Standardization of embryoid body formation from embryonic stem cellsand quality estimation of embryoid bodies

胚胎干细胞拟胚体形成标准化及拟胚体质量评价

• 批准号: 18560751
• 负责人: KUROSAWA Hiroshi
• 金额: $ 2.29万
• 依托单位: University of Yamanashi
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18560751/
• 关键词: ES cell; embryoid body; quantitative PCR; EB formation method; deifferentiation; gene expression; standardization; RT-PCR; 発現遺伝子

The gene expression of EBs was quantitatively analyzed by real-time PCR and the EBs were standardized based on the data of real-time PCR.1. Formation of various types of EBs Various types of EBs were famed in different culture conditions of culture vessel, seeding cell density, oxygen, and nutrient. The EBs were famed by hanging drop method or in low-adherence 96-well multi-plate.2. Gene analysis of EBs by real-time PCRMarker genes (Oct3/4, Rex1, GATA4, GATA6, TTR, AFP, α-MHC, Nkx2.5), which represent differentiation status of EBs, were quantitatively analyzed by real-time PCR.3. Graphical presentation of gene expression pattern of EBs The gene expression pattern of EBs was graphically shown to characterize the differentiation status of EBs.4. Making sure of the relationship between gene expression pattern and actual differentiation observed in the EB differentiation cultureThe EBs highly expressed of a-MHC and Nkx2.5 generate cardiomyocytes with high efficiency. The EBs highly expressed of FIR and AFP have a trend to generate visceral yolk-sac-like structure. It was found that the gene egression pattern in EBs directly reflected the differentiation tendency to be observed in the following culture of EBs.

采用实时荧光定量PCR技术对EBs基因表达进行定量分析，并根据实时荧光定量PCR数据对EBs进行标准化。 1．不同类型EBs的形成不同类型的EBs在不同的培养容器条件下形成。通过悬滴法或低粘附96孔多孔板对接种细胞密度、氧气和营养进行观察。 2．通过实时PCR对代表EB分化状态的（Oct3/4、Rex1、GATA4、GATA6、TTR、AFP、α-MHC、Nkx2.5）进行定量分析。 3．以图形方式显示EB的基因表达模式，以表征EB的分化状态。4．确定EB分化培养物中观察到的基因表达模式与实际分化之间的关系。表达a-MHC和Nkx2.5的细胞高效率地生成心肌细胞，高表达FIR和AFP的EB有生成内脏卵黄囊样结构的趋势，发现EB中的基因排出模式直接反映了分化。在以下 EB 培养中观察到的趋势。

项目成果

Optimization of cystic embryoid body (EB) formation from mouse ES cells and expression of marker genes in differentiating EBs

小鼠 ES 细胞囊性胚状体 (EB) 形成的优化以及分化 EB 中标记基因的表达

• 发表时间: 2007
• 作者: Emiko;Yasuda;Yuji;Seki;Shuiiro;Sakaki. Hiroshi;Kurosawa
• 通讯作者: Kurosawa

Graphical presentation of gene expression pattern in various types of embryoid bodies from mouse ES cells.

小鼠 ES 细胞各种类型胚状体中基因表达模式的图形表示。

• 发表时间: 2007
• 作者: Mikiko;Koike;Shujiro;Sakaki;Yoshifumi;Amano;Hiroshi Kurosawa;Mikiko Koike;Mikiko Koike
• 通讯作者: Mikiko Koike

Graphical presentation of gene expression pattern in various types of embryoid bodies from mouse ES cells

小鼠 ES 细胞各种类型胚状体中基因表达模式的图形表示

• 发表时间: 2007
• 作者: Mikiko;Koike;Shujiro;Sakaki;Yoshifumi;Amano;Hiroshi;Kurosawa
• 通讯作者: Kurosawa

Characterization of embryoid bodies of mouse embryonic stem cells formed under various culture conditions and estimation of differentiation status of such bodies

• DOI: 10.1263/jbb.104.294
• 发表时间: 2007-10-01
• 期刊: JOURNAL OF BIOSCIENCE AND BIOENGINEERING
• 影响因子: 2.8
• 作者: Koike, Mikiko;Sakaki, Shujiro;Kurosawa, Hiroshi
• 通讯作者: Kurosawa, Hiroshi

「研究成果報告書概要(和文)」より

摘自《研究结果报告摘要（日文）》

• 发表时间: 2005
• 作者: Kawauchi;et. al.;Nishimura et al.;Dezawa et al.;Yoshizawa et al.;星野 幹雄;星野 幹雄
• 通讯作者: 星野 幹雄