ESTABLISHMENT OF FEEDER- AND SERUM-FREE CULTURE SYSTEM OF ES CELLS AND ITS APPLICATION TO PROPAGATE ES CELLS FROM INBRED MICE EMBRYOS

ES细胞无饲养层、无血清培养体系的建立及其在近交系小鼠胚胎ES细胞增殖中的应用

• 批准号: 11558099
• 负责人: NIWA Hitoshi
• 金额: $ 5.18万
• 依托单位: RIKEN (THE INSTITUTE OF PHYSICAL AND CHEMICAL RESERCH) (2001)Osaka University (1999-2000)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11558099/
• 关键词: ES cells; serum; feeder cells; proliferation; インスレーター; 胚性幹細胞; 遺伝子発現; 未分化状態; 発現ライブラリー

We found the autocrine activity derived from ES cells to support ES self-renewal in the absence of serum and feeder cells, and named it KSRS. Since we could not identify KSRS by expression cloning and testing known candidates, we tried to purify it from the serum that contains the same activity. Now purification has almost finished and we will analyze the purified fraction by mass-spectrometry.We have already confirrned that this activity can support self-renewal of 129-derived ES cells by culturing them for 1 week with semi-purified fraction followed by blastocyst injection, resulting formation of chimeric mice. Moreover, this serum-free culture condition allowed propagation of ES cells from C57BL6 blastocysts very efficiently in the presence of feeder cells, so now we are trying to remove feeder cells from this condition by choosing appropriate matrix that allows efficient attachment of blastocysts.We investigated about the role of TGF-b superfamily on ES cell growth by modulation of Smad signaling pathway. Since Smad7 overexpression repress proliferation of ES cells and this negative effect can be relieved, the Smad signal would has physiological function to stimulate ES cell proliferation.

我们发现了ES细胞在无血清和饲养层细胞的情况下支持ES自我更新的自分泌活动，并将其命名为KSRS。由于我们不能通过表达克隆和检测已知的候选基因来鉴定KSRS，所以我们试图从含有相同活性的血清中纯化KSRS。目前纯化工作已经基本完成，我们将对纯化的部分进行质谱分析，我们已经证实这种活性可以支持129来源的ES细胞的自我更新，方法是将半纯化的部分培养1周，然后注射囊胚，从而形成嵌合体小鼠。此外，这种无血清培养条件可以使C57BL6胚泡中的ES细胞在有饲养层细胞存在的情况下非常有效地增殖，所以现在我们试图通过选择合适的基质来去除这种饲养层细胞，以便有效地附着囊胚。我们研究了转化生长因子-β超家族通过调节Smad信号通路对ES细胞生长的影响。由于Smad7的过表达抑制了ES细胞的增殖，这一负面影响可以缓解，因此Smad7信号可能具有刺激ES细胞增殖的生理功能。

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