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Human Mesenchymal Stem Cell Proliferation and Differentiation using a 3D-clinostat Culture.    Louis Yuge1, Yumi Kawahara1, Chika Umeda1, Reiko Yoshimoto1, Hidetoshi Tahara2, Teruyuki Kajiume2, Toshinori Ide2, & Katsuko Kataoka2 (1Graduate School of Health Sciences, 2Graduate School of Biomedical Sciences, Hiroshima University, Japan)

   A 3D-clinostat is a multi-directional gravity device for simulating microgravity. By controlled rotation of two axes, a 3D-clinostat minimizes the cumulative gravity vector in cells cultured at the center of the device and makes 10-3 G average over time.

   We cultured human mesenchymal stem cells (hMSC) in the 3D-clinostat (group CL) or in normal 1G environment (group C) in a growth medium for 3 days, and cell pellets were obtained.  FACS analysis after 3 day-culture showed CD 44/CD 29 double positive cells being 12.8-times increased in group CL as compared with group C.  Telomere length of hMSCs did not change during culture periods for both groups, CL and C.   Telomerase activity was undetectable in these hMSC cells.  We examined cytoskeltal change  in both groups, CL and C. Number of focal contacts were much decreased in group CL as compared with group C.  Then, we transplanted the pellets of the 3 day-culture into the cartilage defect made in the intercondylar fossa of the mouse femur.  After 2 weeks, the transplants from group CL formed hyaline cartilage.  On the contrary, non-cartilage tissue containing a few cells, was found in the transplants from group C.  Here we show that hMSC proliferate better and remain more undifferentiated in culture in a 3D-clinostat.  These hMSC which are more viable, and undergo better chondrocyte differentiation after transplantation.   (Supported by “Ground Research Announcement for Space Utilization” and Ministry of Education, Science, Sports, Culture and Technology of Japan)