**4. Conclusion**

Cell-to-cell interaction is important to differentiate varied cells or tissues from stem cells and/ or in embryogenesis. An important component to the stem cell microenvironment is the surrounding matrix, which includes numerous chemical and biophysical cues. The changing local molecular conditions through selecting specific co-culture system might thereby provide promising method to modulate stem cell differentiation. In our researches, we selected the different kinds of cells as a feeder cell to induce the specific differentiation of stem cells. The molecular basis of induction in co-culture system remains to be understood. We tested some factors that have been implicated in the regulation of differentiation, such as GDNF, bFGF, BMP4, or neutralizing antibodies of GDNF. So far, no significant effect factors on the induction were observed. Interestingly, as showed above, the differentiation efficiency of ES cells in coculture system is as high as the efficiency in the multiple-step method with lots of growth factors treatment, or even higher than that. The possible explain for this is that supporting environment provided by co-culture basement cells, such as Sertoli, RPE or BMSCs, or an interaction between co-culture basement cells and ES cells, plays a role in their inducing activity.

In conclusion, we established a simple and effective system for the differentiation of specific cells from ES cells. Further study is warranted to establish selection methods, analyze cell functions, and transplanted for degeneration diseases. However, the co-culture system can serve as a promising method for therapeutic applications and basic research on degeneration diseases.

### **Acknowledgements**

We thank Dr. Kametani Kiyokazo and Ms. Suzuki Kayo (Research Center for Instrumental Analysis of Shinshu University, Matsumoto, Japan) for excellent technical assistance.
