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| 1 | Simplified three-dimensional culture system for long-term expansion of embryonic stem cells显示文摘AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging. METHODS: Mouse embryonic stem cells(ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional(3-D) self-assembling scaffolds and compared with traditional two-dimentional(2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate(PEG-4-Acr) and thiolfunctionalized dextran(Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoB lue(PB) assays. Genetic expression of pluripotency markers(Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D cultureconditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining(Oct4 and Nanog) and western blot analysis(Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers. RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH(1:1 v/v) to a final concentration of 5%(w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels(P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury(mesoderm), NCAM(ectoderm), and GATA4(endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively. CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation. | Christina McKee Mick Perez-Cruet Ferman Chavez G Rasul Chaudhry | 2015 | World Journal of Stem Cells2015，7，7： | 2 |
| 2 | Policy,Politics and Path Dependency:Sport Development in Australia and Finland显示文摘 | Mick G Shane C | 2008 | Sport Management Review2008，11，3： | 1 |
| 3 | Paradoxes of technology:consumer cognizance,emotions,and coping strategies显示文摘 | MICK DAVID G SUSAN F | | 0，，： | 1 |
| 4 | S3-Leitlinie ?Exokrines Pankreaskarzinom” 2007显示文摘 | G Adler T Seufferlein S Bischoff H.-J Brambs S Feuerbach G Grabenbauer S Hahn V Heinemann W Hohenberger J Langrehr M Lutz O Micke H Neuhaus P Neuhaus H Oettle P Schlag R Schmid W Schmiegel K Schlottmann J Werner B Wiedenmann I Kopp | 2007 | Z Gastroenterol2007，，06： | 1 |
| 5 | Visual rhetoric in advertising: text-interpretive, experimental, and reader-response analyses显示文摘 | Mcquarrie E F Mick D G | 1999 | Journal of Consumer Research1999，26，1： | 1 |
| 6 | Protein intake during gestation affects postnatal bovine skeletal muscle growth and relative expression of IGF1 ,IGF1R ,IGF2 and IGF2R显示文摘 | Micke G C Sullivan T M McMillen I C | 2011 | Molecular and Cellular Endocrinology2011，332，12： | 1 |
| 7 | Insulin treatment reduces preprandial plasma ghrelin concentrations in children with type 1 diabetes显示文摘 | Ashraf A Mick G Meleth S | 2007 | Med Sci Monit2007，13，12： | 1 |
| 8 | Rediscovering satisfaction显示文摘 | Fournier S Mick D G | 1999 | Journal of Marketing1999，63，4： | 1 |
| 9 | Policy, politics and path dependency: Sport development in Australia and Finland 显示文摘 | MICK G SHANE C | 2008 | Sport Manage Rev2008，，11： | 1 |
| 10 | A Meaning-Based Model of Ad- vertising Experiences显示文摘 | MICK D G BUHL C | 1992 | Journal of Consumer Research1992，9，3： | 1 |
| 11 | Influence of surface treatment on the in vitro fracture resistance of zirconia-based all ceramic anterior crowns显示文摘 | Sehmitter M Lotze G B6 micke W | 2015 | Dent Mater2015，31，12： | 1 |
| 12 | Exploring antecedents and consequences of customer creativity in a problem-solving context显示文摘 | Burroughs J Mick D G | | 0，，32： | 1 |
| 13 | Expression patterns of a glutamate-binding protein in the rat central nervous system:comparison with N-methyl-D-aspartate receptor subunit1in rat显示文摘 | Sato K Mick G Kiyama H | 1995 | Neuroscience1995，64，2： | 1 |
| 14 | Eeffet of a topically applied whitener on sun damage to Granny Smith appIes 显示文摘 | Sibbett G S Micke W C Mitehell F G | 1991 | Catifornia Agriculture1991，45，1： | 1 |
| 15 | Self-gifts: Phenomenological in- sights from four contexts 显示文摘 | Mick D G DeMoss M | 1990 | Journal of Consumer Re- search1990，17，3： | 1 |
| 16 | Neuropathic pain in spinal cord injury: identification, classification, evaluation 显示文摘 | Calmels P Mick G Perrouin-Verbe B SOFMER (French Society for Physical Medicine and Rehabilitation) | 2009 | Ann Phys Rehabil Med2009，52，： | 1 |
| 17 | Rapid method for the de-termination of organic acids in wine by capillary electrophoresis with indi-rect UV detection显示文摘 | PERES R G MORAES E P MICKE G A | 2009 | Food Control2009，20，： | 1 |
| 18 | Evidence that the 11β-hydroxysteroid dehydrogenase ( 11-HSD1 ) is regulated by pentose pathway flux Studies in rat adipocytes and microsomes 显示文摘 | McCormick K Wang Xudong Mick G | 2006 | J Biol Chem2006，281，： | 1 |
| 19 | Rapid method for the determination of organic acids in wine by capillary electrophoresis with indirect UV detection显示文摘 | Peres R G Moraes E P Micke G A | 2009 | Food Control2009，20，： | 1 |
| 20 | Rapid method for the determination of organic acids in wine by capillary electrophoresis with indirect UV detection显示文摘 | PERES R MORAES E MICKE G | 2009 | Food Control2009，20，6： | 1 |
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