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

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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.