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

我们发现在缺乏血清和饲养细胞的情况下，胚胎干细胞具有支持胚胎干细胞自我更新的自分泌活性，并将其命名为KSRS。由于我们无法通过表达克隆和测试已知的候选物来鉴定KSRS，我们试图从含有相同活性的血清中纯化它。现在纯化差不多完成了，我们将用质谱法分析纯化后的部分。我们已经证实，这种活性可以支持129来源的胚胎干细胞的自我更新，用半纯化的部分培养1周，然后注射囊胚，形成嵌合小鼠。此外，这种无血清培养条件允许C57BL6囊胚中ES细胞在饲养细胞存在的情况下非常有效地繁殖，因此现在我们试图通过选择合适的基质来去除这种条件下的饲养细胞，使囊胚能够有效地附着。我们研究了TGF-b超家族通过调控Smad信号通路在ES细胞生长中的作用。由于Smad7过表达会抑制ES细胞的增殖，且这种负面作用可以缓解，因此Smad信号可能具有刺激ES细胞增殖的生理功能。

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.