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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 | Changes in the epidemiolngic profile of sudden infant death syndrome as rates decline among California infants:1990-1995显示文摘 | Adams E J Chavez G F Steen D | 1998 | Pediatrics1998，102，6： | 1 |
| 3 | Structure of new bioactive triterpenes related to 22-β-hydroxy tingenone显示文摘 | CHAVEZ H VALDIVIS E ESTEVEZ-BRAUN A RAVELO A G | 1998 | Tetrahedron1998，54，13： | 1 |
| 4 | Joint interpretation of gravity and magnetic data over axial symmetric bodies with application to the Darnley Bay anomaly显示文摘 | CHAVEZ R E BAILEY R C GARLAND G D | 1987 | Nwt Canada Geophysical Prospecting1987，35，： | 1 |
| 5 | Genetic and phenotypic variation across a hybrid zone between ecologically divergent tree squirrels (Tamiasciurus) 显示文摘 | Chavez A S Saltzberg C J Kenagy G J | 2001 | Molecular Ecology2001，20，6： | 1 |
| 6 | Internalization of SMmonellatyphimurium into mango pulp and pre- vention of fruit pulp contamination by chlorine and copper ions显示文摘 | Soto M Chavez G Baez M | 2007 | International Journal of En- vironmental Health Research2007，17，6： | 1 |
| 7 | Time of birth and the risk of neonatal death显示文摘 | Gould J B Qin C Chavez G | 2005 | Obstet Gynecol2005，106，20： | 1 |
| 8 | Statistical Method for Sel-t Landsat MSS Ratios 显示文摘 | Chavez P S Berlin G L Sowers L B | 1982 | Journal of Applied Photographic Engineering (S0098-7298)1982，80，： | 1 |
| 9 | Statistical method for selecting Landsat MSS ratios显示文摘 | Chavez P S Berlin G L Sowers L B | 1982 | Journal of Applied Photographic Engineering1982，8，1： | 1 |
| 10 | Dynamic Granger-causal networks of electricity spot prices: A novel approach to market integration 显示文摘 | Castagneto-Gissey G Chavez M De Vico Fallani F | 2014 | Energy Economics2014，44，1： | 1 |
| 11 | Fault detection and classification of analog circuits by means of fuzzy logic based techniques 显示文摘 | TORRALBA A CHAVEZ J FRANQUELO L G | 1990 | Proceedings of IEEE International Symposium on Circuits and Systems1990，，： | 1 |
| 12 | Removal of phenanthrene from soil by co - cultures of bacteria and fungi pregrown on sugarcane bagasse pith 显示文摘 | CHAVEZ G B QUINTERO R Esparza G F | 2008 | Bioresource Technology2008，89，2： | 1 |
| 13 | Searching in metric spaces显示文摘 | Chavez E Navarro G Baeza-Yates R | 2001 | ACM Computing Surveys2001，33，3： | 1 |
| 14 | GS-9620,an oral agonist of Toll-like receptor-7,induces prolonged suppression of hepatitis B virus in chronically infected chimpanzees显示文摘 | Lanford R E Guerra B Chavez D Frey C R Ohmstede C Hesselgesser J Kearney B Moorehead L Subramanian G M McHutchison J G | 2013 | Gastroenterology2013，144，7： | 1 |
| 15 | Cross-listing and Liquidity in Emerging Market Stocks显示文摘 | Silva A C Chavez G A | 2008 | Journal of Banking & Finance2008，32，3： | 1 |
| 16 | Searching in metric spaces显示文摘 | Chavez E Navarro G Baeza-Yates R | 2001 | ACM Computing Surveys2001，33，3： | 1 |
| 17 | Bacteria recovered from teeth with apical periodontitis after antlmicrobial endodontie treatment显示文摘 | CHAVEZ DE PAZ LE DAHLEN G MOLANDER A | 2003 | Int Endod J2003，36，7： | 1 |
| 18 | Linear inversion of gravity data using the spectral expansion method显示文摘 | Chavez R E Garland G D | 1985 | Geophysics1985，50，5： | 1 |
| 19 | Time-dependent spectral analysis of epidemiological time-series withwavelets显示文摘 | Cazelles B Chavez M De Magny G C | 2007 | Journal of the Royal Society Interface2007，4，15： | 1 |
| 20 | Effect of streptozotocin - induced diabetes on activities of cholinesterases in the rat retina显示文摘 | Sanchez - Chavez G Salceda R | 2000 | Iubmb Life2000，49，： | 1 |
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