Development of cystic embryoid bodies with visceral yolk-sac-like structures from mouse embryonic stem cells using low-adherence 96-well plate

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• 作者：Emiko  ; Yasuda¹ ; Yuji  ; Seki¹ ; Takatoshi  ; Higuchi¹ ; Fumio  ; Nakashima² ; Tomozumi  ; Noda² ; Hiroshi  ; Kurosawa¹ ;   ; ^{kurohiro@yamanashi.ac.jp}
• 关键词：ES cells ; Embryoid body ; Cystic EBs ; MPC ; Visceral yolk sac
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：2009
• 出版时间：April 2009
• 年：2009
• 卷：107
• 期：4
• 页码：442-446
• 全文大小：655 K

文摘

Cystic embryoid bodies with visceral yolk-sac-like structure (cystic EB-Vs) are used as a model for the study of early extraembryonic tissue formation containing visceral endoderm-like derivatives. In this study, we optimized the cell density of embryonic stem (ES) cells for developing cystic EB-Vs in a low-adherence 96-well plate. When ES cells were seeded at a density of 4000?cells/well, the cystic EB-Vs were most efficiently developed from ES cells via forming multicellular spherical aggregates called embryoid bodies (EBs). The suspension culture in the low-adherence plate was preferable for developing EBs into cystic EB-Vs rather than the attachment culture in the plate coated with 0.1 % gelatin. The seeding cell density of 4000?cells/well was always superior to 1000?cells/well in the efficiency of cystic EB-V development. Because the high-cell density culture generally raises the limitation of oxygen and nutrient supplies, we investigated the effects of low-oxygen and low-nutrient conditions on the development of cystic EB-Vs. It was found that low oxygen tension was not a factor for promoting the development of cystic EB-Vs. It was suggested that a low-nutrient medium is preferred for developing cystic EB-Vs rather than a sufficient-nutrient medium. In conclusion, the suspension culture in the low-adherence 96-well plate seeded with 4000 ES cells/well was optimum for developing cystic EB-Vs. The low-nutrient condition may be one of the factors for promoting the development of cystic EB-Vs.