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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 | Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein显示文摘 | Ducy P Mckee MD | 1997 | Nature1997，386，： | 1 |
| 3 | Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein显示文摘 | Luo G Ducy P Mckee MD | 1997 | Nature1997，386，6620： | 1 |
| 4 | Trace analy-ses for wastewaters 显示文摘 | Glaser J A Foerst D L McKee G D ei al | 1981 | Environ Sci Technol1981，15，12： | 1 |
| 5 | Improving knowledge, attitudes and beliefs about acute coronary syndrome through an individualized educational intervention: A randomized con- trolled trial显示文摘 | O ~ Brien F McKee G Mooney M | 2014 | Pat edu and coun2014，96，2： | 1 |
| 6 | Spatial configurations for visual hyperacuity显示文摘 | WESTHEMER G MCKEE S P | 1977 | Vision Research1977，17，8： | 1 |
| 7 | Spontaneous calcification of arteries and cartilage in mice lacking matrix Gla protein显示文摘 | Luo G Ducy P McKee M D | 1997 | Nature1997，386，6620： | 1 |
| 8 | Eutectic salt catalysts forgraphite and coal char gasification显示文摘 | McKee D W Spiro C L Kosky P G | 1985 | Fuel1985，64，6： | 1 |
| 9 | The distribution of signs in New Zealand Sign Language 显示文摘 | MCKEE D KENNEDY G D | 2006 | Sign Language Studies2006，6，4： | 1 |
| 10 | An ecosystem perspective of riparian zones-focus on links between land and water显示文摘 | STANLEY V G FREDERICK J S MCKEE W A | 1991 | Bioscience1991，41，8： | 1 |
| 11 | Are there meaningful longitudinal changes in health related quality of life - - SF36, in cardiac rehabil- itation patients? 显示文摘 | Mckee G | 2009 | Eur J Cardiovasc Nurs2009，8，1： | 1 |
| 12 | Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein显示文摘 | Luo G Ducy P Mckee MD Pinero GJ Lover E Behringer RR | 1997 | Nature1997，386，： | 1 |
| 13 | Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein显示文摘 | Luo G Ducy P McKee MD | 1997 | Nature1997，386，： | 1 |
| 14 | Correlations of solution rheology with electrospun fiber formation of linear and branched polyesters 显示文摘 | McKee M G Wilkes G L Colby R H | 2004 | Macromolecules2004，37，： | 1 |
| 15 | Slow- onset and fast- onset symptom presentations in acute coronary syndrome (ACS):new perspectives on prehospital delay in patients with ACS 显示文摘 | O'Donnell S McKee G Mooney M | 2014 | J Emerg Med2014，46，4： | 1 |
| 16 | Spontaneous calcification of arter- ies and cartilagein mice lacking matrix gla protein 显示文摘 | Luo G Ducy P Mckee MD | 1997 | Nature1997，386，： | 1 |
| 17 | Corridors of influence in the dissemination of customer-oriented strategy to customer contact service employees 显示文摘 | Hartline M D MaxhamⅢ J G McKee D 0(2000) | 2000 | Journal of Marketing2000，3550，： | 1 |
| 18 | Corridors of influence in the dissemination of customer-oriented strategy to customer contact service employees 显示文摘 | Hartline M D Maxham III J G Mckee D O | 2000 | Journal of Marketing2000，64，2： | 1 |
| 19 | Spontaneous calcification of ar?teries and cartilage in mice lacking matrix GLA protein显示文摘 | Luo G Ducy P Mckee MD | 1997 | Nature1997，386，6620： | 1 |
| 20 | Spatial configurations for visual hyperacuity 显示文摘 | Westhemer G Mckee S P | 1977 | Vision Research1977，17，8： | 1 |
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