cell-cell adhesion strength, indicated by reduced maximum pulling forces and adhesion energies. Multicellular aggregation and spheroid formation are strongly impaired under acidification or NHE1 overexpression. We show a clear dependence of melanoma cell-cell adhesion on pHe and NHE1 as a modulator. These effects are opposite to cell-matrix interactions that are strengthened by protons extruded via NHE1. We conclude that these opposite effects of NHE1 act synergistically during the metastatic cascade. PMID:28205573

  20. Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

    Science.gov (United States)

    Hertsenberg, Andrew J; Funderburgh, James L

    2016-01-01

    Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

  1. Chignik small boat harbor planning aid report

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — Unless additional salmon use data would indicate otherwise, harbor site 3 is considered the environmentally preferred alternative for construction of a small...

  2. Genetics Home Reference: Floating-Harbor syndrome

    Science.gov (United States)

    ... child may have bones more typical of a child of 2. However, bone age is usually normal by age 6 to 12. Delay in speech development (expressive language delay) may be severe in Floating-Harbor syndrome , ...

  3. Derivation of multipotent mesenchymal precursors from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    2005-06-01

    Full Text Available BACKGROUND: Human embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. Thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue-restricted precursors. METHODS AND FINDINGS: Here, we present culture conditions for the derivation of unlimited numbers of pure mesenchymal precursors from human embryonic stem cells and demonstrate multilineage differentiation into fat, cartilage, bone, and skeletal muscle cells. CONCLUSION: Our findings will help to elucidate the mechanism of mesoderm specification during embryonic stem cell differentiation and provide a platform to efficiently generate specialized human mesenchymal cell types for future clinical applications.

  4. Nucleosome Organization in Human Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    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

  5. 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

  6. 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.

  7. Biological characteristics of cell lines of human dental alveolus

    Institute of Scientific and Technical Information of China (English)

    陈世璋; 黄靖香; 孙明学; 赵斌

    2003-01-01

    Objective To investigate the biological characteristics of cell lines of healthy and diseased human dental alveoli. Methods Primary cell lines from either healthy or diseased human dental alveoli were obtained. Two cell lines, H-258 and H-171 derived from healthy and diseased human tissues respectively, were selected for morphological study and research on their growth and aging, using cell counting, and histochemical and immunohistochemical staining. Results Primary cell lines were successfully established from innormal dental alveoli. After freezing and thawing for three times, cell growth was continued and no morphological alterations were observed. The doubling time was 53.4 hours and mean division index (MDI) was 4‰. Cells were kept normal after twenty generations with no obvious reduction of doubling time and MDI. Of twenty-six primary cell lines derived from healthy human dental alveoli, only three cell lines achieved generation. After freezing and thawing for twice, cultured cells were still alive at a decreased growth speed, with doubling time of 85.9 hours and MDI of 3‰. Both cell lines, H-171 and H-258, shared the characteristics of osteoblast. Conclusions Primary cell lines of diseased human dental alveoli show greater growth potential. All cell lines of dental alveoli share characteristics of osteoblast. The technique we developed may be put into practice for the treatment of abnormal dental alveoli.

  8. Cryopreservation of human embryonic stem cells by vitrification

    Institute of Scientific and Technical Information of China (English)

    周灿权; 麦庆云; 李涛; 庄广伦

    2004-01-01

    Background The efficiency of traditional cryopreservation of human embryonic stem (ES) cells is low, and there have been few attempts to prove new cryopreservation methods effective. This study was designed to evaluate the efficiency of cryopreservation of human ES cells using vitrification method.Methods Human ES cells clumped from an identical cell line were randomly allocated to be cryopreserved by vitrification or by slow freezing. The recovery rates, the growth and differentiation potential of thawed human ES cells were compared between these two groups. The pluripotency of human ES cells after thawing was identified.Results Eighty-one point nine percent (59/72) of human ES cell clumps were recovered after vitrification, while only 22.8% (16/70) were recovered after slow freezing (P<0.01). The colonies after vitrification manifested have not only faster growth but also a lower level of differentiation when compared to colonies subjected to the slow freezing protocol. However, the rates of growth and differentiation in undifferentiated colonies from both groups were identical to the rates in those of non-cryopreserved stem cells after a prolonged culture period. Passage 6 of vitrified human ES cells retained the properties of pluripotent cells, a normal karyotype and expressed the transcription factor OCT-4, stage specific expressed antigen-4 (SSEA-4) and SSEA-3. Teratoma growth of these cells demonstrated the ability to develop into all three germ layers.Conclusions Vitrification is effective in cryopreserving human ES cells. During a prolonged culture, human ES cells retain their pluripotency after cryopreservation.

  9. Identification of skin immune cells in non-human primates.

    Science.gov (United States)

    Adam, Lucille; Rosenbaum, Pierre; Cosma, Antonio; Le Grand, Roger; Martinon, Frédéric

    2015-11-01

    The skin is a valuable target for vaccine delivery because it contains many immune cell populations, notably antigen presenting cells. Skin immune cells have been extensively described in mice and humans but not in non-human primates, which are pertinent models for immunological research in vaccination. The aim of this work was to describe immune cell populations in the epidermis, dermis and skin draining lymph nodes in cynomolgus macaques by a single 12-parameter flow cytometry protocol. Given that skin cells share several markers, we defined a gating strategy to identify accurately immune cells and to limit contamination of one immune cell population by another. The epidermis contained CD1a(+)CD1c(-) Langerhans cells (LCs), CD3(+) T cells and putative NK cells. The dermis contained CD1a(+)CD1c(-) cells, which were similar to LCs, CD1a(+)CD1c(+) dermal dendritic cells (DDCs), CD163(high)CD11b(+) resident macrophages, CD3(+) T cells and putative NK cells. The skin also contained CD66(+) polymorphonuclear cells in some animals. Thus, immune cell populations in the macaque are similar to those in humans despite some differences in phenotype. In skin draining lymph nodes, we identified migratory LCs, CD1a(+)CD1c(+) DDCs and macrophages. The simultaneous identification of these different immune cells with one panel of markers avoids the use of large amounts of precious sample and may improve the understanding of immune mechanisms in the skin after treatment or vaccination.

  10. [In vitro strategies for human gametes production from stem cells].

    Science.gov (United States)

    Tosca, Lucie; Courtot, Anne-Marie; Bennaceur-Griscelli, Annelise; Tachdjian, Gérard

    2011-10-01

    Embryonic stem cells (ESC) are self-renewal and pluripotent cells that are able to differentiate in vitro into several cell types in favourable conditions. Technical protocols for in vitro gametes production have been developed in mice and human species. The functionality of such differentiated cells is not always analysed and an early meiotic arrest is a current observation. These kinds of experimentations have also been tested from human induced pluripotent stem cells (IPSC). However, differentiation ends shortly at the primordial germ cell stage.

  11. Growth in agarose of human cells infected with cytomegalovirus.

    Science.gov (United States)

    Lang, D J; Montagnier, L; Latarjet, R

    1974-08-01

    After infection by human cytomegalovirus (CMV), human diploid fibroblasts could grow in agarose medium for several generations. Clones of infected cells grew for weeks, although in every case they ultimately underwent lysis owing to the cytopathic effect of the virus. Virus was inoculated at high dilution and after UV irradiation in an effort to derive cells infected with noninfectious defective particles still capable of inducing cell stimulation. Dilute or irradiated virus occasionally yielded large colonies of replicating cells, although permanent transformation was not observed. One clone derived from UV-CMV-infected cells was passaged four times before undergoing lysis. During these passages the cells exhibited alterations in morphology and orientation.

  12. Cytotoxinic Mechanism of Hydroxyapatite Nanoparticles on Human Hepatoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    CAO Xian-ying; QI Zhi-tao; DAI Hong-lian; YAN Yu-hua; LI Shi-pu

    2003-01-01

    Stable and single-dispersed HAP nanoparticles were synthesized with chemical method assisted by ultrasonic treatment.HAP nanoparticles were surveyed by AFM and Zataplus.The effect on the Bel-7402 human hepatoma cell lines treated with HAP nanoparticles was investigated by the MTT methods and observation of morphology,and the mechanism was studied in changes of cell cycle and ultrastructure.The result shows that inhibition of HAP nanoparticles on the Bel-7402 human hepatoma cell lines is obviously in vitro.HAP nanoparticles the entered cancer cytoplasm,and cell proliferation is stopped at G1 phase of cell cycle,thus,cancer cells die directly.

  13. Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells.

    Science.gov (United States)

    Vieira Torquato, Heron F; Ribeiro-Filho, Antonio C; Buri, Marcus V; Araújo Júnior, Roberto T; Pimenta, Renata; de Oliveira, José Salvador R; Filho, Valdir C; Macho, Antonio; Paredes-Gamero, Edgar J; de Oliveira Martins, Domingos T

    2017-04-01

    Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6-one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin(+)) and leukemia stem cell population (CD34(+)CD38(-)Lin(-/low)). Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G0/G1 (7μM) and G2 (45μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Detection of EBV, HBV, HCV, HIV-1, HTLV-I and -II, and SMRV in human and other primate cell lines.

    Science.gov (United States)

    Uphoff, Cord C; Denkmann, Sabine A; Steube, Klaus G; Drexler, Hans G

    2010-01-01

    The high prevalence of contaminated cell cultures suggests that viral contaminations might be distributed among cultures. We investigated more than 460 primate cell lines for Epstein-Barr (EBV), hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia/lymphoma virus I and II (HTLV-I/-II), and squirrel monkey retrovirus (SMRV) infections for risk assessment. None of the cell lines were infected with HCV, HIV-1, or HTLV-I/-II. However, one cell line displayed reverse transcriptase activity. Thirty-nine cell lines harbored EBV DNA sequences. Studies on the lytic phase of EBV revealed that five cell lines produce EBV particles and six further cell lines produced EBV upon stimulation. One cell line contained an integrated HBV genome fragment but showed no virus production. Six cell lines were SMRV-infected. Newly established cell lines should be tested for EBV infections to detect B-lymphoblastoid cell lines (B-LCL). B-LCLs established with EBV from cell line B95-8 should be tested for SMRV infections.

  15. The effect of aging on human induced pluripotent stem cells

    Science.gov (United States)

    Sardo, Valentina Lo; Ferguson, William; Erikson, Galina A.; Topol, Eric J; Baldwin, Kristin K; Torkamani, Ali

    2017-01-01

    Induced pluripotent stem cells (iPSCs) are being developed as a source for autologous cell therapies, many of which aim to treat aged patients1–5. To explore the impact of age on iPSC quality, we produced iPSCs from blood cells of 16 donors aged 21–100. We find that while reprogramming resets most of the epigenome, iPSCs retain an epigenetic signature of age that diminishes with passaging. Reprogramming via clonal expansion also exposes somatic mutations present in individual donor cells, which are missed by other methods. We find that exomic mutations in iPSCs increase linearly with age and each iPSC line analyzed carries at least one gene-disrupting mutation, of which several have previously been linked to cancer or dysfunction. Unexpectedly, elderly donors (>90 yrs) harbor fewer mutations than predicted and their distribution suggests that blood in elderly donors derives from a contracted progenitor pool. These studies show that harnessing clonal expansion during reprogramming can uncover age-associated processes relevant to the clinical use of iPSCs. PMID:27941802

  16. Human organomics: a fresh approach to understanding human development using single-cell transcriptomics.

    Science.gov (United States)

    Camp, J Gray; Treutlein, Barbara

    2017-05-01

    Innovative methods designed to recapitulate human organogenesis from pluripotent stem cells provide a means to explore human developmental biology. New technologies to sequence and analyze single-cell transcriptomes can deconstruct these 'organoids' into constituent parts, and reconstruct lineage trajectories during cell differentiation. In this Spotlight article we summarize the different approaches to performing single-cell transcriptomics on organoids, and discuss the opportunities and challenges of applying these techniques to generate organ-level, mechanistic models of human development and disease. Together, these technologies will move past characterization to the prediction of human developmental and disease-related phenomena. © 2017. Published by The Company of Biologists Ltd.

  17. Human dental pulp stem cells: Applications in future regenerative medicine.

    Science.gov (United States)

    Potdar, Pravin D; Jethmalani, Yogita D

    2015-06-26

    Stem cells are pluripotent cells, having a property of differentiating into various types of cells of human body. Several studies have developed mesenchymal stem cells (MSCs) from various human tissues, peripheral blood and body fluids. These cells are then characterized by cellular and molecular markers to understand their specific phenotypes. Dental pulp stem cells (DPSCs) are having a MSCs phenotype and they are differentiated into neuron, cardiomyocytes, chondrocytes, osteoblasts, liver cells and β cells of islet of pancreas. Thus, DPSCs have shown great potentiality to use in regenerative medicine for treatment of various human diseases including dental related problems. These cells can also be developed into induced pluripotent stem cells by incorporation of pluripotency markers and use for regenerative therapies of various diseases. The DPSCs are derived from various dental tissues such as human exfoliated deciduous teeth, apical papilla, periodontal ligament and dental follicle tissue. This review will overview the information about isolation, cellular and molecular characterization and differentiation of DPSCs into various types of human cells and thus these cells have important applications in regenerative therapies for various diseases. This review will be most useful for postgraduate dental students as well as scientists working in the field of oral pathology and oral medicine.

  18. 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.

  19. Identification of human fibroblast cell lines as a feeder layer for human corneal epithelial regeneration.

    Directory of Open Access Journals (Sweden)

    Rong Lu

    Full Text Available There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE and cell growth capacity were evaluated on days 5-14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1 × 10(4 in a 35-mm dish (9.6 cm(2 grew to confluence (about 1.87-2.41 × 10(6 cells in 12-14 days, representing 187-241 fold expansion with over 7-8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction.

  20. A neurological comparative study of the harp seal (Pagophilus groenlandicus) and harbor porpoise (Phocoena phocoena) brain

    DEFF Research Database (Denmark)

    Walløe, Solveig; Eriksen, Nina; Dabelsteen, Torben

    2010-01-01

    The cetacean brain is well studied. However, few comparisons have been done with other marine mammals. In this study, we compared the harp seal (Pagophilus groenlandicus) and the harbor porpoise brain (Phocoena phocoena). Stereological methods were applied to compare three areas of interest...... are the first to provide estimates of the number of neurons and glial cells in the neocortex of the harp seal and harbor porpoise brain and offer new data to the comparative field of mammalian brain evolution....

  1. 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).

  2. Purification and cultivation of human pituitary growth hormone secreting cells

    Science.gov (United States)

    Hymer, W. C.

    1978-01-01

    The maintainance of actively secreting human pituitary growth hormone cells (somatotrophs) in vitro was studied. The primary approach was the testing of agents which may be expected to increase the release of the human growth hormone (hGH). A procedure for tissue procurement is described along with the methodologies used to dissociate human pituitary tissue (obtained either at autopsy or surgery) into single cell suspensions. The validity of the Biogel cell column perfusion system for studying the dynamics of GH release was developed and documented using a rat pituitary cell system.

  3. Persistent donor cell gene expression among human induced pluripotent stem cells contributes to differences with human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Zhumur Ghosh

    Full Text Available Human induced pluripotent stem cells (hiPSCs generated by de-differentiation of adult somatic cells offer potential solutions for the ethical issues surrounding human embryonic stem cells (hESCs, as well as their immunologic rejection after cellular transplantation. However, although hiPSCs have been described as "embryonic stem cell-like", these cells have a distinct gene expression pattern compared to hESCs, making incomplete reprogramming a potential pitfall. It is unclear to what degree the difference in tissue of origin may contribute to these gene expression differences. To answer these important questions, a careful transcriptional profiling analysis is necessary to investigate the exact reprogramming state of hiPSCs, as well as analysis of the impression, if any, of the tissue of origin on the resulting hiPSCs. In this study, we compare the gene profiles of hiPSCs derived from fetal fibroblasts, neonatal fibroblasts, adipose stem cells, and keratinocytes to their corresponding donor cells and hESCs. Our analysis elucidates the overall degree of reprogramming within each hiPSC line, as well as the "distance" between each hiPSC line and its donor cell. We further identify genes that have a similar mode of regulation in hiPSCs and their corresponding donor cells compared to hESCs, allowing us to specify core sets of donor genes that continue to be expressed in each hiPSC line. We report that residual gene expression of the donor cell type contributes significantly to the differences among hiPSCs and hESCs, and adds to the incompleteness in reprogramming. Specifically, our analysis reveals that fetal fibroblast-derived hiPSCs are closer to hESCs, followed by adipose, neonatal fibroblast, and keratinocyte-derived hiPSCs.

  4. On the development of extragonadal and gonadal human germ cells

    Directory of Open Access Journals (Sweden)

    A. Marijne Heeren

    2016-02-01

    Full Text Available Human germ cells originate in an extragonadal location and have to migrate to colonize the gonadal primordia at around seven weeks of gestation (W7, or five weeks post conception. Many germ cells are lost along the way and should enter apoptosis, but some escape and can give rise to extragonadal germ cell tumors. Due to the common somatic origin of gonads and adrenal cortex, we investigated whether ectopic germ cells were present in the human adrenals. Germ cells expressing DDX4 and/or POU5F1 were present in male and female human adrenals in the first and second trimester. However, in contrast to what has been described in mice, where ‘adrenal’ and ‘ovarian’ germ cells seem to enter meiosis in synchrony, we were unable to observe meiotic entry in human ‘adrenal’ germ cells until W22. By contrast, ‘ovarian’ germ cells at W22 showed a pronounced asynchronous meiotic entry. Interestingly, we observed that immature POU5F1+ germ cells in both first and second trimester ovaries still expressed the neural crest marker TUBB3, reminiscent of their migratory phase. Our findings highlight species-specific differences in early gametogenesis between mice and humans. We report the presence of a population of ectopic germ cells in the human adrenals during development.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. 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 ...

  7. 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.

  8. 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.

  9. Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

    OpenAIRE

    2009-01-01

    Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS ...

  10. Modelling familial dysautonomia in human induced pluripotent stem cells

    OpenAIRE

    Lee, Gabsang; Studer, Lorenz

    2011-01-01

    Induced pluripotent stem (iPS) cells have considerable promise as a novel tool for modelling human disease and for drug discovery. While the generation of disease-specific iPS cells has become routine, realizing the potential of iPS cells in disease modelling poses challenges at multiple fronts. Such challenges include selecting a suitable disease target, directing the fate of iPS cells into symptom-relevant cell populations, identifying disease-related phenotypes and showing reversibility of...

  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. Generation of human-induced pluripotent stem cells.

    Science.gov (United States)

    Park, In-Hyun; Lerou, Paul H; Zhao, Rui; Huo, Hongguang; Daley, George Q

    2008-01-01

    Pluripotent cells, such as embryonic stem cells, are invaluable tools for research and can potentially serve as a source of cell- and tissue-replacement therapy. Rejection after transplantation of cells and tissue derived from embryonic stem cells is a significant obstacle to their clinical use. Recently, human somatic cells have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Human iPS cells are a potential source of patient-specific pluripotent stem cells that would bypass immune rejection. iPS cells can also be used to study diseases for which there are no adequate human in vitro or animal models. In this protocol, we describe how to establish primary human fibroblasts lines and how to derive iPS cells by retroviral transduction of reprogramming factors. Overall, it takes 2 months to complete reprogramming human primary fibroblasts starting from biopsy.

  13. Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

    Science.gov (United States)

    Goodwin, Thomas J.

    2010-01-01

    Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

  14. The Isolation and Characterization of Human Prostate Cancer Stem Cells

    Science.gov (United States)

    2015-05-01

    IGF1, SOX15, BMPR1B, TGFBR1, etc), which fall into distinct GO categories including SC, development, stress response, and wound healing (unpublished...prostate cancer through the elucidation of the role of cancer stem cells in the pathogenesis of the disease. During the past year, we have made the...studies, ii) in vitro co-culture of human prostate cancer cells (established cell lines and primary patient samples) with human prostate fibroblasts

  15. Isolation, identification and differentiation of human embryonic cartilage stem cells.

    Science.gov (United States)

    Fu, Changhao; Yan, Zi; Xu, Hao; Zhang, Chen; Zhang, Qi; Wei, Anhui; Yang, Xi; Wang, Yi

    2015-07-01

    We isolated human embryonic cartilage stem cells (hECSCs), a novel stem cell population, from the articular cartilage of eight-week-old human embryos. These stem cells demonstrated a marker expression pattern and differentiation potential intermediate to those of human embryonic stem cells (hESCs) and human adult stem cells (hASCs). hECSCs expressed markers associated with both hESCs (OCT4, NANOG, SOX2, SSEA-3 and SSEA-4) and human adult stem cells (hASCs) (CD29, CD44, CD90, CD73 and CD10). These cells also differentiated into adipocytes, osteoblasts, chondrocytes, neurons and islet-like cells under specific inducing conditions. We identified N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) as an inducer of chondrogenic differentiation in hECSCs. Similar results using N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) were obtained for two other types of human embryonic tissue-derived stem cells, human embryonic hepatic stem cells (hEHSCs) and human embryonic amniotic fluid stem cells (hEASCs), both of which exhibited a marker expression pattern similar to that of hECSCs. The isolation of hECSCs and the discovery that N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) induces chondrogenic differentiation in different stem cell populations might aid the development of strategies in tissue engineering and cartilage repair.

  16. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

    Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

  17. Vasoprotective effects of human CD34+ cells: towards clinical applications

    Directory of Open Access Journals (Sweden)

    Lerman Amir

    2009-07-01

    Full Text Available Abstract Background The development of cell-based therapeutics for humans requires preclinical testing in animal models. The use of autologous animal products fails to address the efficacy of similar products derived from humans. We used a novel immunodeficient rat carotid injury model in order to determine whether human cells could improve vascular remodelling following acute injury. Methods Human CD34+ cells were separated from peripheral buffy coats using automatic magnetic cell separation. Carotid arterial injury was performed in male Sprague-Dawley nude rats using a 2F Fogarty balloon catheter. Freshly harvested CD34+ cells or saline alone was administered locally for 20 minutes by endoluminal instillation. Structural and functional analysis of the arteries was performed 28 days later. Results Morphometric analysis demonstrated that human CD34+ cell delivery was associated with a significant reduction in intimal formation 4 weeks following balloon injury as compared with saline (I/M ratio 0.79 ± 0.18, and 1.71 ± 0.18 for CD34, and saline-treated vessels, respectively P Conclusion Delivery of human CD34+ cells limits neointima formation and improves arterial reactivity after vascular injury. These studies advance the concept of cell delivery to effect vascular remodeling toward a potential human cellular product.

  18. 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.

  19. 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.

  20. Isolation and characterization of a novel arenavirus harbored by Rodents and Shrews in Zhejiang province, China.

    Science.gov (United States)

    Li, Kun; Lin, Xian-Dan; Wang, Wen; Shi, Mang; Guo, Wen-Ping; Zhang, Xiao-He; Xing, Jian-Guang; He, Jin-Rong; Wang, Ke; Li, Ming-Hui; Cao, Jian-Hai; Jiang, Mu-Liu; Holmes, Edward C; Zhang, Yong-Zhen

    2015-02-01

    To determine the biodiversity of arenaviruses in China, we captured and screened rodents and shrews in Wenzhou city, Zhejiang province, a locality where hemorrhagic fever diseases are endemic in humans. Accordingly, arenaviruses were detected in 42 of 351 rodents from eight species, and in 12 of 272 Asian house shrews (Suncus murinus), by RT-PCR targeting the L segment. From these, a single arenavirus was successfully isolated in cell culture. The virion particles exhibited a typical arenavirus morphology under transmission electron microscopy. Comparison of the S and L segment sequences revealed high levels of nucleotide (>32.2% and >39.6%) and amino acid (>28.8% and >43.8%) sequence differences from known arenaviruses, suggesting that it represents a novel arenavirus, which we designated Wenzhou virus (WENV). Phylogenetic analysis revealed that all WENV strains harbored by both rodents and Asian house shrews formed a distinct lineage most closely related to Old World arenaviruses.

  1. Human embryonic stem cells differentiate into functional renal proximal tubular-like cells.

    Science.gov (United States)

    Narayanan, Karthikeyan; Schumacher, Karl M; Tasnim, Farah; Kandasamy, Karthikeyan; Schumacher, Annegret; Ni, Ming; Gao, Shujun; Gopalan, Began; Zink, Daniele; Ying, Jackie Y

    2013-04-01

    Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular-like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells.

  2. Growth suppressive efficacy of human lak cells against human lung-cancer implanted into scid mice.

    Science.gov (United States)

    Teraoka, S; Kyoizumi, S; Suzuki, T; Yamakido, M; Akiyama, M

    1995-06-01

    The purpose of our study was to determine the efficacy of immunotherapy using human lymphokine activated killer (LAK) cells against a human-lung squamous-cell carcinoma cell line (RERF-LC-AI) implanted into severe combined immunodeficient (SCID) mice. A statistically significant growth suppressive effect on RERF-LC-AI implanted into SCID mice was observed when human LAK cells were administered into the caudal vein of the mice treated with a continuous supply (initiated prior to LAK cells injection) of rIL-2. The human LAK cells stained with PKH 2, a fluorescent dye, for later detection using flow cytometry were administered into the caudal vein of RERF-LC-AI bearing SCID mice; the cells persisted for 7 days in the implanted lung cancer tissue and in the mouse peripheral blood, but for 5 days in the mouse spleen. The number of infiltrated human LAK cells in each tissue increased dose-dependently with the number of injected cells. The results indicate that the antitumor effect most likely occurred during the early implantation period of the human LAK cells. These results demonstrate the applicability of this model to the in vivo study of human lung cancer therapy.

  3. Deep sequencing reveals low incidence of endogenous LINE-1 retrotransposition in human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Hubert Arokium

    Full Text Available Long interspersed element-1 (LINE-1 or L1 retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs. However, by using an engineered reporter construct over-expressing L1, another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications, it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here, we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs. Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition, we used a novel sequencing strategy. As opposed to conventional sequencing direction, we sequenced from the 3' end of L1Hs to the genomic DNA, thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells, presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore, these insertions could not be detected in iPSCs by PCR, likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects.

  4. Deep sequencing reveals low incidence of endogenous LINE-1 retrotransposition in human induced pluripotent stem cells.

    Science.gov (United States)

    Arokium, Hubert; Kamata, Masakazu; Kim, Sanggu; Kim, Namshin; Liang, Min; Presson, Angela P; Chen, Irvin S

    2014-01-01

    Long interspersed element-1 (LINE-1 or L1) retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs). However, by using an engineered reporter construct over-expressing L1, another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications, it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here, we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs). Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition, we used a novel sequencing strategy. As opposed to conventional sequencing direction, we sequenced from the 3' end of L1Hs to the genomic DNA, thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells, presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore, these insertions could not be detected in iPSCs by PCR, likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects.

  5. Use of human pluripotent stem cells to study and treatretinopathies

    Institute of Scientific and Technical Information of China (English)

    Karim Ben M’Barek; Florian Regent; Christelle Monville

    2015-01-01

    Human cell types affected by retinal diseases (such asage-related macular degeneration or retinitis pimentosa)are limited in cell number and of reduced accessibility. As aconsequence, their isolation for in vitro studies of diseasemechanisms or for drug screening efforts is fastidious.Human pluripotent stem cells (hPSCs), either of embryonicorigin or through reprogramming of adult somatic cells,represent a new promising way to generate models ofhuman retinopathies, explore the physiopathologicalmechanisms and develop novel therapeutic strategies.Disease-specific human embryonic stem cells were thefirst source of material to be used to study certain diseasestates. The recent demonstration that human somaticcells, such as fibroblasts or blood cells, can be geneticallyconverted to induce pluripotent stem cells together withthe continuous improvement of methods to differentiatethese cells into disease-affected cellular subtypes opensnew perspectives to model and understand a largenumber of human pathologies, including retinopathies.This review focuses on the added value of hPSCs for thedisease modeling of human retinopathies and the study oftheir molecular pathological mechanisms. We also discussthe recent use of these cells for establishing the validationstudies for therapeutic intervention and for the screeningof large compound libraries to identify candidate drugs.

  6. Proteomic Analysis of Human Blastocoel Fluid and Blastocyst Cells

    DEFF Research Database (Denmark)

    Linnert Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen;

    the cells of the blastocyst are exposed. The ICM is the starting point for the development of undifferentiated human embryonic stem cells (hESCs), which posses the potential to develop into any cell type present in the adult human body [1,2]. This ability makes hESCs a potential source of cells...... for regenerative medicine, such as in the treatment of diabetes, Parkinson’s disease, blindness, and spinal cord injury. In the context of developing regenerative medicine based on hESCs, it remains a challenge to employ safe, xenofree and defined culture conditions. The blastocoel fluid is per se the in vivo......The human blastocyst consists of 100-200 cells that are organized in an outer layer of differentiated trophectoderm (TE) cells lining the blastocyst cavity into which the undifferentiated inner cell mass (ICM) protrudes. The cavity of the blastocyst is filled with blastocoel fluid to which all...

  7. Proteomic Analysis of Human Blastocoel Fluid and Blastocyst Cells

    DEFF Research Database (Denmark)

    Linnert Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen

    the cells of the blastocyst are exposed. The ICM is the starting point for the development of undifferentiated human embryonic stem cells (hESCs), which posses the potential to develop into any cell type present in the adult human body [1,2]. This ability makes hESCs a potential source of cells......The human blastocyst consists of 100-200 cells that are organized in an outer layer of differentiated trophectoderm (TE) cells lining the blastocyst cavity into which the undifferentiated inner cell mass (ICM) protrudes. The cavity of the blastocyst is filled with blastocoel fluid to which all...... for regenerative medicine, such as in the treatment of diabetes, Parkinson’s disease, blindness, and spinal cord injury. In the context of developing regenerative medicine based on hESCs, it remains a challenge to employ safe, xenofree and defined culture conditions. The blastocoel fluid is per se the in vivo...

  8. Human induced pluripotent stem cells: A disruptive innovation.

    Science.gov (United States)

    De Vos, J; Bouckenheimer, J; Sansac, C; Lemaître, J-M; Assou, S

    2016-01-01

    This year (2016) will mark the 10th anniversary of the discovery of induced pluripotent stem cells (iPSCs). The finding that the transient expression of four transcription factors can radically remodel the epigenome, transcriptome and metabolome of differentiated cells and reprogram them into pluripotent stem cells has been a major and groundbreaking technological innovation. In this review, we discuss the major applications of this technology that we have grouped in nine categories: a model to study cell fate control; a model to study pluripotency; a model to study human development; a model to study human tissue and organ physiology; a model to study genetic diseases in a dish; a tool for cell rejuvenation; a source of cells for drug screening; a source of cells for regenerative medicine; a tool for the production of human organs in animals.

  9. Genome editing: a robust technology for human stem cells.

    Science.gov (United States)

    Chandrasekaran, Arun Pandian; Song, Minjung; Ramakrishna, Suresh

    2017-09-01

    Human pluripotent stem cells comprise induced pluripotent and embryonic stem cells, which have tremendous potential for biological and therapeutic applications. The development of efficient technologies for the targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. Genome editing of stem cells is possible with the help of synthetic nucleases that facilitate site-specific modification of a gene of interest. Recent advances in genome editing techniques have improved the efficiency and speed of the development of stem cells for human disease models. Zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system are powerful tools for editing DNA at specific loci. Here, we discuss recent technological advances in genome editing with site-specific nucleases in human stem cells.

  10. Phenotype and functions of memory Tfh cells in human blood.

    Science.gov (United States)

    Schmitt, Nathalie; Bentebibel, Salah-Eddine; Ueno, Hideki

    2014-09-01

    Our understanding of the origin and functions of human blood CXCR5(+) CD4(+) T cells found in human blood has changed dramatically in the past years. These cells are currently considered to represent a circulating memory compartment of T follicular helper (Tfh) lineage cells. Recent studies have shown that blood memory Tfh cells are composed of phenotypically and functionally distinct subsets. Here, we review the current understanding of human blood memory Tfh cells and the subsets within this compartment. We present a strategy to define these subsets based on cell surface profiles. Finally, we discuss how increased understanding of the biology of blood memory Tfh cells may contribute insight into the pathogenesis of autoimmune diseases and the mode of action of vaccines.

  11. Human embryonic stem cell lines derived from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhen Fu FANG; Fan JIN; Hui GAI; Ying CHEN; Li WU; Ai Lian LIU; Bin CHEN; Hui Zhen SHENG

    2005-01-01

    Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF,Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

  12. 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.

  13. Mimulone-induced autophagy through p53-mediated AMPK/mTOR pathway increases caspase-mediated apoptotic cell death in A549 human lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Hyun-Kyu An

    Full Text Available Anticancer properties and mechanisms of mimulone (MML, C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3 puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA, pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy.

  14. Mimulone-Induced Autophagy through p53-Mediated AMPK/mTOR Pathway Increases Caspase-Mediated Apoptotic Cell Death in A549 Human Lung Cancer Cells

    Science.gov (United States)

    Lee, Ji-Won; Park, Mi-Hyun; Moon, Hyung-In; Park, Shin-Ji; Baik, Ji-Sue; Kim, Cheorl-Ho; Lee, Young-Choon

    2014-01-01

    Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy. PMID:25490748

  15. Radiation response of mesenchymal stem cells derived from bone marrow and human pluripotent stem cells

    OpenAIRE

    Islam, Mohammad S; Stemig, Melissa E.; Takahashi, Yutaka; Hui, Susanta K.

    2014-01-01

    Mesenchymal stem cells (MSCs) isolated from human pluripotent stem cells are comparable with bone marrow-derived MSCs in their function and immunophenotype. The purpose of this exploratory study was comparative evaluation of the radiation responses of mesenchymal stem cells derived from bone marrow- (BMMSCs) and from human embryonic stem cells (hESMSCs). BMMSCs and hESMSCs were irradiated at 0 Gy (control) to 16 Gy using a linear accelerator commonly used for cancer treatment. Cells were harv...

  16. The effects of TSH on human vascular endothelial cells and smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    田利民

    2014-01-01

    Objective To study the effect of thyroid-stimulating hormone(TSH)on human vascular endothelial cells and smooth muscle cells and to explore the roles of TSH in the development of atherosclerosis.Methods Human vascular endothelial cells and smooth muscle cells were cultured in vitro.MTT method was used to assay the effect of TSH on cell viability.Real-time PCR was used

  17. Applied Developmental Biology: Making Human Pancreatic Beta Cells for Diabetics.

    Science.gov (United States)

    Melton, Douglas A

    2016-01-01

    Understanding the genes and signaling pathways that determine the differentiation and fate of a cell is a central goal of developmental biology. Using that information to gain mastery over the fates of cells presents new approaches to cell transplantation and drug discovery for human diseases including diabetes. © 2016 Elsevier Inc. All rights reserved.

  18. Establishment of human embryonic stem cell line from gamete donors

    Institute of Scientific and Technical Information of China (English)

    LI Tao; ZHOU Can-quan; MAI Qing-yun; ZHUANG Guang-lun

    2005-01-01

    Background Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. Methods Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed.Results Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types.Conclusions HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

  19. Generation of dendritic cells and macrophages from human induced pluripotent stem cells aiming at cell therapy.

    Science.gov (United States)

    Senju, S; Haruta, M; Matsumura, K; Matsunaga, Y; Fukushima, S; Ikeda, T; Takamatsu, K; Irie, A; Nishimura, Y

    2011-09-01

    This report describes generation of dendritic cells (DCs) and macrophages from human induced pluripotent stem (iPS) cells. iPS cell-derived DC (iPS-DC) exhibited the morphology of typical DC and function of T-cell stimulation and antigen presentation. iPS-DC loaded with cytomegalovirus (CMV) peptide induced vigorous expansion of CMV-specific autologous CD8+ T cells. Macrophages (iPS-MP) with activity of zymosan phagocytosis and C5a-induced chemotaxis were also generated from iPS cells. Genetically modified iPS-MPs were generated by the introduction of expression vectors into undifferentiated iPS cells, isolation of transfectant iPS cell clone and subsequent differentiation. By this procedure, we generated iPS-MP expressing a membrane-bound form of single chain antibody (scFv) specific to amyloid β (Aβ), the causal protein of Alzheimer's disease. The scFv-transfectant iPS-MP exhibited efficient Aβ-specific phagocytosis activity. iPS-MP expressing CD20-specific scFv engulfed and killed BALL-1 B-cell leukemia cells. Anti-BALL-1 effect of iPS-MP in vivo was demonstrated in a xeno-transplantation model using severe combined immunodeficient mice. In addition, we established a xeno-free culture protocol to generate iPS-DC and iPS-MP. Collectively, we demonstrated the possibility of application of iPS-DC and macrophages to cell therapy.

  20. Stepwise development of MAIT cells in mouse and human.

    Directory of Open Access Journals (Sweden)

    Emmanuel Martin

    2009-03-01

    Full Text Available Mucosal-associated invariant T (MAIT cells display two evolutionarily conserved features: an invariant T cell receptor (TCRalpha (iTCRalpha chain and restriction by the nonpolymorphic class Ib major histocompatibility complex (MHC molecule, MHC-related molecule 1 (MR1. MR1 expression on thymus epithelial cells is not necessary for MAIT cell development but their accumulation in the gut requires MR1 expressing B cells and commensal flora. MAIT cell development is poorly known, as these cells have not been found in the thymus so far. Herein, complementary human and mouse experiments using an anti-humanValpha7.2 antibody and MAIT cell-specific iTCRalpha and TCRbeta transgenic mice in different genetic backgrounds show that MAIT cell development is a stepwise process, with an intra-thymic selection followed by peripheral expansion. Mouse MAIT cells are selected in an MR1-dependent manner both in fetal thymic organ culture and in double iTCRalpha and TCRbeta transgenic RAG knockout mice. In the latter mice, MAIT cells do not expand in the periphery unless B cells are added back by adoptive transfer, showing that B cells are not required for the initial thymic selection step but for the peripheral accumulation. In humans, contrary to natural killer T (NKT cells, MAIT cells display a naïve phenotype in the thymus as well as in cord blood where they are in low numbers. After birth, MAIT cells acquire a memory phenotype and expand dramatically, up to 1%-4% of blood T cells. Finally, in contrast with NKT cells, human MAIT cell development is independent of the molecular adaptor SAP. Interestingly, mouse MAIT cells display a naïve phenotype and do not express the ZBTB16 transcription factor, which, in contrast, is expressed by NKT cells and the memory human MAIT cells found in the periphery after birth. In conclusion, MAIT cells are selected by MR1 in the thymus on a non-B non-T hematopoietic cell, and acquire a memory phenotype and expand in the

  1. Tumor-initiating label-retaining cancer cells in human gastrointestinal cancers undergo asymmetric cell division.

    Science.gov (United States)

    Xin, Hong-Wu; Hari, Danielle M; Mullinax, John E; Ambe, Chenwi M; Koizumi, Tomotake; Ray, Satyajit; Anderson, Andrew J; Wiegand, Gordon W; Garfield, Susan H; Thorgeirsson, Snorri S; Avital, Itzhak

    2012-04-01

    Label-retaining cells (LRCs) have been proposed to represent adult tissue stem cells. LRCs are hypothesized to result from either slow cycling or asymmetric cell division (ACD). However, the stem cell nature and whether LRC undergo ACD remain controversial. Here, we demonstrate label-retaining cancer cells (LRCCs) in several gastrointestinal (GI) cancers including fresh surgical specimens. Using a novel method for isolation of live LRCC, we demonstrate that a subpopulation of LRCC is actively dividing and exhibits stem cells and pluripotency gene expression profiles. Using real-time confocal microscopic cinematography, we show live LRCC undergoing asymmetric nonrandom chromosomal cosegregation LRC division. Importantly, LRCCs have greater tumor-initiating capacity than non-LRCCs. Based on our data and that cancers develop in tissues that harbor normal-LRC, we propose that LRCC might represent a novel population of GI stem-like cancer cells. LRCC may provide novel mechanistic insights into the biology of cancer and regenerative medicine and present novel targets for cancer treatment. Copyright © 2012 AlphaMed Press.

  2. Human stem cells and articular cartilage regeneration.

    Science.gov (United States)

    Inui, Atsuyuki; Iwakura, Takashi; Reddi, A Hari

    2012-11-05

    The regeneration of articular cartilage damaged due to trauma and posttraumatic osteoarthritis is an unmet medical need. Current approaches to regeneration and tissue engineering of articular cartilage include the use of chondrocytes, stem cells, scaffolds and signals, including morphogens and growth factors. Stem cells, as a source of cells for articular cartilage regeneration, are a critical factor for articular cartilage regeneration. This is because articular cartilage tissue has a low cell turnover and does not heal spontaneously. Adult stem cells have been isolated from various tissues, such as bone marrow, adipose, synovial tissue, muscle and periosteum. Signals of the transforming growth factor beta superfamily play critical roles in chondrogenesis. However, adult stem cells derived from various tissues tend to differ in their chondrogenic potential. Pluripotent stem cells have unlimited proliferative capacity compared to adult stem cells. Chondrogenesis from embryonic stem (ES) cells has been studied for more than a decade. However, establishment of ES cells requires embryos and leads to ethical issues for clinical applications. Induced pluripotent stem (iPS) cells are generated by cellular reprogramming of adult cells by transcription factors. Although iPS cells have chondrogenic potential, optimization, generation and differentiation toward articular chondrocytes are currently under intense investigation.

  3. Human Stem Cells and Articular Cartilage Regeneration

    Directory of Open Access Journals (Sweden)

    A. Hari Reddi

    2012-11-01

    Full Text Available  The regeneration of articular cartilage damaged due to trauma and posttraumatic osteoarthritis is an unmet medical need. Current approaches to regeneration and tissue engineering of articular cartilage include the use of chondrocytes, stem cells, scaffolds and signals, including morphogens and growth factors. Stem cells, as a source of cells for articular cartilage regeneration, are a critical factor for articular cartilage regeneration. This is because articular cartilage tissue has a low cell turnover and does not heal spontaneously. Adult stem cells have been isolated from various tissues, such as bone marrow, adipose, synovial tissue, muscle and periosteum. Signals of the transforming growth factor beta superfamily play critical roles in chondrogenesis. However, adult stem cells derived from various tissues tend to differ in their chondrogenic potential. Pluripotent stem cells have unlimited proliferative capacity compared to adult stem cells. Chondrogenesis from embryonic stem (ES cells has been studied for more than a decade. However, establishment of ES cells requires embryos and leads to ethical issues for clinical applications. Induced pluripotent stem (iPS cells are generated by cellular reprogramming of adult cells by transcription factors. Although iPS cells have chondrogenic potential, optimization, generation and differentiation toward articular chondrocytes are currently under intense investigation.

  4. 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.

  5. Abnormal number cell division of human thyroid anaplastic carcinoma cell line, SW 1736

    Directory of Open Access Journals (Sweden)

    Keiichi Ikeda

    2015-12-01

    Full Text Available Cell division, during which a mother cell usually divides into two daughter cells during one cell cycle, is the most important physiological event of cell biology. We observed one-to-four cell division during imaging of live SW1736 human thyroid anaplastic carcinoma cells transfected with a plasmid expressing the hybrid protein of green fluorescent protein and histone 2B (plasmid eGFP-H2B. Analysis of the images revealed a mother cell divided into four daughter cells. And one of the abnormally divided daughter cells subsequently formed a dinucleate cell.

  6. Proteins differentially expressed in human beta-cells-enriched pancreatic islet cultures and human insulinomas

    DEFF Research Database (Denmark)

    Terra, Letícia F; Teixeira, Priscila C; Wailemann, Rosangela A M

    2013-01-01

    In view of the great demand for human beta-cells for physiological and medical studies, we generated cell lines derived from human insulinomas which secrete insulin, C-peptide and express neuroendocrine and islet markers. In this study, we set out to characterize their proteomes, comparing them t...

  7. Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

    Science.gov (United States)

    Lopez, Ana D.; Kayali, Ayse G.; Hayek, Alberto; King, Charles C.

    2014-01-01

    For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells. PMID:24895054

  8. Isolation, culture, and imaging of human fetal pancreatic cell clusters.

    Science.gov (United States)

    Lopez, Ana D; Kayali, Ayse G; Hayek, Alberto; King, Charles C

    2014-05-18

    For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation(1-9). However in vitro, genesis of insulin producing cells from human fetal ICCs is low(10); results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust(11-17). A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro(11-22), far fewer exist for ICCs(10,23,24). Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue(6). Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

  9. 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.

  10. Melatonin Sensitizes H1975 Non-Small-Cell Lung Cancer Cells Harboring a T790M-Targeted Epidermal Growth Factor Receptor Mutation to the Tyrosine Kinase Inhibitor Gefitinib

    Directory of Open Access Journals (Sweden)

    Miyong Yun

    2014-08-01

    Full Text Available Background/Aims: The use of tyrosine kinase inhibitors (TKIs to target active epidermal growth factor receptor (EGFR-harbouring mutations has been effective in patients with advanced non-small-cell lung cancer (NSCLC. However, the use of TKIs in NSCLS patients with somatic EGFR mutations, particularly T790M, causes drug resistance. Thus, in the present study, we investigated overcoming resistance against the TKI gefitinib by combination treatment with melatonin in H1975 NSCLC cells harbouring the T790M somatic mutation. Methods: H1975 and HCC827 cells were treated with melatonin in combination with gefitinib, and cell viability, cell cycle progression, apoptosis, and EGFR, AKT, p38, Bcl-2, Bcl-xL, caspase 3 and Bad protein levels were examined. Results: Treatment with melatonin dose-dependently decreased the viability of H1975 cells harbouring the T790M somatic mutation compared to HCC827 cells with an EGFR active mutation. Melatonin-mediated cell death resulted in decreased phosphorylation of EGFR and Akt, leading to attenuated expression of survival proteins, such as Bcl-2, Bcl-xL and survivin, and activated caspase 3 in H1975 cells, but not in HCC827 cells. However, we did not observe a significant change in expression of cell cycle proteins, such as cyclin D, cyclin A, p21 and CDK4 in H1975 cells. Surprisingly, co-treatment of gefitinib with melatonin effectively decreased the viability of H1975 cells, but not HCC827 cells. Moreover, co-treatment of H1975 cells caused consistent down-regulation of EGFR phosphorylation and induced apoptosis compared to treatment with gefitinib or melatonin alone. Conclusions: Our findings demonstrate that melatonin acts as a potent chemotherapeutic agent by sensitising to gefitinib TKI-resistant H1975 cells that harbour a EGFR T790M mutation.

  11. Melatonin sensitizes H1975 non-small-cell lung cancer cells harboring a T790M-targeted epidermal growth factor receptor mutation to the tyrosine kinase inhibitor gefitinib.

    Science.gov (United States)

    Yun, Miyong; Kim, Eun-Ok; Lee, Duckgue; Kim, Ji-Hyun; Kim, Jaekwang; Lee, Hyemin; Lee, Jihyun; Kim, Sung-Hoon

    2014-01-01

    The use of tyrosine kinase inhibitors (TKIs) to target active epidermal growth factor receptor (EGFR)-harbouring mutations has been effective in patients with advanced non-small-cell lung cancer (NSCLC). However, the use of TKIs in NSCLS patients with somatic EGFR mutations, particularly T790M, causes drug resistance. Thus, in the present study, we investigated overcoming resistance against the TKI gefitinib by combination treatment with melatonin in H1975 NSCLC cells harbouring the T790M somatic mutation. H1975 and HCC827 cells were treated with melatonin in combination with gefitinib, and cell viability, cell cycle progression, apoptosis, and EGFR, AKT, p38, Bcl-2, Bcl-xL, caspase 3 and Bad protein levels were examined. Treatment with melatonin dose-dependently decreased the viability of H1975 cells harbouring the T790M somatic mutation compared to HCC827 cells with an EGFR active mutation. Melatonin-mediated cell death resulted in decreased phosphorylation of EGFR and Akt, leading to attenuated expression of survival proteins, such as Bcl-2, Bcl-xL and survivin, and activated caspase 3 in H1975 cells, but not in HCC827 cells. However, we did not observe a significant change in expression of cell cycle proteins, such as cyclin D, cyclin A, p21 and CDK4 in H1975 cells. Surprisingly, co-treatment of gefitinib with melatonin effectively decreased the viability of H1975 cells, but not HCC827 cells. Moreover, co-treatment of H1975 cells caused consistent down-regulation of EGFR phosphorylation and induced apoptosis compared to treatment with gefitinib or melatonin alone. Our findings demonstrate that melatonin acts as a potent chemotherapeutic agent by sensitising to gefitinib TKI-resistant H1975 cells that harbour a EGFR T790M mutation. © 2014 S. Karger AG, Basel.

  12. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

    OpenAIRE

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34+ hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex...

  13. Human Soluble TRAIL Protein Inducing Apoptosis in Osteosarcoma Cell

    Institute of Scientific and Technical Information of China (English)

    ZHU Shaobo; YU Aixi; ZHANG Zhongning; WU Gang

    2007-01-01

    This study is to examine the effect of human recombinant soluble TRAIL (TNF-related apoptosis-inducing ligand) protein inducing apoptosis in MG-63 human osteosarcoma cells. The inhibitive rates of TRAIL to MG-63 cells were detected by MTT assay. The apoptosis induced by TRAIL in MG-63 human osteosarcoma cells was analyzed with FACS and TUNEL and the apoptotic bodies were observed by transmission electron microscope. MTT assay showed that the inhibitive rates of 500, 1 000,2 000 and 4 000 ng/mL TRAIL for 24 h were 10.1%, 24.3%,50.6% and 97.7% respectively. Flow cytometric analysis showed that after MG-63 cells were treated with 2 μg/mL TRAIL for 6 h,obvious apoptotic peak would immediately appear before diploid peak. Human soluble TRAIL protein can quickly kill MG-63 osteosarcoma cells selectively, and may have potential value for clinical treatment of osteosarcoma.

  14. Generation and application of human iPS cells

    Institute of Scientific and Technical Information of China (English)

    CUI Ghun; RAO LingJun; CHENG LinZhao; XIAO Lei

    2009-01-01

    Human embryonic stem (ES) cells are capable of unlimited proliferation and maintenance of pluripo-tency in vitro; these properties may lead to potential applications in regenerative medicine.However,immune rejection hampers the allogenic application of human ES cells.Over-expression of several specific transcription factors has been used to reprogram human adult cells into induced pluripotent stem (iPS) cells,which are similar to hESCs in many aspects.The iPS technique makes it possible to produce patient-specific pluripotent stem cells for transplantation therapy without immune rejection.However,some challenges remain,including viral vector integration into the genome,the existence of exogenous oncogenic factors,and low induction efficiency.Here,we review recent advances in human iPS methodology,as well as remaining challenges and its potential applications.

  15. Effect of Human Cytomegalovirus Infection on Nerve Growth Factor Expression in Human Glioma U251 Cells

    Institute of Scientific and Technical Information of China (English)

    HAI-TAO WANG; BIN WANG; ZHI-JUN LIU; ZHI-QIANG BAI; LING LI; HAI-YAN LIU; DONG-MENG QIAN; ZHI-YONG YAN; XU-XIA SONG

    2009-01-01

    Objectives To explore the change of endogenic nerve growth factor (NGF) expression in human glioma cells infected with human cytomegalovirus (HCMV). Methods U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. Results The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group (P<0.05). Conclusion HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.

  16. The Adhesion of Lactobacillus salivarius REN to a Human Intestinal Epithelial Cell Line Requires S-layer Proteins

    Science.gov (United States)

    Wang, Ran; Jiang, Lun; Zhang, Ming; Zhao, Liang; Hao, Yanling; Guo, Huiyuan; Sang, Yue; Zhang, Hao; Ren, Fazheng

    2017-01-01

    Lactobacillus salivarius REN, a novel probiotic isolated from Chinese centenarians, can adhere to intestinal epithelial cells and subsequently colonize the host. We show here that the surface-layer protein choline-binding protein A (CbpA) of L. salivarius REN was involved in adherence to the human colorectal adenocarcinoma cell line HT-29. Adhesion of a cbpA deletion mutant was significantly reduced compared with that of wild-type, suggesting that CbpA acts as an adhesin that mediates the interaction between the bacterium and its host. To identify the molecular mechanism of adhesion, we determined the crystal structure of a truncated form of CbpA that is likely involved in binding to its cell-surface receptor. The crystal structure identified CbpA as a peptidase of the M23 family whose members harbor a zinc-dependent catalytic site. Therefore, we propose that CbpA acts as a multifunctional surface protein that cleaves the host extracellular matrix and participates in adherence. Moreover, we identified enolase as the CbpA receptor on the surface of HT-29 cells. The present study reveals a new class of surface-layer proteins as well as the molecular mechanism that may contribute to the ability of L. salivarius REN to colonize the human gut. PMID:28281568

  17. Derivation of novel human ground state naive pluripotent stem cells.

    Science.gov (United States)

    Gafni, Ohad; Weinberger, Leehee; Mansour, Abed AlFatah; Manor, Yair S; Chomsky, Elad; Ben-Yosef, Dalit; Kalma, Yael; Viukov, Sergey; Maza, Itay; Zviran, Asaf; Rais, Yoach; Shipony, Zohar; Mukamel, Zohar; Krupalnik, Vladislav; Zerbib, Mirie; Geula, Shay; Caspi, Inbal; Schneir, Dan; Shwartz, Tamar; Gilad, Shlomit; Amann-Zalcenstein, Daniela; Benjamin, Sima; Amit, Ido; Tanay, Amos; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

    2013-12-12

    Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation

  18. Mesenchymal Stem Cell Levels of Human Spinal Tissues.

    Science.gov (United States)

    Harris, Liam; Vangsness, C Thomas

    2017-09-06

    .: Systematic Review. .: The aim of this study was to investigate, quantify, compare and compile the various mesenchymal stem cell tissue sources within human spinal tissues to act as a compendium for clinical and research application. .: Recent years have seen a dramatic increase in academic and clinical understanding of human mesenchymal stem cells (MSCs). Previously limited to cells isolated from bone marrow, the past decade has illicited the characterization and isolation of human MSCs from adipose, bone marrow, synovium, muscle, periosteum, peripheral blood, umbilical cord, placenta and numerous other tissues. As researchers explore practical applications of cells in these tissues, the absolute levels of MSCs in specific spinal tissue will be critical to guide future research. .: The PubMED, MEDLINE, EMBASE and Cochrane databases were searched for articles relating to the harvest, characterization, isolation and quantification of human mesenchymal stem cells from spinal tissues. Selected articles were examined for relevant data, categorized according to type of spinal tissue, and when possible, standardized to facilitate comparisons between sites. .: Human mesenchymal stem cell levels varied widely between spinal tissues. Yields for Intervertebral disc demonstrated roughly 5% of viable cells to be positive for MSC surface markers. Cartilage endplate cells yielded 18,500- 61,875 cells/ 0.8 mm thick sample of cartilage end plate. Ligamentum flavum yielded 250,000- 500,000 cells per gram of tissue. Annulus fibrosus FACS treatment found 29% of cells positive for MSC marker Stro-1. Nucleus pulposus yielded mean tissue samples of 40,584-234,137 MSCs/gram of tissue. .: Numerous tissues within and surrounding the spine represent a consistent and reliable source for the harvest and isolation of human mesenchymal stem cells. Among the tissues of the spine, the annulus fibrosus and ligamentum flavum each offer considerable levels of mesenchymal stem cells, and may

  19. Radiogenic transformation of human mammary epithelial cells in vitro

    Science.gov (United States)

    Yang, T. C.; Georgy, K. A.; Tavakoli, A.; Craise, L. M.; Durante, M.

    1996-01-01

    Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

  20. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Stefania Bruno

    2016-01-01

    Full Text Available Human liver stem cells (HLSCs are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs, and dendritic cells (DCs in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2 and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs, HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

  1. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation.

    Science.gov (United States)

    Bruno, Stefania; Grange, Cristina; Tapparo, Marta; Pasquino, Chiara; Romagnoli, Renato; Dametto, Ennia; Amoroso, Antonio; Tetta, Ciro; Camussi, Giovanni

    2016-01-01

    Human liver stem cells (HLSCs) are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs), and dendritic cells (DCs) in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2) and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs), HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

  2. 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.

  3. Isolation and culture of human umbilical vein endothelial cells (HUVEC).

    Science.gov (United States)

    Cheung, Ambrose L

    2007-02-01

    Human-derived endothelial cells can now be routinely harvested from human umbilical veins. Studies with human umbilical vein endothelial cells (HUVEC) have been conducted with cells from passage 2 to 5. It is now also possible to cryopreserve primary and early-passaged HUVEC for future propagation and for forwarding to an end user by express courier. Stored HUVEC have been stably retrieved even after several years. These retrieval techniques have facilitated the deployment of HUVEC for many studies, including those for homeostasis, inflammatory disorders, atherosclerosis, cancer, and microbial adhesion and invasion. In this unit, we will delineate the procedure for harvesting, propagation, and storage of HUVEC.

  4. Chemical Conversion of Human Fibroblasts into Functional Schwann Cells

    Directory of Open Access Journals (Sweden)

    Eva C. Thoma

    2014-10-01

    Full Text Available Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

  5. Stem cell markers in the heart of the human newborn

    Directory of Open Access Journals (Sweden)

    Armando Faa

    2016-07-01

    Full Text Available The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. Several recent studies now show that the different cell types that characterize the adult human heart arise from a common ancestor. Human cardiac stem cells differentiate into cardiomyocytes, and, in lesser extent, into smooth muscle and endothelial cells. The characterization of human cardiac stem cells (CSCs has important clinical implications. In recent years, CD117 (c-kit has been reported to mark a subtype of stem/progenitor cells in the human heart, with stem cell-like properties, including the ability to self-renewal and clonogenicity multipotentiality. Proceedings of the 2nd International Course on Perinatal Pathology (part of the 11th International Workshop on Neonatology · October 26th-31st, 2015 · Cagliari (Italy · October 31st, 2015 · Stem cells: present and future Guest Editors: Gavino Faa, Vassilios Fanos, Antonio Giordano

  6. Calorimetric signatures of human cancer cells and their nuclei

    Energy Technology Data Exchange (ETDEWEB)

    Todinova, S. [Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113 (Bulgaria); Stoyanova, E. [Department of Molecular Immunology, Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, Tzarigradsko shose Blvd. 73, Sofia 1113 (Bulgaria); Krumova, S., E-mail: sakrumo@gmail.com [Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113 (Bulgaria); Iliev, I. [Institute of Experimental Morphology, Pathology and Anthropology with Museum, Acad. G. Bonchev Str., Bl. 25, Sofia 1113 (Bulgaria); Taneva, S.G. [Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113 (Bulgaria)

    2016-01-10

    Graphical abstract: - Highlights: • Two temperature ranges are distinguished in the thermograms of cells/nuclei. • Different thermodynamic properties of cancer and normal human cells/nuclei. • Dramatic reduction of the enthalpy of the low-temperature range in cancer cells. • Oxaliplatin and 5-FU affect the nuclear matrix proteins and the DNA stability. - Abstract: The human cancer cell lines HeLa, JEG-3, Hep G2, SSC-9, PC-3, HT-29, MCF7 and their isolated nuclei were characterized by differential scanning calorimetry. The calorimetric profiles differed from normal human fibroblast (BJ) cells in the two well distinguished temperature ranges—the high-temperature range (H{sub T}, due to DNA-containing structures) and the low-temperature range (L{sub T}, assigned to the nuclear matrix and cellular proteins). The enthalpy of the L{sub T} range, and, respectively the ratio of the enthalpies of the L{sub T}- vs. H{sub T}-range, ΔH{sub L}/ΔH{sub H}, is strongly reduced for all cancer cells compared to normal fibroblasts. On the contrary, for most of the cancer nuclei this ratio is higher compared to normal nuclei. The HT-29 human colorectal cancer cells/nuclei differed most drastically from normal human fibroblast cells/nuclei. Our data also reveal that the treatment of HT-29 cancer cells with cytostatic drugs affects not only the DNA replication but also the cellular proteome.

  7. 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.

  8. 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...

  9. 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.

  10. Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Xuan Liu

    2015-05-01

    Full Text Available Hexavalent chromium (Cr(VI is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI induction. In chromate-reducing ability test, HepG2-YieF cells that harbored the reductase showed significantly higher reducing ability of Cr(VI than HepG2 control cells. This result was further supported by the evidence of increased Cr(VI-removing ability of crude cell extract of HepG2-YieF. Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI stress. Subcellular localization of YieF was determined by tracing GFP-YieF fusion protein that was detected in both nucleus and cytoplasm by laser confocal microscopy. Altogether, this study successfully demonstrated that the expression of a prokaryotic Cr(VI-reducing gene yieF endowed mammalian cell HepG2 with enhanced chromate resistance, which brought new insight of Cr(VI detoxification in mammalian cells.

  11. Chromate Reductase YieF from Escherichia coli Enhances Hexavalent Chromium Resistance of Human HepG2 Cells.

    Science.gov (United States)

    Liu, Xuan; Wu, Gaofeng; Zhang, Yanli; Wu, Dan; Li, Xiangkai; Liu, Pu

    2015-05-26

    Hexavalent chromium (Cr(VI)) is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction. In chromate-reducing ability test, HepG2-YieF cells that harbored the reductase showed significantly higher reducing ability of Cr(VI) than HepG2 control cells. This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF. Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress. Subcellular localization of YieF was determined by tracing GFP-YieF fusion protein that was detected in both nucleus and cytoplasm by laser confocal microscopy. Altogether, this study successfully demonstrated that the expression of a prokaryotic Cr(VI)-reducing gene yieF endowed mammalian cell HepG2 with enhanced chromate resistance, which brought new insight of Cr(VI) detoxification in mammalian cells.

  12. Rho GTPase expression in human myeloid cells.

    Directory of Open Access Journals (Sweden)

    Suzanne F G van Helden

    Full Text Available Myeloid cells are critical for innate immunity and the initiation of adaptive immunity. Strict regulation of the adhesive and migratory behavior is essential for proper functioning of these cells. Rho GTPases are important regulators of adhesion and migration; however, it is unknown which Rho GTPases are expressed in different myeloid cells. Here, we use a qPCR-based approach to investigate Rho GTPase expression in myeloid cells.We found that the mRNAs encoding Cdc42, RhoQ, Rac1, Rac2, RhoA and RhoC are the most abundant. In addition, RhoG, RhoB, RhoF and RhoV are expressed at low levels or only in specific cell types. More differentiated cells along the monocyte-lineage display lower levels of Cdc42 and RhoV, while RhoC mRNA is more abundant. In addition, the Rho GTPase expression profile changes during dendritic cell maturation with Rac1 being upregulated and Rac2 downregulated. Finally, GM-CSF stimulation, during macrophage and osteoclast differentiation, leads to high expression of Rac2, while M-CSF induces high levels of RhoA, showing that these cytokines induce a distinct pattern. Our data uncover cell type specific modulation of the Rho GTPase expression profile in hematopoietic stem cells and in more differentiated cells of the myeloid lineage.

  13. Human embryonic stem cell derivation and directed differentiation.

    Science.gov (United States)

    Trounson, A

    2005-01-01

    Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much

  14. Generating trunk neural crest from human pluripotent stem cells

    OpenAIRE

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

    2016-01-01

    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 typi...

  15. Hematopoietic Development from Human Induced Pluripotent Stem Cells

    OpenAIRE

    2009-01-01

    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. circ...

  16. Lymphoreticular cells in human brain tumours and in normal brain.

    OpenAIRE

    1982-01-01

    The present investigation, using various rosetting assays of cell suspensions prepared by mechanical disaggregation or collagenase digestion, demonstrated lymphoreticular cells in human normal brain (cerebral cortex and cerebellum) and in malignant brain tumours. The study revealed T and B lymphocytes and their subsets (bearing receptors for Fc(IgG) and C3) in 5/14 glioma suspensions, comprising less than 15% of the cell population. Between 20-60% of cells in tumour suspensions morphologicall...

  17. In Vitro Generation of IL-35-expressing Human Wharton's Jelly-derived Mesenchymal Stem Cells Using Lentiviral Vector.

    Science.gov (United States)

    Amari, Afshin; Ebtekar, Massoumeh; Moazzeni, Seyed Mohammad; Soleimani, Masoud; Mohammadi Amirabad, Leila; Tahoori, Mohammad Taher; Massumi, Mohammad

    2015-08-01

    Human Wharton's Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) are easily available cells without transplant rejection problems or ethical concerns compared to bone-marrow-derived MSCs for prospective clinical applications. These cells display immunosuppressive properties and may be able to play an important role in autoimmune disorders. Regulatory T-cells (Treg) are important to prevent autoimmune disease development. Interleukin 35 (IL-35) induces the proliferation of Treg cell populations and reduces the activity of T helper 17 (Th17) and T helper 1 (Th1) cells, which play a central role in initiation of inflammation and autoimmune disease. Recent studies identified IL-35 as a new inhibitory cytokine required for the suppressive function of Treg cells. We created IL-35-producing hWJ-MSCs as a good vehicle for reduction of inflammation and autoimmune diseases. We isolated hWJ-MSCs based on explant culture. HWJ-MSCs were transduced at MOI=50 (Multiplicity of Infection) with lentiviral particles harboring murine Interleukin 35 (mIL-35). Expression of IL-35 in hWJ-MSCs was quantified by an IL-35 ELISA kit. IL-35 bioactivity was analyzed by inhibiting the proliferation of mouse splenocytes using CFSE cell proliferation kit. Frequency of CD4+CD25+CD127 low/neg Foxp3+ Treg cells was measured by flow cytometry. There was an up to 85% GFP positive transduction rate, and the cells successfully released a high level of mIL-35 protein (750 ng/ml). IL-35 managed to inhibit CD4+ T cell proliferation with PHA, and improved the frequency of Treg cells. Our data suggest that transduced hWJ-MSCs overexpressing IL-35 may provide a useful approach for basic research on gene therapy for autoimmune disorders.

  18. Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

    Science.gov (United States)

    Liévin-Le Moal, Vanessa

    2013-01-01

    SUMMARY Hosts are protected from attack by potentially harmful enteric microorganisms, viruses, and parasites by the polarized fully differentiated epithelial cells that make up the epithelium, providing a physical and functional barrier. Enterovirulent bacteria interact with the epithelial polarized cells lining the intestinal barrier, and some invade the cells. A better understanding of the cross talk between enterovirulent bacteria and the polarized intestinal cells has resulted in the identification of essential enterovirulent bacterial structures and virulence gene products playing pivotal roles in pathogenesis. Cultured animal cell lines and cultured human nonintestinal, undifferentiated epithelial cells have been extensively used for understanding the mechanisms by which some human enterovirulent bacteria induce intestinal disorders. Human colon carcinoma cell lines which are able to express in culture the functional and structural characteristics of mature enterocytes and goblet cells have been established, mimicking structurally and functionally an intestinal epithelial barrier. Moreover, Caco-2-derived M-like cells have been established, mimicking the bacterial capture property of M cells of Peyer's patches. This review intends to analyze the cellular and molecular mechanisms of pathogenesis of human enterovirulent bacteria observed in infected cultured human colon carcinoma enterocyte-like HT-29 subpopulations, enterocyte-like Caco-2 and clone cells, the colonic T84 cell line, HT-29 mucus-secreting cell subpopulations, and Caco-2-derived M-like cells, including cell association, cell entry, intracellular lifestyle, structural lesions at the brush border, functional lesions in enterocytes and goblet cells, functional and structural lesions at the junctional domain, and host cellular defense responses. PMID:24006470

  19. Cardiac Progenitor Cell Extraction from Human Auricles

    KAUST Repository

    Di Nardo, Paolo

    2017-02-22

    For many years, myocardial tissue has been considered terminally differentiated and, thus, incapable of regenerating. Recent studies have shown, instead, that cardiomyocytes, at least in part, are slowly substituted by new cells originating by precursor cells mostly embedded into the heart apex and in the atria. We have shown that an elective region of progenitor cell embedding is represented by the auricles, non-contractile atria appendages that can be easily sampled without harming the patient. The protocol here reported describes how from auricles a population of multipotent, cardiogenic cells can be isolated, cultured, and differentiated. Further studies are needed to fully exploit this cell population, but, sampling auricles, it could be possible to treat cardiac patients using their own cells circumventing rejection or organ shortage limitations.

  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. Dark cells in human oral leukoplakias

    Energy Technology Data Exchange (ETDEWEB)

    Klein-Szanto, A.J.P. (Oak Ridge National Lab., TN); Sega, M.; Banoczy, J.; Albrecht, M.

    1982-01-01

    Dark basal keratinocytes, characterized by a strong affinity for basic dyes and by electron density of cytoplasm and nucleus, could be recognized in eleven oral leukoplakias. The percentage of dark cells was higher in the group comprising leukoplakias verrucosa, and erosiva (28% of the basal cells) than in the leukoplakia simplex group (10%). The presence of these cells is a good indicator of the degree of histological dysplasia and correlates well with the preneoplastic potential of these lesions.

  3. Cell sources for in vitro human liver cell culture models.

    Science.gov (United States)

    Zeilinger, Katrin; Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

    2016-09-01

    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described.

  4. Staphylococcal SSL5 Binding to Human Leukemia Cells Inhibits Cell Adhesion to Endothelial Cells and Platelets

    Directory of Open Access Journals (Sweden)

    Annemiek M. E. Walenkamp

    2010-01-01

    Full Text Available Bacterial proteins provide promising tools for novel anticancer therapies. Staphylococcal superantigen-like 5 (SSL5 was recently described to bind P-selectin glycoprotein ligand-1 (PSGL-1 on leukocytes and to inhibit neutrophil rolling on a P-selectin surface. As leukocytes and tumor cells share many characteristics in migration and dissemination, we explored the potential of SSL5 as an antagonist of malignant cell behavior. Previously, it was demonstrated that rolling of human HL-60 leukemia cells on activated endothelial cells was mediated by P-selectin. In this study, we show that SSL5 targets HL-60 cells. Binding of SSL5 was rapid and without observed toxicity. Competition of SSL5 with the binding of three anti-PSGL-1 antibodies and P-selectin to HL-60 cells identified PSGL-1 as the ligand on HL-60 cells. Presence of sialyl Lewis x epitopes on PSGL-1 was crucial for its interaction with SSL5. Importantly, SSL5 not only inhibited the interaction of HL-60 cells with activated endothelial cells but also with platelets, which both play an important role in growth and metastasis of cancers. These data support the concept that SSL5 could be a lead in the search for novel strategies against hematological malignancies.

  5. Analysis of Intracellular Calcium Signaling in Human Embryonic Stem Cells.

    Science.gov (United States)

    Péntek, Adrienn; Pászty, Katalin; Apáti, Ágota

    2016-01-01

    Measurement of changes in intracellular calcium concentration is one of the most common and useful tools for studying signal transduction pathways or cellular responses in basic research and drug screening purposes as well. Increasing number of such applications using human pluripotent stem cells and their derivatives requires development of calcium signal measurements for this special cell type. Here we describe a modified protocol for analysis of calcium signaling events in human embryonic stem cells, which can be used for other pluripotent cell types (such as iPSC) or their differentiated offspring as well.

  6. 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.

  7. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

    Science.gov (United States)

    Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F; Psathaki, Olympia E; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R; Schlenke, Peter; Zaehres, Holm

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.

  8. Antibody-Independent Function of Human B Cells Contributes to Antifungal T Cell Responses.

    Science.gov (United States)

    Li, Rui; Rezk, Ayman; Li, Hulun; Gommerman, Jennifer L; Prat, Alexandre; Bar-Or, Amit

    2017-03-08

    Fungal infections (e.g., Candida albicans) can manifest as serious medical illnesses, especially in the elderly and immune-compromised hosts. T cells are important for Candida control. Whether and how B cells are involved in antifungal immunity has been less clear. Although patients with agammaglobulinemia exhibit normal antifungal immunity, increased fungal infections are reported following B cell-depleting therapy, together pointing to Ab-independent roles of B cells in controlling such infections. To test how human B cells may contribute to fungal-associated human T cell responses, we developed a novel Ag-specific human T cell/B cell in vitro coculture system and found that human B cells could induce C. albicans-associated, MHC class II-restricted responses of naive T cells. Activated B cells significantly enhanced C. albicans-mediated Th1 and Th17 T cell responses, which were both strongly induced by CD80/CD86 costimulation. IL-6(+)GM-CSF(+) B cells were the major responding B cell subpopulation to C. albicans and provided efficient costimulatory signals to the T cells. In vivo B cell depletion in humans resulted in reduced C. albicans-associated T responses. Of note, the decreased Th17, but not Th1, responses could be reversed by soluble factors from B cells prior to depletion, in an IL-6-dependent manner. Taken together, our results implicate an Ab-independent cytokine-defined B cell role in human antifungal T cell responses. These findings may be particularly relevant given the prospects of chronic B cell depletion therapy use in lymphoma and autoimmune disease, as patients age and are exposed to serial combination therapies.

  9. Random mitotic activities across human embryonic stem cell colonies.

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Q.; Duggan, R.; Dasa, S.; Li, F.; Chen, L. (Biosciences Division)

    2010-08-01

    A systemic and quantitative study was performed to examine whether different levels of mitotic activities, assessed by the percentage of S-phase cells at any given time point, existed at different physical regions of human embryonic stem (hES) cell colonies at 2, 4, 6 days after cell passaging. Mitotically active cells were identified by the positive incorporation of 5-bromo-2-deoxyuridine (BrdU) within their newly synthesized DNA. Our data indicated that mitotically active cells were often distributed as clusters randomly across the colonies within the examined growth period, presumably resulting from local deposition of newly divided cells. This latter notion was further demonstrated by the confined growth of enhanced green florescence protein (EGFP) expressing cells amongst non-GFP expressing cells. Furthermore, the overall percentage of mitotically active cells remained constantly at about 50% throughout the 6-day culture period, indicating mitotic activities of hES cell cultures were time-independent under current growth conditions.

  10. Human airway xenograft models of epithelial cell regeneration

    Directory of Open Access Journals (Sweden)

    Puchelle Edith

    2000-10-01

    Full Text Available Abstract Regeneration and restoration of the airway epithelium after mechanical, viral or bacterial injury have a determinant role in the evolution of numerous respiratory diseases such as chronic bronchitis, asthma and cystic fibrosis. The study in vivo of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to restore a functional respiratory epithelium after several weeks. Recently, human tracheal xenografts have been developed in immunodeficient severe combined immunodeficiency (SCID and nude mice. In this review we recall that human airway cells implanted in such conditioned host grafts can regenerate a well-differentiated and functional human epithelium; we stress the interest in these humanized mice in assaying candidate progenitor and stem cells of the human airway mucosa.

  11. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Murison, G.; Chubb, C.B.H.; Maeda, S.; Gemmell, M.A.; Huberman, E.

    1987-08-01

    Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

  12. Inhibition of fatty acid metabolism reduces human myeloma cells proliferation.

    Directory of Open Access Journals (Sweden)

    José Manuel Tirado-Vélez

    Full Text Available Multiple myeloma is a haematological malignancy characterized by the clonal proliferation of plasma cells. It has been proposed that targeting cancer cell metabolism would provide a new selective anticancer therapeutic strategy. In this work, we tested the hypothesis that inhibition of β-oxidation and de novo fatty acid synthesis would reduce cell proliferation in human myeloma cells. We evaluated the effect of etomoxir and orlistat on fatty acid metabolism, glucose metabolism, cell cycle distribution, proliferation, cell death and expression of G1/S phase regulatory proteins in myeloma cells. Etomoxir and orlistat inhibited β-oxidation and de novo fatty acid synthesis respectively in myeloma cells, without altering significantly glucose metabolism. These effects were associated with reduced cell viability and cell cycle arrest in G0/G1. Specifically, etomoxir and orlistat reduced by 40-70% myeloma cells proliferation. The combination of etomoxir and orlistat resulted in an additive inhibitory effect on cell proliferation. Orlistat induced apoptosis and sensitized RPMI-8226 cells to apoptosis induction by bortezomib, whereas apoptosis was not altered by etomoxir. Finally, the inhibitory effect of both drugs on cell proliferation was associated with reduced p21 protein levels and phosphorylation levels of retinoblastoma protein. In conclusion, inhibition of fatty acid metabolism represents a potential therapeutic approach to treat human multiple myeloma.

  13. Connexin mutant embryonic stem cells and human diseases

    Institute of Scientific and Technical Information of China (English)

    Kiyomasa; Nishii; Yosaburo; Shibata; Yasushi; Kobayashi

    2014-01-01

    Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin(Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells(ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.

  14. Therapeutic potentials of human embryonic stem cells in Parkinson's disease

    National Research Council Canada - National Science Library

    Newman, Mary B; Bakay, Roy A E

    2008-01-01

    .... The isolation, differentiation, and long-term cultivation of human embryonic stem cells and the therapeutic research discovery made in relation to the beneficial properties of neurotrophic and neural...